Proceedings of the 28th Annual Southwest Nutrition and Management Conference

Tempe, Arizona February 21 & 22, 2013 Sponsored by: The University of Arizona Department of Animal Sciences

Coordinating Committee Moe Bakke Lance Baumgard Robert Collier Ken Eng Mitch Etchebarne Jerry Kennedy Jim Loughead Rick Lundquist Chel Moore Naji Nassereddine Fred Owens John Smith Lawson Spicer Matthew VanBaale Ueli Zaugg

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Table of Contents
Prince Agri Products, Inc. - Pre-Conference Symposium
DCAD Revisted: Prepartum Use to Optimize Health and Lactational Performance David Beede, Ph.D. Michigan State University..1 Mastitis and Dairy Replacement Heifers: Management Practices to Reduce the Odds Stephen Nickerson, Ph.D. University of Georgia .......19 The Physiology of Stress and Effects on Immune Health in Ruminants Jeff Carroll, Ph.D. USDA/ARS..........35 Predicting Transition Cow Health and Performance Use of Blood and Fecal Biomarkers for Herd-Level Evaluation and Diagnostics Thomas Overton, Ph.D., Cornell University..45

Southwest Nutrition and Management Conference

Economics of Feeds in Dairy Rations Normand St. Pierre, Ph.D. Ohio State University......57 Managing Heat Stress and its Impact on Cow Behavior John Smith, Ph.D. University of Arizona.68 Early Weaning Beef Calves to Improve Herd Productivity: Implications on Cow and Calf Nutrition John Arthington, Ph.D. University of Florida...80 BH in the Rumen: The Gateway to Seeing Benefits from Fat Supplements Thomas Jenkins, Ph.D. Clemson University..94 Why Cows Die on Dairies Franklyn Garry, Ph.D. Colorado State University.....110
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An Update on Hypocalcemia on Dairy Farms Garrett Oetzel, Ph.D. University of Wisconsin-Madison.117 Performance of Calves in Different Calf Feeding Systems Mark Hill, Ph.D. Provimi Nutrition.........128 Update on Milk Fat and Human Health Adam Lock, Ph.D. Cornell University.141 Use of Probiotics in Dairy Rations During Heat Stress Laun W. Hall University of Arizona....153

DCAD Revisited: Prepartum Use to Optimize Health and Lactational Performance J. A. Shire and D. K. Beede Department of Animal Science Michigan State University Corresponding author: beede@msu.edu
SUMMARY Normal calcium (Ca) status is extremely important through the transition period to launch a healthy and successful lactation. From recent evidence, routine monitoring to identify subclinical hypocalcemia (defined by early postpartum blood serum Ca of less than 8.6 mg/dl) would be appropriate in herds with unacceptable periparturient health disorders. Based on available analyses the four-element equation for dietary cation-anion difference [DCAD = (Na+ + K+) (Cl- + S2-)] is most appropriate to formulate a reduced DCAD of the close-up ration. The most effective first step to lower the DCAD is to reduce K+ and Na+ as much as possible before supplementing anions. DCAD of -10 mEq/100 g is recommended; a somewhat lower DCAD (-15 mEq/100 g) may be advantageous to help accommodate biological variation among animals within the closeup group. Close-up diets with 1.0% Ca and 0.35% Mg are sufficient in properly supplemented anionic diets to support normal periparturient Ca status. Various vitamin D-related processes, including regulation of periparturient Ca status, might be improved with supplementation of vitamin D throughout the close-up period. The body of evidence accumulated over the last 40+ years highlights the direct and indirect importance of periparturient Ca status and how Ca status can be improved by manipulation of close-up DCAD to improve lactational performance. Future research should consider: a) timing of vitamin D supplementation prior to calving; b) factors that contribute to vitamin D receptor regulation in the transition period; and, c) performance of heifers fed anionic diets. INTRODUCTION The importance of successfully transitioning dairy cows from pregnancy to lactation has been known for decades. Periparturient hypocalcemia can be detrimental to both cows and dairy producers. If preventive measures to manage blood calcium (Ca) around parturition are not taken, a cascade of metabolic problems can lead to secondary health issues, impaired immune function, reduced milk production, and poor reproductive performance. Elliot Block (1984) was first in North America to report that reducing the dietary cation-anion difference (DCAD) before calving could benefit early postpartum Ca status and aid transition into lactation. In the late 1980s and early 1990s the so-called anionic salts were supplemented in close-up diets. Success was variable mainly because of lack of understanding about how to deliver this new technology. Often prepartum feed intake was reduced with the salts. Consequently, commercial 1

anion supplements apparently causing fewer feed intake problems became available. Nonetheless, anion supplementation still does not work as well as desired in some cases. This paper takes a brief look back at the development of the DCAD strategy for transition dairy cows, suggests some possible reasons why it is not more universally efficacious, and addresses current approaches to monitor and reduce risk of hypocalcemia as well as new ideas to consider when implementing the prepartum DCAD strategy. HYPOCALCEMIA Subclinical Hypocalcemia Hypocalcemia is a critical concern due to the enormous physiological demands for Ca at calving and the start of lactation (Oetzel et al., 1988; Horst et al., 1997). Calcium demand of a cow at initiation of lactation is about double compared with when she was non-lactating and pregnant. For example, the Ca requirement of a 1600-pound pregnant dry cow increases from about 11 g/d to support the conceptus to about 23 g/d after calving to support milk production (Goff and Horst, 1997a). In order to suffice, a cow must bring at least 30 g of Ca/d into the plasma pool in very short order by intestinal absorption and(or) bone resorption (Horst et al., 1997). This is a formidable physiological task. Normal blood plasma Ca concentration is tightly regulated and generally kept between 8.5 to 10 mg/dl (2.1 to 2.5 mM; Cahn and Line, 2005). In the periparturient period with enormous physiological demands, blood Ca may drop below this range; though is not characteristically considered subclinical until blood plasma Ca drops to the range of 8.0 to 5.5 mg/dl (2.00 to 1.38 mM), but with homeostasis still maintained (Goff, 2008). Recent findings suggest that the subclinical range might be widened to include blood serum Ca concentrations of 8.6 to 5.5 mg/dl to better identify cows at risk for metritis (Martinez et al., 2012). The point at which a cow is truly hypocalcemic and biological functions become impaired varies among individual cows; their capacity to maintain homeostasis and Ca status as influenced by several factors is discussed subsequently. A possible on-farm strategy might be to more routinely monitor and identify subclinical hypocalcemia as serum Ca concentrations of less than 8.6 mg/dl in herds with unacceptable incidences of periparturient health disorders. Clinical Hypocalcemia Milk fever (clinical hypocalcemia) defines an extreme decrease in blood plasma Ca below a certain point around time of calving resulting in homeostatic failure. The point at which it is considered clinical was suggested to be below 5.5 mg/dl (Goff, 2008). Milk fever may or may not be accompanied by other signs such as poor appetite, recumbency, and lethargy (Horst et al., 1997). A study conducted by the USDA National Animal Health Monitoring System (2002) estimated a 5% incidence rate of milk fever in the United States (Reinhardt et al., 2011). Failure to maintain muscle tone and contractility of the gastrointestinal tract and the cardiovascular system quickly leads to death in the majority of milk fever cases (Cahn and Line, 2005). Not all cows that have subclinical hypocalcemia develop milk fever, but it is important that diagnostic and preventive measures are taken before the cows health declines and subsequent problems arise.

Predisposing Factors Reasons for susceptibility to hypocalcemia vary and may be multi-factorial. One of the most commonly noted differences in susceptibility is parity. Incidence of subclinical hypocalcemia (< 8.0 mg plasma Ca /dl) typically rises with increasing parity, affecting 25% of heifers and almost half of cows of second and greater parity (Reinhardt et al., 2011). It is thought that heifers especially are less susceptible because they have greater bone depletion/repletion activity and are more able to mobilize bone Ca from their Ca reserves than are later parity cows (van Mosel et al., 1993). Additionally, later parity cows produce more colostrum and milk making the demand for Ca greater. A similar relationship with parity was noted with clinical milk fever affecting less than 1% of heifers, but surpassing 6% for cows of third and greater parity (Reinhardt et al., 2011). This greater milk fever risk and incidence is in part due to parity as well as history of previous milk fever. History of milk fever seems to be a large determinant of whether or not a cow develops hypocalcemia and milk fever at subsequent parturitions. This is presumably due to a decreased ability of these particular cows to respond immediately to biological signals and increase vitamin D receptor (VDR) numbers in a timely manner (Goff et al., 1995a). Blood Ca and vitamin D3 concentrations are obviously important components in Ca mobilization, bone resorption, and intestinal absorption (Jones, 2008). However, deficiencies in dietary Ca, vitamin D, or biologically active 1, 25 dihydroxyvitamin D are not thought to be the casual reasons for hypocalcemia. For instance, low Ca diets were used successfully to reduce incidence of hypocalcemia (Goings et al., 1974; Thilsing-Hansen et al., 2002) and active vitamin D plasma concentrations usually are considered sufficient in afflicted cows (Goff et al., 1989). A possible explanation for the metabolic cause of hypocalcemia is slow VDR upregulation, and(or) overall lower VDR numbers between breeds (e.g., Jersey vs. Holstein; Goff et al., 1995b). Jerseys have fewer overall VDR compared with Holsteins (Goff et al., 1995b) and Jerseys are more susceptible to milk fever (Lean et al., 2006). Conversely, Holsteins have similar numbers of receptors compared with Brown Swiss (Lisegang et al., 2008) and both breeds typically have less incidence of milk fever than Jerseys. The regulation of VDR could be a very plausible explanation for differences among breeds and whether or not a particular cow of a particular parity or breed develops hypocalcemia and to what severity the disorder manifests. DIETARY CATION-ANION DIFFERENCE History and Current Use The ability to reduce the incidence of periparturient hypocalcemia through addition of mineral acids to the diet was first documented by Ender and Dishington (1970). Shortly thereafter they showed that multiple dietary anionic salts were effective in stimulating increased blood Ca (Ender et al., 1971; Dishington, 1975). Block (1984) introduced the dietary cation-anion balance theory to transition cow research in North America. He demonstrated that providing more dietary fixed anions than cations via anionic salts in the close-up period increased blood Ca in early postpartum cows. Today, this strategy is commonly known as dietary cation-anion difference (DCAD), which more specifically addresses the calculation of DCAD in ration formulation (Sanchez and Beede, 1991).

The DCAD can be calculated as several variants of an equation involving more or less of the seven dietary essential macromineral elements: milliequivalents (mEq) of [(Na+ + K+ + Mg++ Ca2+) - (Cl- + S2- + P3-)/100g of dietary DM]. As part of a ration formulation and management strategy, a low or negative DCAD is supplied in the diet for 2 to 3 wk prepartum (Block, 1984; Oetzel et al., 1988). An effective DCAD reduction changes the acid-base status of the cow within 36 hr (Goff and Horst, 1998). However, because of variation in actual compared with expected calving day (e.g., 8 to10 d, S.D.) sufficient time of dietary provision must be used to accommodate the majority of cows in the close-up group. Anion supplementation effectively reduces the incidence of periparturient hypocalcemia and milk fever by increasing the concentration of blood Ca (Block, 1984; Oetzel et al., 1988). Early on, the so-called anionic salts (e.g., chloride and sulfate salts of ammonium, calcium or magnesium) were used. Certain anionic salts were thought to be more palatable than others (e.g., greater DMI when magnesium sulfate compared with calcium chloride was supplemented; Oetzel and Barmore, 1993). In the USDA survey, an estimated 27% of U.S. dairy operations used anionic salts (as specifically worded in the survey) to decrease DCAD in an effort to reduce incidence of periparturient hypocalcemia (USDA, 2007). Use of commercial products to supplement anions rather than anionic salts is much more popular today than in the past due to their ease of use and often the added benefit as a source of protein. The use of palatable carriers and addition of flavoring in commercial sources may help dilute or mask the presumably bitter taste thought to accompany pure anionic salt sources. The first-line, most efficacious strategy for lowering DCAD is to reduce the strong cations (i.e., K+ and Na+) before adding anion sources. About 47% of U.S. dairy operations specifically aimed to lower dietary K in efforts to reduce DCAD of prepartum diets (USDA, 2007). DCAD Equations There are a variety of ways to calculate DCAD depending on which macromineral elements (fixed ions) are considered important (Ender et al., 1971; Goff, 2004). Most equations include Na, K, Cl, and S, but variations on this come from addition of Ca and Mg, estimated coefficients of absorption for the different mineral elements, and the inclusion or exclusion of S and P. Overall, the most widely used equation in the industry is that originally used by Ender et al. (1971) [DCAD = (Na+ + K+) (Cl- + S2-)]. Based solely on the acidifying effect of dietary S in urine and blood, Spanghero (2004) suggested that S had negligible influence on the cows acid base balance in the basic four-element equation. Thus, the simpler three-element equation [DCAD = (Na+ + K+) (Cl-)] was suggested. However, increased S in the diet was associated with decreased incidence of milk fever and the source of S (as part of an organic compound or as supplemental sulfates) could be a factor (Lean et al., 2006). Note that supplementation of elemental sulfur (e.g., the flowers of sulfur) is worthless as it does not provide absorbable S nor does it have acidifying properties as part of the DCAD calculation. The meta-analysis of Lean et al. (2006) assessed the ability of four relevant DCAD equations to both affect acid-base status and to predict milk fever of Holstein, Norwegian and Swedish Red and White, and Jersey cows. It is important to note that using an equation to predict the effects of macromineral elements on milk fever is not necessarily the same as using an equation to predict overall acid-base status. For example, Jerseys have a greater susceptibility to milk fever than Holsteins (Lean et al., 2006), while Mg inclusion in the diet reduced risk of milk fever but made the DCAD value more positive. A criterion for data inclusion in the study of Lean et al. 4

(2006) was that they could calculate DCAD from information given in the original reports; however, lactation number, which is a recognized risk factor for milk fever (Reinhardt et al., 2011) was not in the model. The analysis also did not consider Ca in the DCAD equation noting that diets with both low Ca (0.5 %) and high Ca (> 1.0 %) were effective in preventing milk fever. Additionally, in their analysis there was a time by treatment interaction on the efficacy of Ca inclusion rate and lack of sufficient data with dietary Ca inclusion greater than 1.1%. Results indicate that [DCAD = (Na+ + K+) (Cl- + S2-)] was the most effective in describing acid-base status as well as predicting reduced incidence of milk fever. Similarly, a metaanalysis by Charbonneau et al. (2006) concluded that [DCAD = (Na + + K+) (Cl- + 0.6S2-)] correlated with milk fever incidence and acid-base status prediction. The 0.6 coefficient was based on findings of Tucker et al. (1991a) and later supported by Goff et al. (2004) indicating that S had less acidifying effect compared with Cl. Diet composition and desired outcome (i.e., risk prevention or acidifying ability) should be important considerations when determining which equation to use. Based on all of the available information the four-element equation seems most acceptable. Whether or not the 0.6 coefficient for S should be incorporated likely depends upon the sources of supplemental S in a particular diet. Mechanism of Action Metabolic acidosis or alkalosis results from the change in electrical charge of biological fluids due to either more anions (i.e., acidosis) or more cations (i.e., alkalosis) entering the blood. The change in electrical charge of the blood results in transfer of H+ ions and changes in blood pH (i.e., acid-base balance; Goff, 2008). Blood pH is tightly regulated within a narrow normal physiological range, keeping arterial blood pH between 7.35 to 7.45 and venous blood at a slightly lower pH (Nagy et al., 2001). Though blood pH can be reduced through use of negative DCAD, these changes may not be significant (Charbonneau et al., 2006; Grnberg et al., 2011). A meta-analysis indicated a significant negative relationship between decreasing DCAD (from +30 to 0 mEq/100g) and decreasing blood pH; however, the change in blood pH was very small and not statistically significant (Charbonneau et al., 2006). Compared with sulfate salts, chloride anionic salts are thought to have a greater acidifying effect in blood (Goff et al., 2004; Charbonneau et al., 2006). A more easily accessible proxy for monitoring systemic acid-base status in the field is urine pH. Typically if urine pH is below 7.0 then systemic acidosis has been generated (Jardon, 1995). Charbonneau et al. (2006) suggested that inducing acidosis beyond the point where urine pH drops below 6.8 is not warranted in field application of the DCAD strategy because risk of hypocalcemia was not further reduced. Typical anionic salts fed in close-up rations with negative DCAD include chloride and sulfate salts of calcium, magnesium, and ammonium. Hydrochloric acid was most effective at lowering urine pH compared with the anionic salts (Goff et al., 2004). The exact mechanism by which a low or negative DCAD aids in the reduction of hypocalcemia during the periparturient period is not completely understood. It apparently has to do with two independent mechanisms. First, the mildly acidic systemic environment created by lowered DCAD stimulates osteoclasts to start mineralizing bone Ca to correct the acidosis. Under hypocalcemic conditions parathyroid hormone (PTH) is secreted. This hormone assists in the subsequent formation of the active 1, 25 dihdroxyvitamin D hormone (1,25 [OH] 2 D). Through 5

both PTH and biologically active vitamin D, bone resorption of Ca is stimulated (Horst and Reinhardt, 1983). Additionally, there is increased renal tubular resorption of Ca and increased efficiency of Ca absorption in the small intestine (Goff and Horst, 2003). Together these mechanisms and newly available Ca sources contribute to the return of Ca homeostasis. Further connection between anion supplementation and Ca status is thought to be due to the decrease in blood pH by anions (Goff, 2008). When both pH is low and PTH is active, osteoclastic activity is even greater and thus Ca resorption increases significantly (Bushinsky, 2001). It is commonly accepted that the reason for this increased activity is that the creation of an acidic metabolic environment allows increased tissue responsiveness to PTH. When blood pH is more alkaline (>7.35), PTH cannot tightly bind to its receptors on the bone surface and in renal tissue. However, when blood is at its normal physiological pH of 7.35, PTH is tightly bound to its receptors and can therefore better stimulate its target cells (Bushinsky, 2001; Goff, 2008). Optimal DCAD for Close-up Cows It is not necessary for the prepartum DCAD to be negative in order to stimulate homeostatic acid-base mechanisms and increase blood Ca. Lowering DCAD from +30 to 0 mEq/100g resulted in a decrease of urine pH from a normal of 8.09 to 7.01 (Charbonneau et al., 2006). Furthermore, this mild systemic acidosis resulted in significant increase (~11%) in total blood Ca concentration immediately postpartum and reduced incidence of milk fever from 16.4 to 3.2%. In another study, cows fed close-up diets with a negative DCAD (-10 to -12 mEq/100 g) with supplemental calcium chloride and calcium sulfate (personal communication with authors) or a commercial supplement did not have increased blood Ca after calving compared with cows fed a control ration (+22 mEq/100g) (DeGroot et al., 2012). It was suggested that systemic acidic conditions decrease the ability of insulin to bind to its receptor (Whittaker et al., 1982). This causes reduced insulin sensitivity resulting in decreased glucose uptake and utilization (Bigner et al., 1996). Because of the shift in energy demand at onset of lactation, loss of utilizable glucose can be detrimental to both milk production and cow health. Grnberg et al. (2011) did not observe an effect on insulin response or sensitivity when mild systemic acidosis was induced through use of anion supplementation (-9 mEq/100 g). Similarly, Ramos-Nieves et al. (2009) and DeGroot et al. (2010) found no difference in plasma glucose concentrations during the periparturient period in relation to DCAD prepartum (-15 mEq/100 g and -12 mEq/100 g, respectively). The influence of anion supplementation on DMI varies among studies. Both Block (1984) and Oetzel et al. (1988) found no changes in prepartum DMI when anion sources were provided (-13 mEq/100 g and -7.6 mEq/100 g, respectively). DeGroot et al. (2010) also observed changed acid-base status without compromising prepartum DMI in multiparous cows, but observed increased postpartum DMI (2.1 kg/cow per d) for cows receiving anion supplementation prepartum. These findings differ from a meta-analysis that indicated a decrease in prepartum DMI of 1.3 kg/cow per d when DCAD was reduced from +30 to 0 mEq/100g (Charbonneau et al., 2006). Ramos-Nieves et al. (2009) also reported a significant decrease in DMI (15.6 vs. 14.4 kg/cow per d) when prepartum anion supplementation was provided (+10 vs. -15 mEq/100, respectively). This discrepancy could be due to the amount and(or) source of anions 6

supplemented to reach a desired DCAD. Sodium and K are recognized as strong cation contributors with greater dietary concentrations being linked to increased incidence of milk fever (Goff and Horst, 1997b; Lean et al., 2006). For most effective DCAD formulation, dietary Na and K should be reduced as much as practically possible before adding supplemental anions. In order to avoid possible adverse effects of supplemental anions on DMI while still achieving mild systemic acidosis, a DCAD of -10 mEq/100 g is suggested. Using a somewhat lower DCAD (e.g., -15 mEq/100 g) may be advantageous to help accommodate biological variation among individual cows (i.e., fluctuations in daily DMI and differences in biological ability to maintain acid-base balance). Management of Heifers (Primiparous Cows) Although primiparous cows do not typically experience as many or the severity of Ca-related metabolic problems during transition as multiparous cows, both pregnant heifers and multiparous cows are often in the same group and fed the same close-up ration. The USDA (2007) survey suggested that about 20% of dairy farms use anion supplementation in close-up rations for heifers. A possible adverse effect of providing a negative DCAD to heifers is reduced DMI (Tucker et al., 1991b; Moore et al., 2000); but, other negative effects are largely not reported. DeGroot et al. (2010) demonstrated that using various anion sources (commercial supplements, or calcium sulfate and calcium chloride) to create a negative DCAD during the close-up period did not result in any obvious negative side effects in pregnant heifers. Acidbase balance was effectively altered with heifers having lower prepartum urine pH compared with multiparous cows (6.24 and 6.80 respectively). Similar to the multiparous cows, the prepartum DMI of heifers was not impacted and early postpartum DMI increased (1.4 kg/cow per d). Overall, there is insufficient evidence to say whether or not heifers/primiparous cows will be impacted negatively in major ways if provided a negative DCAD diet in late gestation. The lowered prepartum urine pH (i.e., affected acid-base status) of heifers could be an unnecessary consequence considering there was no significant change in postpartum plasma Ca concentrations. Overall management of heifers must still be considered when deciding on anion supplementation. Improper monitoring of mixed parity close-up groups may lead to competition among animals and reduced DMI of typically smaller heifers. DCAD and Milk Yield Response Prepartum anion supplementation improved lactation performance in some studies. Block (1984) reported a 6.8% increase in milk yield of cows given anionic salts (DCAD: -13 mEq/100 g) in the close-up period with no reported change in postpartum DMI. Likewise, high producing multiparous cows provided diets supplemented with anions (-10 to 12 mEq/100 g) prepartum increased milk yield by 18% (6.5 kg/cow per d) compared with cows fed a control diet (+22 mEq/100 g; DeGroot et al., 2010). This increase was likely due to greater DMI of cows fed anions prepartum. Differences in milk yield are not always found. No changes in milk yield, milk composition, or postpartum DMI were found when comparing prepartum anionic vs. cationic diets (-15 vs. +15 mEq/100g by Moore et al., 2000; or, -15 vs. + 10 mEq/100g by Ramos-Nieves et al., 2009). Similarly, milk yield and postpartum DMI did not differ among cows in a pasture system provided a prepartum diet with a DCAD of +50 vs. +7 mEq/100g (Roche et al., 2003). The improvement in some studies from feeding a low or negative DCAD

prepartum on postpartum milk yield is most likely due to improved periparturient Ca status and health, and improved postpartum DMI. MACROMINERALS Cations Whether or not feeding less or more dietary Ca than the cows requirement in close-up diets is still questioned. To truly restrict Ca intake below requirement to affect Ca homeostasis, less than 20 g/cow per d is required (Thilsing-Hansen et al., 2002) or possibly less than 15 g/cow per d (Goings et al., 1974). The current NRC (2001) recommendation for absorbed Ca for a pregnant Holstein dry cow (1600 lb BW) is approximately 11 g/d (or about 20 - 30 g of total dietary Ca/cow per d). By markedly restricting dietary Ca prepartum, hypocalcemic-like conditions are induced prior to calving. This results in increased fractional absorption of Ca from the small intestine, renal reabsorption, and bone resorption (Goff and Horst, 2003). Because these mechanisms are stimulated before calving, increased Ca in blood at calving can result (Goings et al., 1974; Thilsing-Hansen et al., 2002). The relatively high Ca content of some forages and byproduct feeds can make dietary restriction of Ca difficult. Furthermore, the concentration of Ca in the diet is not as important a factor in occurrence of milk fever as other macromineral cations such as K, Na and Mg (Goff and Horst, 1997b; Lean et al., 2006). Whether or not there is an interaction between DCAD and Ca has been questioned (Goff and Horst, 1997b; Oba et al., 2011; Goff and Horst, 2012). In a classic study, Goff and Horst (1997b) were the first to directly assess the effects of increasing dietary Ca and K (that is increasing DCAD) on hypocalcemia. They found no change in incidence of milk fever of multiparous Jersey cows with 0.5% vs. 1.5% Ca in the close-up diet. However, hypocalcemia increased when greater concentrations of Na and K were incorporated into the diet regardless of Ca concentration. This response demonstrated clearly that the overall effect of DCAD, independent of dietary Ca, greatly influenced occurrence of hypocalcemia. The more influential effect of Na and K compared with Ca on incidence of milk fever was later confirmed by meta-analysis (Lean et al., 2006). They also observed increased milk fever when close-up diets had greater Ca (0.5 vs. 1.0%). An even more significant increase in milk fever was observed when cows were fed close-up diets with >1.5% Ca. In their study, length of time of feeding high Ca diets and few data with high Ca close-up diets may be contributing factors explaining why dietary Ca did not affect incidence of milk fever. Higher dietary Ca might be beneficial if fed longer than 20 d (Lean et al., 2006), though a 14-d period was a long enough to show a beneficial effect of high Ca in another study (Oba et al., 2011). From these studies it appears that a Ca inclusion rate of 1.0% in close-up diets is sufficient to maintain periparturient blood Ca provided that an anionic diet is fed. Calcium and DCAD The aforementioned difference in response to DCAD rather than dietary Ca could be explained in a couple of ways. Limiting Ca in a positive DCAD diet causes some degree of hypocalcemia and stimulates PTH to increase blood Ca before calving (Thilsing-Hansen et al., 2002). On the other hand, the mild metabolic acidosis that results from a negative DCAD increases hypercalciurea (Vagnoni and Oetzel, 1998). Thus, feeding greater Ca with anion 8

supplementation could aid in maintaining blood Ca concentrations around calving. Oba et al. (2011) examined both of these concepts by supplementing diets with moderate or low Ca (0.9% vs. 0.3%) with DCAD of -6.4 or +9.1 mEq/100 g and challenging cows with intravenous EDTA (a blood Ca-chelator) infusion. The time for cows to recover to 90% of their pre-challenge blood ionized-Ca concentration was shorter for those fed +9.1 mEq/100 g DCAD with 0.3% dietary Ca and for those fed -6.4 mEq/100 g DCAD with 0.9% Ca; whereas, the time for cows to reach their pre-challenged ionized-Ca status was longer for those fed +9.1 mEq/100 g DCAD with 0.9% Ca as well as those fed the -6.4 mEq/100 g DCAD with 0.3% Ca. A significant interaction of dietary Ca% by DCAD on time to recover to 90% of pre-challenge ionized blood Ca also was detected. Conversely, Goff and Horst (2012) found no advantage in the cows ability to increase blood Ca when fed 1.5% Ca compared with 0.5% Ca while on negative DCAD diets (-6.1 and -6.8 mEq/100 g). Also, Chan et al. (2006) noted no change in blood Ca with anion supplementation (-6 mEq/100 g) and either moderate (0.99%) or high (1.5%) dietary Ca. These findings support the idea that the DCAD in the close-up diet is much more influential on development of hypocalcemia than dietary Ca concentration per se. Perhaps the differences in recovery time of blood ionized-Ca in response to DCAD with varying dietary Ca found by Oba et al. (2011) was actually due to differences in DCAD. Theoretically, the treatment group (+9.1 mEq/100 g with 0.9% Ca) that had the greatest recovery time also would have the greatest DCAD value due to increased Ca inclusion. Calcium Status and Milk Yield Besides troublesome health effects associated with periparturient hypocalcemia (Curtis et al., 1983), lactation performance can be impacted by Ca status in early lactation. Kimya et al. (2005) reported increased milk production of both primiparous and multiparous cows (1.7 and 3.9 kg/cow per d, respectively) when blood Ca status was maintained on a low Ca diet (0.46%). Additionally, milk Ca, P, and Mg were not affected by changes in close-up Ca concentration (0.46% vs. 0.86%) for any parity. In another study where normal blood Ca was maintained around parturition and moderate or high Ca was fed prepartum (0.99% vs. 1.5%) there was no difference in milk yield or composition (Chan et al., 2006). Interestingly, Jawor et al. (2012) reported increased milk yield of hypocalcemic cows during the first few weeks of lactation. The biggest difference was in third lactation cows where milk yield remained greater for hypocalcemic compared with normocalcemic cows through 280 DIM before declining. However, other researchers observed no differences in the milk yield of hypocalcemic compared with normocalcemic cows (Martinez et al., 2012). In another study, milk yield was reduced by hypocalcemia (-3.2 kg of milk/cow per d; Chapinal et al., 2012). These differences in lactational performance among various studies demonstrate the importance of proactive approaches for monitoring and treating hypocalcemia. Magnesium and Calcium Magnesium is extremely important in homeostatic mechanisms to correct hypocalcemia. Magnesium deficiency impairs PTH secretion and PTH receptor response resulting in blunted Ca mobilization and hypocalcemia (Johannsson and Raisz, 1983); thus, exacerbating and predisposing hypocalcemic conditions around parturition. Cows that do not maintain normocalcemia around parturition had reduced blood Mg concentrations as well (Martinez et al., 2012). Normal plasma Mg concentration is 1.8 to 2.4 mg/dl (0.75 mM to 1.0 mM; Goff, 2008). 9

Whereas, Mg did not have a strong influence on predicting acid-base response to varying DCAD (Charbonneau et al., 2006; Lean et al., 2006) dietary Mg concentration is thought to influence occurrence of milk fever. An increase in dietary Mg concentration (0.3 to 0.4%) greatly reduced milk fever incidence (Lean et al., 2006). The recommended concentration of dietary Mg is 0.35 to 0.40% (Goff, 2008). An interaction between high dietary Ca and Mg absorption may exist. Close-up cows fed 1.36% Ca and 0.18% Mg had lower apparent Mg digestibility and urinary Mg excretion compared with cows provided 0.45 or 0.90% Ca (Kronqvist et al., 2011). It should be noted that prepartum DCAD was positive (~20 mEq/100g); based on urinary Mg excretion all cows in this study were subclinically hypomagnesmic. In another study, blood Mg was not changed when 55 to 91 g of Ca/cow per d (0.46 to 0.86% Ca) were provided with 0.18% dietary Mg (Kamiya et al., 2005). In that study the DCAD was not reported and there was no increase in blood PTH or bone resorption markers. VITAMIN D Vitamin D Activation and Receptor Regulation Vitamin D metabolites such as 1,25 (OH)2 D3 have a variety of important physiological roles such as cellular differentiation, Ca signaling (Jones, 2008), and immunological functions (Kimura et al., 2006; Nelson et al., 2010). Failure of these biological functions due to vitamin D deficiency can exacerbate and complicate the effects of hypocalcemia. As a result, risk of secondary health problems such as mastitis, displaced abomasum (Goff, 2008), and poor reproductive performance (Ward et al., 1970) are increased greatly. Vitamin D is needed when plasma Ca drops below its homeostatic range. Through Ca and PTH signaling the vitamin (hormone) is released from its stores and transported to the liver via binding proteins (Jones, 2008). In the liver vitamin D is hydroxylated, forming 25 hydroxyvitamin D (25OHD). This newly formed vitamin D metabolite is then transported through blood to the kidney where it is fully activated by another hydroxylase and converted to the active form 1,25 (OH)2 D (Jones, 2008). The role of active vitamin D in intestinal Ca absorption is crucial (Goff and Horst, 2003; Jones, 2008). There are a couple of theories as to how vitamin D status might influence hypocalcemia. Questioned initially was whether or not hypocalcemia could be caused by deficient vitamin D activation to form the active 1,25 (OH) 2 D hormone. This was rare with hypocalcemia incidence rate due to low 1,25 (OH)2 D occurring in less than 10% of cows (Goff et al., 1989). However, cows afflicted with milk fever in successive parturitions apparently do have delayed 1,25 (OH)2 D production and response. These cows also have greater plasma PTH in the days surrounding parturition compared with cows that have not had a prior incidence of milk fever (Goff et al., 1989). Interestingly, there may be a compounding negative influence of PTH on vitamin D receptors (VDR; Reinhardt and Horst, 1990). Goff et al. (1995a) also observed a slight decrease in VDR at time of calving when PTH activity would be elevated. How exactly PTH and vitamin D metabolites influence each other and their receptors is not clearly understood. Perhaps by providing enough vitamin D in the prepartum period to stimulate 1,25

10

(OH)2 D production and VDR up-regulation, the negative effects of PTH at calving would not be so detrimental to VDR numbers and 1,25 (OH) 2 D stimulation. Further action of vitamin D is thought to be indirectly involved in the occurrence of hypocalcemia through VDR regulation around time of parturition (Goff et al., 1995a; Goff et al., 1995b). For vitamin D to control Ca absorption efficiently it is important that VDRs are present in adequate numbers and are effectively stimulated. It was thought that dairy cows with clinical milk may have inadequate numbers of prepartum receptors for 1,25 (OH) 2 D, the active hormone needed for Ca uptake. Goff et al. (1995a) showed a significant increase in colon VDR numbers in pregnant compared with non-pregnant Jersey cows, suggesting preparation for lactational demands. However, there was no significant change in receptor numbers in the time immediately around parturition. A slight decrease in receptor numbers at time of calving was observed; although the reason for this decrease was unclear as complications with hypocalcemia were an issue. Also observed was a delayed increase in VDR numbers of cows with a previous history of milk fever; but, eventually typical numbers were reached. Liesegang et al. (2008) explored the idea of decreased VDR function in third and greater lactation Holstein and Brown Swiss cows compared with first and second lactation cows. There was no difference in intestinal VDR numbers between breeds or as lactation number increased. Importantly, none of the cows experienced periparturient hypocalcemia and all cows were in mid to late lactation at time of sampling. The entire impact of VDRs around parturition is still unclear. There is an obvious link between VDR existence and hypocalcemia (Beckman and DeLuca, 2002), though how they are regulated during the cows transition period is not entirely clear. Other factors affecting VDRs are thought to include DCAD (Goff et al., 1995a), VDR gene alterations (Deiner et al., 2012), VDR anatomical locale (Beckman and DeLuca, 2002), and glucocorticoids (Hidalgo et al., 2010). However, definitive evidence of the role of VDRs in hypocalcemia of dairy cows has not been fully elucidated. Controlling Hypocalcemia and Milk Fever The ability to decrease incidence of hypocalcemia in dairy cows with greater susceptibility by supplementing vitamin D during the close-up period is well documented. Julien et al. (1976) demonstrated that cows of third and greater lactation with a prior history of milk fever were less likely to develop milk fever during their subsequent lactation if injected with 10 million IU of vitamin D3 1 wk prepartum. Furthermore, Hibbs and Conrad (1960) were able to increase protection against milk fever in the more susceptible third and greater lactation Jersey cows by as much as 38% by supplementing vitamin D (15 and 30 million IU/cow per d) as part of the TMR or in capsule form for a week before calving. However, this protective effect of vitamin D was not always observed (Taylor et al., 2008) and supplemental vitamin D benefits to cows thought to be less susceptible is not well understood. These differences in response may be due to a variety of reasons such as history of milk fever (Hibbs et al., 1976), parity, level of milk production, or length of period of Ca stress (Muir et al., 1968). A low or negative DCAD during the close-up period may very well play an important part in the effectiveness of vitamin D as well (Wilkens et al., 2012).

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Supplementation Whereas it is relatively unlikely for vitamin D to be deficient in most modern dairy farms, there is reason to believe that supplemental vitamin D3 may be beneficial. Vitamin D2 and D3 can be supplied orally or by intramuscular injection. Injection was thought to be more effective as the vitamin would not be degraded in the rumen if given orally (Sommerfeldt et al., 1979). However, Hymller and Jensen (2010) reported no ruminal degradation of orally supplemented vitamin D2 or D3. Drawbacks to using injectable vitamin D are optimal timing relative to actual parturition, labor, and increased possibility of toxicosis. Because vitamin D is a fat-soluble vitamin, it can accumulate in body tissues with frequent or large dose injections and result in vitamin D toxicosis (Littledike and Horst, 1980). Tolerance of supplementary vitamin D by injection can be 100 times less than to oral delivery (Littledike and Horst, 1980). Vitamin D can be supplemented in its various plant and animal-derived forms (Sommerfeldt et al., 1983; Taylor et al., 2008; Wilkens et al., 2012). It was suggested that vitamin D3 supplementation is more effective than vitamin D2 because vitamin D3 more efficiently increased vitamin D blood metabolites (Sommerfeldt et al., 1983; Hymller and Jensen, 2010). Vitamin D3 is thought to be more effective at increasing vitamin D blood metabolites due to differences in absorption mechanisms for D2 and D3 in the intestine (Sommerfeldt et al., 1983; Hymller and Jensen, 2010). Furthermore, Hymller and Jensen (2011) suggested vitamin D 2 supplementation might have a direct negative influence on D3 utilization in the body. Thus, supplementing with vitamin D2 could be misleading when looking at the overall effects of vitamin D supplementation on vitamin D plasma metabolite concentrations. Because vitamin D3 is the naturally occurring form in animals and has the most biological activity, vitamin D 3 supplementation is considered best. The most efficacious amount of vitamin D3 needed by close-up and lactating dairy cows is not clear. In general, 30 IU/kg of BW per d is recommended for cows in late pregnancy and early lactation. For a 1600 lb cow, this is roughly equal to 20,000 IU/cow per d (NRC, 2001). Due to the greater Ca demand of high producing cows, 30,000 to 40,000 IU/cow per d may improve Ca absorption and milk production (NRC, 2001). Appropriate timing of administration of vitamin D supplementation relative to actual calving date can be difficult. Additionally, length of time of supplementation prior to calving to help prevent hypocalcemia is unclear. Research referenced previously found positive results by D3 injection for about 1 wk before calving (Julien et al., 1976; Hibbs and Conrad, 1960). Taylor et al. (2008) were able to increase blood 25 hydroxyvitamin D (25-OHD) after calving by orally supplementing 600,000 IU of 25-OHD/cow per d in gel capsules within 6 d of calving, but there was no increase in blood Ca around parturition. It is likely that for vitamin D supplementation to work optimally all dietary and physiological conditions (i.e., acid-base status, dietary Ca, VDRs) must be in alignment. Perhaps dietary supplementation of vitamin D at recommended doses for a longer period of time (e.g., 20,000 IU/d for entire close-up period) would effectively stimulate and up-regulate VDRs thus allowing for more efficient Ca absorption. This could allow adequate time for vitamin D activation and subsequent stimulation of important biological processes.

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RELATIONSHIPS WITH HEALTH AND REPRODUCTION Periparturient Metabolic Disorders Calcium is a major cellular signal needed for a range of biological functions (Jones, 2008). A classic path analysis by Curtis et al. (1983) showed significant associations between poor Ca status and muscle contraction-associated problems (i.e., dystocia and displaced abomasum) and ketosis. Cows experiencing hypocalcemia spend 10% more time standing but less time at feed and water in the days just before calving (Jawor et al., 2012). With decreased intake and insufficient Ca for proper gut motility, risk of displaced abomasum postpartum increased (Curtis et al., 1983). Furthermore, hypocalcemia predisposes cows to increased concentrations of metabolites associated with ketosis. Both primiparous and multiparous cows fed a close-up diet with a negative DCAD had decreased prepartum and postpartum blood non-esterified fatty acid (NEFA) concentrations (DeGroot et al., 2010). The deleterious side effects of greater blood NEFA concentrations and increased liver triglyceride accumulation might be reduced as a result of the negative DCAD prepartum and suggests a possible reason to provide a negative DCAD diet to close-up heifers (DeGroot et al., 2010). Others did not observe changes in peripartum NEFA concentrations of cows provided an anion DCAD; perhaps changes in NEFA at this time are more likely due to reduced DMI rather than the anionic diet per se (Ramos-Nieves et al., 2009). Cows that develop hypocalcemia have greater NEFA concentrations than those that maintain normal blood Ca; and, as a result those hypocalcemic cows are at greater risk of developing ketosis (Reinhardt et al., 2011). Lower postpartum ketone -hydroxybutyrate (BHBA) concentrations were observed for cows that maintained normocalcemia (DeGroot et al., 2010; Martinez et al., 2012). Reproduction In the USDA (2007) survey, approximately 79% of cows in the U. S. were culled for poor reproductive performance. Through better control of periparturient Ca status, subsequent reproductive management and success might be improved. Evidence to support this possibility was first reported by Ward et al. (1970) who observed a decrease in number of days to first ovulation postpartum by feeding 200 g Ca/d prepartum compared with 100 g/d. Additionally, vitamin D supplementation decreased both days to first estrus and days to conception (Ward et al., 1970). Calcium status is not thought to alter estrous cyclicity directly (Ward et al., 1970; Martinez et al., 2012); however, cows that experience difficulty at parturition (i.e., dystocia, stillbirth, and retained placenta) have reduced normal cyclicity (Martinez et al., 2012). Furthermore, cows that maintain normal Ca status around parturition are more likely to have increased pregnancy rate and reduced days open (Martinez et al., 2012). Also, Chapinal et al. (2012) reported a link between incidence of periparturient disease and pregnancy rate. Cows identified as being at greater risk for clinical diseases (i.e., increased blood NEFA and BHBA, and(or) hypocalcemia) postpartum and dystocia had lower odds of pregnancy at first artificial insemination (AI). Cows that maintained normal Ca status around parturition were 1.5 times more likely to conceive at first AI.

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Immunity Relationships between blood Ca concentrations and the cows ability to combat infections have been established (Kimura et al., 2006; Nelson et al., 2010; Martinez et al., 2012). Calcium is an important second messenger in the immune response. In an attempt to adjust the depleted Ca pool during times of hypocalcemia, peripheral mononuclear cells release Ca from their stores. In a short time these Ca stores are depleted and this limits the immune cells ability to send Ca as a signal when triggered as an immune response (Kimura et al., 2006). Martinez et al. (2012) linked greater risk of metritis with drops in serum Ca concentrations. Four times as many cows developed metritis if they displayed both birthing problems (e.g., dystocia, twinning, stillbirths, or retained placenta) and subclinical hypocalcemia compared with cows that did not have birthing difficulties and maintained normal Ca status. Inadequate vitamin D in the transition period compounds problems associated with hypocalcemia by limiting the cows ability to increase Ca absorption and resorption (Goff and Horst, 2003); thus, prolonging hypocalcemia and subsequent health problems. It has been understood for a while that active vitamin D has a large role in immune function as a signal hormone for monocytes (Reinhardt and Hustmyer, 1987). More recently, vitamin D has been shown to be a crucial component of cow monocyte function (Nelson et al., 2010). Previously this relationship between cow monocytes and vitamin D was not known. It is now understood that monocytes not only have the ability to respond to active vitamin D, but also actually produce the hormone (Nelson et al., 2010). The amount of vitamin D needed for appropriate immune responses is unknown; it is simply assumed that the NRC (2001) recommended daily intake is sufficient for all known biological needs. Given these recent findings, further investigation into supplementation of vitamin D and increased dietary Ca in rations of sick and(or) cows predisposed to hypocalcemia could prove beneficial. CONCLUSIONS Subclinical hypocalcemia and milk fever doubtless are detrimental disorders that affect mineral element status, periparturient health, and lactational and reproductive performance of too many dairy cows. Calcium status around parturition can be affected by a variety of factors surveyed in this paper. Utilizing a low or negative DCAD in the close-up ration is a tried and often effective way to increase blood Ca in the periparturient period. However, more proactive approaches (i.e., reconsideration of normal blood Ca range, vitamin D supplementation, more enhancement of immune function by modulating Ca status, diets tailored specifically to late pregnant heifers, targeted approaches for predisposed cows) may prove efficacious and should be considered. Together these findings highlight both the direct and indirect importance of Ca status around parturition and how subsequent milk production and future performance and profitability can be affected. REFERENCES Beckman, M.J. and H.F. DeLuca. 2002. Regulation of renal vitamin D receptor is important determinant of 1-alpha, 25-dihydroxyvitamin D(3) levels in vivo. Arch. Biochem. Biophys. 401:44-52. 14

Bigner, D.R., J.P. Goff, M.A. Faust, J.L. Burton, H.D. Tyler, and R.L. Horst. 1996. Acidosis effects on insulin response during glucose tolerance tests in Jersey cows. J. Dairy Sci. 79:2182-2188. Block, E. 1984. Manipulating dietary anions and cations for prepartum dairy cows to reduce incidence of milk fever. J. Dairy Sci. 67:2939-2948. Bushinsky, D.A. 2001. Acid-base imbalance and the skeleton. Eur. J. Nutr. 40:238-244. Cahn C.M., and S. Line. 2005. The Merck Veterinary Manual. 9th ed. Whitehouse Station, NJ. Chan, P.S., J.W. West, and J.K. Bernard. 2006. Effect of prepartum dietary calcium on intake and serum and urinary mineral concentrations of cows. J. Dairy Sci. 89:704-713. Chapinal, N., S.J. LeBlanc, M.E. Carson, K.E. Leslie, S. Godden, M. Capel, J.E.P. Santos, M.W. Overton, and T.F. Duffield. 2012. The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance. J. Dairy Sci. 95:1301-1309. Charbonneau, E., D. Pellerin, and G.R. Oetzel. 2006. Impact of lowering dietary cation-anion difference in non-lactating dairy cows: A meta-analysis. J. Dairy Sci. 89:537-548. Curtis, C.R., H.N. Erb, C.J. Sniffen, R.D. Smith, P.A. Powers, M.C. Smith, M.E. White, R.B. Hillman, and E.J. Pearson. 1983. Association of parturient hypocalcemia with eight periparturient disorders in Holstein cows. J. Amer. Vet. Med. Assoc. 183:559 561. DeGroot, M.A., E. Block, and P.D. French. 2010. Effect of prepartum anionic supplementation on periparturient feed intake, health, and milk production. J. Dairy Sci. 93:5268-5279. Deiner, C., M. Reiche, D. Lassner, D. Grienitz, S. Twardzoik, A. Moesch, P. Wenning, and H. Martens. 2012. Alleic variations in coding regions of the vitamin D receptor gene in dairy cows and potential susceptibility to periparturient hypocalcaemia. J Dairy Res. 79:423-428. Dishington, I.W. 1975. Prevention of milk fever (hypocalcemic paresis puerparalis) by dietary salt supplementation. Acta. Vet. Scand. 16:503. Ender, F., and I.W. Dishington. 1970. Etiology and prevention of paresis puerperalis in dairy cows. Page 71 in Parturient hypocalcemia. J.J.B. Anderson, ed. Academic Press, New York, NY. Ender, F., I.W. Dishington, and A. Helgebostad. 1971. Calcium balance studies in dairy cows under experimental induction and prevention of hypocalcaemic paresis puerperalis. Z. Tierphysio. Tierernhr., Futtermittelkd. 28:233. Goff, J.P., T.A. Reinhardt, R.L. Horst. 1989. Recurring hypocalcemia of bovine parturient paresis is associated with failure to produce 1,25 dihydroxyvitamin D. J. Endocrinology 125:49-53. Goff J.P., T.A. Reinhardt, and R.L. Horst. 1995a. Milk fever and dietary cation-anion balance effects on concentration of vitamin D receptor in tissue of periparturient dairy cows. J. Dairy Sci. 78:2388-2394. Goff, J.P., T.A. Reinhardt, D.B. Beitz, and R.L. Horst. 1995b. Breed affects tissue vitamin D receptor concentration in periparturient dairy cows: a milk fever risk factor? J. Dairy Sci. 78(Suppl. 1):184. (Abstr). Goff, J.P. and R.L. Horst. 1997a. Physiological changes at parturition and their relationship to metabolic disorders. J. Dairy Sci. 80:1260-1268. Goff, J.P and R.L. Horst. 1997b. Effects of the addition of potassium or sodium, but not calcium, to prepartum rations on milk fever in Dairy Cows. J. Dairy Sci. 80:176-186. Goff, J.P., and R.L. Horst. 1998. Use of hydrochloric acid as a source of anions for prevention of milk fever. J. Dairy Sci. 81:2874-2880. 15

Goff, J.P. and R.L. Horst. 2003. Role of acid-base physiology on the pathogenesis of parturient hypocalcemia (milk fever) the DCAD Theory in Principal and Practice. Acta. Vet. Scand. Suppl. 97:51-56. Goff, J.P., R. Ruiz, and R.L. Horst. 2004. Relative acidifying activity of anionic salts commonly used to prevent milk fever. J. Dairy Sci. 87:1245 1255. Goff, J.P. 2008. The monitoring, prevention and treatment of milk fever and subclinical hypocalcemia in dairy cows. Vet. J. 176:50-57 Goff, J.P., and R.L. Horst. 2012. Comparison of low versus high calcium anionic diets for prevention of hypocalcemia and milk fever. J. Dairy Sci. 95(Suppl. 2):444. Goings, R.L., N.L. Jacobson, D.C. Beitz, E.T. Littledike , and K.D. Wiggers. 1974. Prevention of parturient paresis by a prepartum calcium deficient diet. J. Dairy Sci. 57:1184-1188. Grnberg, W., S.S. Donkin, and P.D. Constable. 2011. Periparturient effects of feeding a low dietary cation-anion difference diet on acid-base, calcium and phosphorus homeostasis and on intravenous glucose tolerance test in high-producing dairy cows. J. Dairy Sci. 94:727-745. Hibbs, J.W. and H. R. Conrad. 1960. Studies of milk fever in dairy cows. VI. Effect of three prepartal dosage levels of vitamin D on milk fever incidence. J. Dairy Sci. 43:11241128. Hibbs, J.W. and H.R. Conrad. 1976. Milk Fever in Dairy Cows. VII. Effect of continuous vitamin D feeding on incidence of milk fever 1. J. Dairy Sci. 76:1944-1946. Hidalgo, A.A., D.L. Trump, and C.S. Johnson. 2010. Glucocorticoid regulation of the vitamin D receptor. J Steroid Biochem. Mol. Biol. 121:372-375. Horst, R.L. and T.A. Reinhardt.1983. Vitamin D metabolism in ruminants and its relevance to the periparturient cow. J. Dairy Sci. 66:661-678. Horst, R.L., J.P. Goff, and T.A. Reinhardt. 1997. Strategies for preventing milk fever in dairy cattle. J. Dairy Sci. 80:1269-1280. Hymller, L. and S.K. Jensen. (2010). Stability in the rumen and effect on plasma status of single oral doses of vitamin D and vitamin E in high-yielding dairy cows. J. Dairy Sci. 93:548-5757. Hymller L. and S.K. Jensen. 2011. Vitamin D2 impairs utilization of vitamin D3 in highyielding dairy cows in a cross-over supplementation regimen. J. Dairy Sci. 94:33623366. Jardon, P.W. 1995. Using urine pH to monitor anionic salt programs. Compend. Contin. Educ. Pract. Vet. 17:860866. Jawor, P.E., J.M. Huzzey, S.J. LeBlanc, and M.A.G. von Keyserlingk. 2012. Associations of subclinical hypocalcemia at calving with milk yield, and feeding drinking and standing behaviors around parturition in Holstein cows. J. Dairy Sci. 95:1240-1248. Jones, G. 2008. Pharmocokinetics of Vitamin D toxicity. Am J Clin Nutr. 88(Suppl). 582S586S. Johannsson, A.J., and L.G. Raisz. 1983. Effects of low medium magnesium concentration on bone resorption in response to parathyroid hormone and 1,25-dihydroxyvitamin D in organ culture. Endocrinology 113:2294-2298. Julien, W.E., H.R. Conrad, J.W. Hibbs, and W.L. Crist. 1976. Milk Fever in Dairy Cows. VIII. Effect of injected vitamin D3 and calcium and phosphorus intake on incidence1. J. Dairy Sci. 76:431-436.

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Kamiya, Y., M. Kamiya, M. Tanaka, and S. Shioya. 2005. Effects of calcium intake and parity on plasma minerals and bone turnover around parturition. Anim. Sci. Journal. 76:325330. Kimura, K., T.A. Reinhardt, and J.P. Goff. 2006. Parturition and hypocalcemia blunts calcium signals in immune cells of dairy cattle. J. Dairy Sci. 89:2588:2595. Kronqvist, C., R. Emanuelson, R. Sprndly, and K. Holtenius. 2011. Effects of prepartum dietary calcium level on calcium and magnesium metabolism in periparturient dairy cows. J. Dairy Sci. 94:1365-1373. Lean, I.J., P.J. DeGaris, D.M. McNell, and E. Block. 2006. Hypocalcemia in dairy cows: metaanalysis and dietary cation-anion difference theory revisited. J. Dairy Sci. 89:669-684. Liesegang, A., K. Singer, and A. Boos. 2008. Vitamin D receptor amounts across different segments of the gastrointestinal tract in Brown Swiss and Holstein Friesian cows of different age. J. Anim. Physiol. Anim. Nutr. 92:316-323. Littledike, E.T., and R.L.Horst.1980.Problems with vitamin D injections for prevention of milk fever: Toxicity of large doses and increased incidence of small doses. J. Dairy Sci. 63(Suppl.1):89. Martinez. N., C.A. Risco, F.S. Lima, R.S. Bisinotto, L.F. Greco, E.S. Ribeiro, F. Maunsell, K.N. Galvo, J.E.P. Santos. 2012. Evaluation of peripartal calcium status, energetic profile, and neutrophil function in dairy cows at low or high risk of developing uterine disease. J. Dairy Sci. 95:7158-7152. Moore, S.J., M.J. VandeHaar, B.K. Sharma, T.E. Pilbeam, D.K. Beede, H.F. Bucholtz, J.S. Liesman, R.L. Horst, and J.P. Goff. 2000. Effects of altering dietary cation-anion difference on calcium and energy metabolism in peripartum cows. J. Dairy Sci. 83:2095-2104. Muir, L.A., J.W. Hibbs, and H.R. Conrad. 1968. Effect of vitamin D on the ability of cows to mobilize blood calcium. J. Dairy Sci. 67:1046-1050. Nagy, O., G. Kovc, H. Seidel, T. Weissov. 2001. The effect of arterial blood sampling sites on blood gases and acid-base balance parameters in calves. Acta. Vet. Hung. 49:331-340. National Research Council (NRC). 2001. Nutrient Requirements of Dairy Cattle. 7 th ed. Washington, D.C. Nelson, C.D., T.A. Reinhardt, T.C. Thacker, D.C. Beitz, and J.D. Lippolis. 2010. Modulation of the bovine innate immune response by production of 1,25 -dihydroxyvitamin D3 in bovine monocytes. J. Dairy Sci. 93:1041-1049. Oba, M., A.E. Oakley, and G.F. Tramblay. 2011. Dietary Ca concentration to minimize the risk of hypocalcemia in dairy cows is affected by the dietary cation-anion difference. Anim. Feed Sci.Tech. 164:147-153. Oetzel, G.R., J.D. Olson, C.R. Curtis, and M.J. Fettman. 1988. Ammonium chloride and ammonium sulfate for prevention of parturient paresis in dairy cows. J. Dairy Sci. 71:3302-3309. Oetzel G.R., and J.A. Barmore. 1993. Intake of a concentrate mixture containing various anionic salts fed to pregnant, nonlacting, dairy cows1. J. Dairy Sci. 76:1617-1623. Ramos-Nieves J.M., B.J. Thering, M.R. Waldron, P.W. Jardon, and T.R. Overton. 2009. Effects of anion supplementation to low-potassium prepartum diets on macromineral status and performance of periparturient dairy cows. J. Dairy Sci. 92:5677-5691. Reinhardt, T.A., and F.G. Hustmyer. 1987. Role of vitamin D in the immune system. J. Dairy Sci. 70:952-962.

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Reinhardt, T.A., and R.L. Horst, 1990. Parathyroid hormone down regulates 1,25 dihydroxyvitamin D receptors (VDR) and VDR mRNA in vitro and blocks homologous up-regulation in vivo. Endocrinology 127:942-948. Reinhardt, T.A., J.D. Lippolis, B.J. McCluskey, J.P. Goff, and R.L. Horst. 2011. Prevalence of subclinical hypocalcemia in dairy herds. Vet. J. 188:122-124. Roche, J.R., D. Dalley, P. Moate, C. Grainger, M. Rath, and F. OMara. 2003. A low dietary cation-anion difference precalving and calcium supplementation post calving increase plasma calcium but not milk production in a pasture-based system. J. Dairy Sci. 88:2658-2666. Sanchez, W.K., and D.K. Beede. 1991. Interrelationships of dietary Na, K and Cl and cationanion difference in lactation rations. In Proc. Florida Rum. Nutr. Conf. University of Florida, Gainesville. p.31. Sommerfeldt, J.L., R.L. Horst, E.T. Littledike, and D. C. Beitz. 1979. In vitro degradation of cholecalciferol in rumen fluid. J.Dairy Sci. 62(Suppl. 1):192193. Sommerfeldt, J.L., J.L. Napoli, E.T. Littledike, D.C. Beitz, and R.L. Horst. 1983. Metabolism of orally administered [3H] ergocalciferol and [3H] cholecalciferol by dairy calves. J.Nutr.113:2595 2600. Spanghero, M. 2004. Prediction of urinary and blood pH in non-lactating dairy cows fed anionic diets. Anim. Feed Sci. Tech. 116:83-92. Taylor, M.S., K.V. Knolton, M.L. McGilliard, W.M. Seymour, and J.H. Herbein. 2008. Blood mineral, hormone and osteocalcin responses of multiparous Jersey cows to an oral dose of 25-hydroxyvitamin D3 or vitamin D3 before parturition. J. Dairy Sci. 91:2408-2416. Thilsing-Hansen, T., R.J. Jrgensen, and S. stergaard. 2002. Milk fever control principles: a review. Acta. Vet. Scand. 43:1-19. Tucker, W.B., J.F. Hogue, D.F. Waterman, T.S. Swenson, Z. Xin, R.W. Hemken, J.A. Jackson, G.D. Adams, and L.J. Spicer. 1991a. Role of sulfur and chloride in the dietary cationanion balance equation for lactating dairy cattle. J. Dairy Sci. 69:1205-1213 Tucker, W., Z. Xin, and R. W. Hemkin. 1991b. Influence of calcium chloride on systemic acidbase status and calcium metabolism in dairy heifers. J. Dairy Sci. 74:1401-1407. USDA. 2007. Animal and Plant Health Inspection Service (APHIS). Veterinary Services. National Animal Health Monitoring System (NAHMS), Part 1: Reference of dairy cattle health and management practices in the United States. Vagoni, D.B., and G.R. Oetzel. 1998. Effects of dietary cation-anion difference on the acid-base status of dry dairy cows. J. Dairy Sci. 81:1643-1652. van Mosel M., A.T. Vant Klooster, F. van Mosel, and J.V.D. Kuilen. 1993. Effects of reducing dietary [(Na+ + K+) (Cl- + SO4)] on the rate of calcium mobilization by dairy cows at parturition. Res. Vet. Sci. 54:1-9. Ward, G., G.B. Marion, C.W. Campbell, and J.R. Dunham. 1970. Influences of calcium intake and vitamin D supplementation on reproductive performance of dairy cows. J. Dairy Sci. 51:204-206. Wilkens, M.R., I. Oberheide, B. Schrder, E. Azem, W. Steinberg, and G. Breves. 2012. Influence of the combination of 25-hydroxyvitamin D3 and a diet negative in cationanion difference on peripartal calcium homeostasis of dairy cows. J. Dairy Sci. 95:151164. Whittaker, J., C. Cuthbert, V.A. Hammond, and K.G. Alberti. 1982. The effects of metabolic acidosis in vivo on insulin binding to isolated rat adipocytes. Metabolism. 31:553-557.

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Mastitis and Dairy Replacement Heifers: Management Practices to Reduce the Odds
S.C. Nickerson1, V.J. Eubanks1, J.D. Chapman2, L.O. Ely,1, K.P. Zanzalari 2, F.M. Kautz1, D.J. Hurley1, N.E. Forsberg3, and Y.Q. Wang3 1 University of Georgia, Athens, GA, 2 Prince Agri Products Inc., Quincy, IL, 3 OmniGen Research LLC, Corvallis, OR. Corresponding author: scn@uga.edu SUMMARY Prevalence of mastitis in unbred, breeding age, and pregnant dairy heifers is higher than formerly realized. Infected mammary quarters, especially those with Staph. aureus IMI, exhibit reduced mammary gland secretory potential, marked leukocyte infiltration, and the accompanying inflammation. Both nonlactating and lactating commercial antibiotic infusion products have been used successfully to cure existing infections and reduce SCC, and nonlactating therapy prevents new IMI with environmental streptococci. However, the goal is to prevent new infections from occurring in these young dairy animals through management strategies aimed at vaccination, use of teat seals, fly control, and dietary supplementation. Results of commercial vaccine trials illustrate that immunization will reduce Staph. aureus mastitis in heifers at calving between approximately 45 and 60%, with reductions in SCC of 50%. Likewise, a fly control program for heifers will decrease incidence of Staph. aureus mastitis by up to 83%. Lastly, dietary supplementation to boost the immune systems of heifers has been shown to reduce incidence of mastitis at calving, lower SCC, and increase milk yield. As global milk quality standards become more stringent, management practices based on curing existing infections and preventing new IMI in heifers will ensure that these young dairy animals enter the milking herd free of mastitis and with low SCC. Such practices should be considered for incorporation into dairy herd health programs in herds suffering from a high prevalence of heifer mastitis, especially that caused by Staph. aureus. Not only do these practices reduce new infections in first calf heifers at parturition, they also reduce the introduction of Staph. aureus to the milking herd. INTRODUCTION Replacement heifers are critical to dairy herd productivity because they represent the future milking and breeding stock of all dairy operations. The goal should be to provide an environment for heifers to develop their full lactation potential at the desired age with minimal expense. Animal health and well-being play vital roles in achieving this potential, and one disease that can influence future productivity is heifer mastitis caused by Staphylococcus aureus, the coagulase-negative staphylococci (CNS), and the environmental streptococci. The presence of mastitis in young dairy heifers is generally not observed until freshening or until the first clinical flare-up in early lactation. Thus, animals may carry intramammary infections (IMI) for a year or more before they are diagnosed with mastitis, resulting in reduced milk yield (Boddie et al., 1987). The greatest development of milk-producing tissue in the udder occurs during the first pregnancy, so it is important to protect the heifer mammary gland from pathogenic microorganisms to ensure maximum milk production during the first and future lactations. 19

PREVALANCE IF MASTITIS IN UNBRED AND PREGNANT HEIFERS The initial focus on heifer mastitis began in the mid 1980s after several dairy producers in Louisiana complained to university researchers that a large percentage of their heifers were freshening with clinical mastitis. Subsequent study of breeding age animals in a research herd revealed that IMI were diagnosed as early as 6 months of age, and that infections persisted throughout pregnancy and into lactation (Boddie et al., 1987). A follow-up study of 4 commercial herds demonstrated that IMI were found in 96.9% of heifers and in 74.6% of mammary quarters (Trinidad et al., 1990a). Although the vast majority of udders were visually healthy, 30% of udders and 15.1% of quarters showed clinical symptoms of mastitis as evidenced by clots, flakes, and blood; only rarely were quarters swollen and enlarged. Staph. aureus was isolated from 14.7% of quarters. This microorganism was also isolated from 25% of quarters with clinical symptoms. Staph. aureus causes severe damage to mammary tissue, and infections are very difficult to eliminate in lactating cows. Other organisms isolated from secretions and percentage frequencies were Staph. chromogenes (43.1%). Staph. hyicus (24.3%), other staphylococcal spp. (3.6%), Strep. dysgalactiae (0.4%), Strep. spp. (3.3%), Nocardia species (0.4%), and mixed isolates containing staphylococci and streptococci (5.1%). Figure 1. Although the majority of breeding age dairy heifers are healthy (a), up to 30% show clinical symptoms of mastitis such as ropey, clotted secretions (b); however, enlarged, swollen quarters are rare (c), thus, this disease is difficult to diagnose via visual observation of the mammary gland.

MAMMARY LEUKOCYTE RESPONSE TO INTRAMAMMARY INFECTION In lactating cows, the milk somatic cell count (SCC) is used as a measure of milk quality. This count is composed mainly of leukocytes (macrophages, lymphocytes, and neutrophils, Figure 1) and is considered an important parameter for assessing mammary health status (e.g. inflammation). It is well known that milk yield decreases as the SCC and the incidence of mastitis increases. Thus, SCC in breeding age and pregnant heifer mammary gland secretions have been analyzed to measure the degree of inflammation and potential reductions in future milk yield. In a study by Boddie et al. (1987), the mean SCC of quarters from unbred heifers infected with Staph. chromogenes, Staph. hyicus, and Staph. aureus were 7.8, 8.5, and 9.2 x 106/ml, respectively, whereas the mean SCC of uninfected quarters was 3.5 x 10 6. Approximately 13% of quarter secretions sampled prepartum contained Staph. aureus, and after 20

freshening, the SCC of these quarters averaged 578 x 10 3/ml, a cell count associated with a loss of greater than 4.4 lb of milk/day. Figure 2. The somatic cell count includes macrophages (M), lymphocytes (L), and neutrophils (N).

N M L

The volume of mammary secretion is very low in breeding-age animals; thus, somatic cells become concentrated, resulting in high SCCs even in uninfected quarters. Such elevated SCC over a long period of time suggests that mammary tissues in affected quarters are in a state of chronic inflammation, which could adversely affect development of milk-producing tissues and negatively affect future milk yield. In response to infection, neutrophils become the major leukocyte type that infiltrates mammary tissue from the vascular system to phagocytose and kill mastitis-causing bacteria. The migration of neutrophils across the mammary epithelium causes mechanical as well as chemical damage to the milk secretory cells resulting in decreased yield (Akers and Nickerson, 2011). MAMMARY TISSUE RESPONSE TO PRESENCE OF INFECTION Histologic observations of mammary tissue samples from uninfected quarters of heifers by Trinidad et al. (1990a) showed that milk-producing alveoli were small; the epithelial lining was composed of a single layer of cuboidal cells surrounding a small luminal space with little or no stained secretory product (Figure 3a). Interalveolar connective tissue area comprised approximately half of the observed tissue area. Mammary tissues infected with Staph. aureus, on the other hand, exhibited large amounts of interalveolar connective tissues and reductions in epithelial and luminal areas (Figure 3b), suggesting reduced secretory activity. Indeed, results of morphometric analysis showed that percentages of alveolar epithelium and lumen in quarters infected with Staph. aureus were lower (P < .05) than those in uninfected quarters. Quarters infected with Staph. aureus also showed a greater percentage (P < .05) of interalveolar stroma than did uninfected quarters. Additionally, quarters infected with Staph. aureus exhibited 21

significantly greater infiltration of leukocytes (mainly lymphocytes and neutrophils) compared with uninfected tissues. The greatest development of secretory tissues in young heifers occurs during the first pregnancy, and these tissues are affected adversely by bacterial infection and inflammation, leading to deposition of connective tissue instead of milk secretory tissue, which results in a reduction of future milk yield. Figure 3a. Portion of mammary parenchymal tissue typical of that obtained from uninfected quarters exhibiting small milk-producing alveoli (A) with empty, ovoid lumens (1), lumens with some secretions (2), and interalveolar connective tissue stroma (S). x180. Figure 3b. Parenchymal tissue from a quarter infected with Staph. aureus exhibiting a large percentage of interalveolar connective tissue stroma (S), fewer numbers of alveoli (A), and limited alveolar luminal areas (L). D = Duct. x180.

USE OF ANTIBIOTIC THERAPY TO CONTROL MAMMARY INFECTIONS Curing Existing Cases of Mastitis with Nonlactating Cow Antibiotic Therapy Because of the high level of infection found in breeding age and pregnant heifers, especially mastitis caused by Staph. aureus, antimicrobial therapy should be considered. In an initial study to evaluate the effectiveness of treatment, several heifers from 4 commercial herds were randomly selected to receive a single intramammary treatment of a penicillin and dihydrostreptomycin product into all 4 mammary quarters (Trinidad et al., 1990b). Treatments 22

were made at approximately 60 days prior to calving. Results showed that 97.1% of treated heifers (n = 35) were infected with some type of mastitis at the time of treatment, but, at calving, only 40% remained infected. Of the untreated control heifers (n = 38), 100% were infected at initial sampling, and at calving, mastitis was reduced only slightly to 97.4%. Staph. aureus was isolated from 11 quarters of 6 treated heifers before antibiotic infusion (45.8%), but at calving, this organism was isolated from only 1 quarter of 1 heifer (4.2%). In the control group, 18 quarters of 10 heifers were infected with Staph. aureus at time of treatment (45%). At calving, 6 of the control heifers still had Staph. aureus mastitis in 11 quarters (55%). Thus, the overall incidence of IMI caused by Staph. aureus was reduced over 90% and SCC were reduced by 50%. Production data collected over the first 2 months of lactation demonstrated that Staph. aureus-infected heifers receiving nonlactating cow therapy during pregnancy produced an average of 5.5 lb more milk per day (or 10% more milk) than Staph. aureus-infected herd mates that did not receive treatment. Other advantages include a longer productive life and higher income due to lower SCC and higher quality milk premiums. Subsequent studies (Owens et al., 1991, 1994) confirmed results of the initial investigation above. Heifers that were infected with Staph. aureus were infused 8-12 weeks prepartum with one dose of 300 mg of a cephapirin benzathine product into all 4 mammary quarters and were compared with untreated controls infected with Staph. aureus. Results demonstrated cure rates between 87 and 100%. After antibiotic infusion, SCC in infected quarters that cured decreased from 15 x 106/ml to 4 x 106/ml 1 week later, and to 700 x 103/ml at calving, whereas SCC in untreated controls at calving were 5 x 106/ml. Treated heifers in which Staph. aureus IMI were cured yielded 11% more milk during the first 2 months of lactation. One of these studies also demonstrated that prepartum treatment served as a prophylactic against new cases of environmental strep mastitis (Owens et al., 1994), reducing the new IMI rate at calving by 93%. Thus, use of nonlactating cow therapy was effective in preventing new IMI as well as curing existing infections. When Is the Best Time to Administer Nonlactating Cow Therapy? To answer this question, a 2-year study was conducted (Owens et al., 2001) in which 233 Jersey heifers were quarter sampled shortly after they were confirmed pregnant and at 4-week intervals thereafter. At the initial sampling, 56.5% of quarters were infected with some type of organism, and 15.4% of quarters were infected with Staph. aureus. After the initial sampling, animals were treated with a one-time infusion of 1 of 5 different nonlactating cow infusion products during the first (0 - 90 days), second (91 - 180 days), or third (181 - 270 days) trimester of pregnancy. Results showed that cure rates for the 5 products were high, ranging from 67 to 100%, and significantly higher than the spontaneous cure rate (25%) observed in untreated control quarters; no differences were observed among the three treatment time periods during gestation. Thus, the timing of treatment is best determined by what is most convenient for the management practices of a particular dairy. For example, heifers could be treated: 1) at time of artificial insemination; 2) during routine rectal palpation to determine pregnancy status; or 3) when moved to a new location in preparation for calving. Treatment should be administered no less than 45 days prior to expected calving date to prevent antibiotic residues. When administering treatment, it is important to 1) restrain the heifer in a headlock and/or squeeze chute, and if necessary further restrain the animal by tailing; 2) sanitize the teat orifice with cotton balls or pads soaked in 70% alcohol; 3) use the partial insertion technique 23

when inserting the tip of the syringe cannula (only 2-3 mm) into the teat canal; and 4) dip teats in a germicide to kill any contaminating bacteria. See Figure 4. The treatment of heifers during pregnancy with a nonlactating cow product is advantageous because: 1) the cure rate is higher than during lactation, especially against Staph. aureus; 2) there are no milk losses during therapy; 3) the risk of antibiotic residues is minimal; 4) SCC at calving is reduced; 5) new IMI with environmental streptococci is prevented; and 6) milk production is increased by approximately 10% in successfully treated animals. Figure 4. a) Securing the animal in a headlock system while feeding helps to immobilize the heifer. b) Teats should be sanitized prior to treatment. c) When infusing antibiotic, only the tip of the syringe cannula should be inserted into the teat orifice. d) After therapy, teats should be immersed in an effective germicide.

Efficacy of Lactating Cow Products in Curing IMI Lactating cow products also have been used successfully in heifers when treating infections caused by the environmental streptococci and CNS immediately prior to calving. Studies on this subject have been performed in late gestation 1 - 3 weeks before calving. Although treating animals 1 week prior to calving is successful, antibiotic residues often result, so most trials have focused on treating 2-3 weeks prepartum. For example, Oliver et al. (2004) conducted a trial to determine if therapy with penicillinnovobiocin or pirlimycin hydrochloride 2 weeks prepartum was effective in curing IMI and thereby reducing the level of mastitis during early lactation. Approximately 73% of Holstein heifers were infected 14 days before expected calving; the majority of IMI were due to CNS (44%) and Staph. aureus (30%). At calving, the cure rate was 76% after treatment with penicillin-novobiocin, and 59% following treatment with pirlimycin. In this same study, 96% of Jersey heifers were infected 14 days before calving; the majority of IMI were due to CNS (61%), Strep. spp. (19%), and Staph. aureus (8%). At calving, the cure rate was 75% after treatment with penicillin-novobiocin, and 87% following treatment with pirlimycin. Thus, prepartum therapy of heifer mammary glands with penicillin-novobiocin or pirlimycin hydrochloride was effective in reducing the percentage of heifers infected with mastitis pathogens during early lactation. 24

As a part of the above trial, milk production and somatic cell count data from 82 control heifers and 111 heifers treated with antibiotics before calving were evaluated (Oliver et al., 2003). Milk production was about 10% higher in heifers treated prepartum with antibiotics. Additionally, treated heifers had significantly lower SCCs than control heifers (~52,000 vs. 81,000). Thus, prepartum antibiotic treatment to reduce the rate of mastitis in heifers during early lactation was economically beneficial. The studies on prepartum treatment with lactating cow therapy administered 7 - 21 days before calving have shown treatment to be effective for quarters infected with CNS and Strep. uberis, but waiting until this time to treat chronic Staph. aureus mastitis might be too late. A mammary gland that has been infected with Staph. aureus for several months to a year will not develop normally, and treatment during the immediate prepartum period would most likely be of little benefit in curing infections or salvaging mammary tissue. At this point, the tissue damage would have already been done, and affected quarters should have been treated earlier in gestation to: 1) cure existing infections; 2) reduce chronic inflammation; and 3) allow mammary tissue to develop normally during the later stages of pregnancy. Results of these trials demonstrated that nonlactating and lactating cow antimicrobial treatment of heifers, known to be at risk for developing IMI, is advantageous because the cure rate is much higher than that obtained when treating infections during lactation. In addition, most studies show that SCC are lower, there is no milk loss due to therapy, risk of antibiotic residue at calving is minimal, and future milk production is increased in heifers cured of IMI. However, the goal in controlling mastitis is to prevent new infections, and management strategies to enhance disease prevention are discussed below. MANAGING HEIFERS TO PREVENT MAMMARY INFECTIONS Role of Vaccination in Mastitis Control Although antimicrobial therapy is successful in curing existing cases of mastitis, the goal from a herd management perspective is to prevent new infections from occurring, and vaccination has been attempted as a prophylactic measure. The purpose of vaccination is to increase circulating antibodies directed against certain mastitis pathogens to protect against bacterial invasion. Researchers in Louisiana evaluated a commercially available Staph. aureus vaccine in young dairy animals (Nickerson et al., 1999). This product is the only Staph. aureus vaccine on the market in the US (Lysigin, Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO, USA). At 6 months of age, 35 Jersey heifers were vaccinated following manufacturers instructions using a 5-ml dose intramuscularly in the semimembranosus muscle (upper thigh) of the rear leg (Figure 5), and 14 days later, animals received a booster dose, which was repeated at 6-month intervals. Another 35 heifers served as unvaccinated controls. Note: Injections were administered per label instructions; however, it is generally recommended to avoid muscle tissues in the leg, thereby minimizing potential abscess formation; injection into the neck region is preferable. Results demonstrated that: 1) the number of quarters exhibiting chronic Staph. aureus mastitis during pregnancy was reduced 43.1% in vaccinates compared with controls; 2) rate of new IMI during pregnancy was reduced 44.8%; 3) rate of new IMI at freshening was reduced 44.7%; and 4) the SCC was reduced by 50% in vaccinates compared with controls. 25

Figure 5. Vaccination (5 cc) into the left semimembranosus muscle of the rear leg using an 18-gauge needle.

In a subsequent, more in depth study using the same vaccine (Lysigin ), 106 Holstein heifers from a dairy herd in Virginia were evaluated (Nickerson et al., 2009). Previous microbiological culture of heifer mammary secretions indicated that approximately 35% of the animals were infected with Staph. aureus. At 6 to 18 months of age, 53 heifers were vaccinated and boosted as above, and the other 53 heifers served as unvaccinated controls. The purpose was to determine if vaccination reduced the level of Staph. aureus at calving as observed in the Louisiana trial. Vaccine efficacy data showed that the percentage of heifers with Staph. aureus IMI at freshening was lower in vaccinates (13.3%) compared with controls (34.0%); a reduction of 60.9%. Likewise, an examination of health records showed that the percentage of heifers that were culled or died during the trial was reduced by approximately one-third by vaccination: 16.9% in vaccinates and 24.5% in controls. Somatic cell counts in samples collected during first week of lactation from uninfected heifers for vaccinates and controls were 66,095 and 132,754/ml, respectively; a 50.2% reduction; and for infected heifers, SCC were 441,764 and 892,176/ml, respectively; a 50.5% reduction. An examination of the 305-day lactation milk yield for the 1st lactation of both vaccinated and unvaccinated control heifers demonstrated an approximate 10% increase in production in vaccinates vs. controls (24,250 vs. 22,046 lb, respectively) or a difference of 2,204 lb. Likewise, the 305-day pounds of both fat and protein were higher in vaccinates than controls (fat: 899 vs. 747 kg, respectively; protein: 727 vs. 694 kg, respectively). An examination of the number of days in milk for the first lactation demonstrated that vaccinates lactated 13 days longer than the unvaccinated controls (349 vs. 336 days). In addition, average first lactation SCC were 11,000 cells/ml lower in vaccinates compared with controls (49,000 vs. 60,000/ml). Results of this Virginia investigation demonstrated that vaccinating dairy heifers according to the prescribed protocol with a commercial USDA licensed Staph. aureus bacterin, Lysigin, reduced the number of new Staph. aureus intramammary infections at time of calving by 60.9%, lowered the SCC by 50%, and decreased the culling rate by approximately one-third. In 26

addition, overall milk yield, production of fat and protein, and number of days in milk were greater in vaccinated heifers compared with controls. This prevention strategy may represent a major control mechanism for managing Staph. aureus in the future, especially as new antigens and adjuvants are added to vaccine preparations. Efficacy of an Infusible Teat Seal for Preventing IMI Because bacteria breach the teat canal to cause infection, products have been developed to serve as barrier to bacterial entry or seal the teat canal against infection. Such products form a physical plug in the distal teat cistern and teat canal (Figure 6). In one study involving 255 pregnant heifers, mammary quarters were treated approximately 1 month prepartum with an infusible teat seal composed of bismuth subnitrate. Results showed that this procedure 1) reduced the risk of new IMI with any organism by 74%, 2) reduced the prevalence of post calving IMI by 65%; 3) reduced the risk of Strep. uberis infection in quarters with an IMI precalving by 70%; and 4) reduced the incidence of clinical mastitis from which a pathogen was isolated by 70% in quarters detected with an IMI pre-calving (Parker et al., 2007). In a subsequent trial, 1,067 bred heifers were treated approximately 1 month prepartum with teat seal, and results demonstrated that treatment 1) reduced the risk of clinical mastitis by 68%, and 2) reduced the risk of IMI due to Strep. uberis by 84% (Parker et al., 2008). Although teat seal contains no antibiotic and cannot cure existing infections, this product is effective in preventing new cases of mastitis, especially those caused by Strep. uberis. It is emphasized that to be effective, teat seal should be administered approximately 30 days prior to calving and that strict teat end hygiene be followed when applying teat seal products. Figure 6. Radiograph of a teat infused with teat seal, illustrating the teat seal material in the distal teat cistern and in the teat canal (arrows).

The Role of Horn Flies in the Development of Heifer Mastitis

27

Historically, the major association between flies and intramammary infections has been with the development of summer mastitis, in which the biting fly, Hydrotoea irritans, is the proven vector. Summer mastitis is an isolated seasonal problem primarily in July, August, and September in heifers and dry cows of northern Europe, and may be controlled by insecticidal sprays. In the US, fly control is used to reduce these insect pests on farm premises, and subsequently reduce animal stress, but its application as an adjunct management practice for preventing new cases of mastitis and reducing SCC has not been considered or embraced by producers. An initial survey performed at Louisiana State University showed that prevalence of mastitis in bred heifers was significantly lower in dairy herds that used some form of fly control for their lactating cows, dry cows, and heifers compared with herds applying no fly control (Figure 7) (Nickerson et al., 1995). The greatest reductions were in numbers of Staph. aureus and the environmental streptococci, both major mastitis pathogens in adult cows associated with elevations in SCC. The particular species of fly associated with mastitis is the blood-sucking horn fly (Haematobia irritans). This species is commonly found on the backs of dairy animals (Figure 8a), preferring a dark hair coat, but will also attack the teats, leading to the development of mastitis, especially among dairy heifers. Results of the survey above (Nickerson et al., 1995) also demonstrated that bred heifers having teats with bite lesions and scabs caused by horn flies exhibited a 70% frequency of intramammary infection compared with a 40% frequency in heifers with normal teats free of lesions. Such infections are always associated with elevated SCC in excess of 5 million/ml in these young animals. Horn flies tend to attack front teats rather than rear teats. See Figure 8b below illustrating horn flies and lesions on heifer teats. Figure 7. Prevalence of mastitis in Louisiana dairy herds with and without a fly control program.

100 90 80 70 60 50 40 30 20 10 0

With fly control

W/o fly control

100

55.2 41.4 32.9 20.7 5.6 CNS 3.7 Total 44.4

Percent

S. aureus Env. Streps Mastitis-causing pathogen

28

Figure 8. a) Horn flies on the back of a Holstein heifer; note preference for dark hair color vs. white hair. b) Udder of a 10-month-old heifer illustrating horn flies (arrows) and lesions on teat ends.

Since that first survey, researchers have proven through DNA studies that the horn fly is not only responsible for teat lesions on heifers, but is indeed a vector in the transmission of mastitiscausing bacteria such as Staph. aureus, from heifer to heifer, especially during the warm and humid months of the year (Owens at al., 1998). Such mastitic heifers serve as sources of IMI for transmission to the entire lactating and nonlactating herds. How Can Flies Be Controlled to Manage Heifer Mastitis? Once it was established that the horn fly was a vector in the transmission of mastitis-causing bacteria, the next step was to develop management practices to reduce flies and lower the prevalence of intramammary infections. Insecticide-impregnated tags placed on the tail switch in close proximity to the udder during the spring and summer months were successful in reducing horn fly populations by 60% as well as the incidence of mastitis during the first 2 months after placement. However, after 2 months, tags fell off and replacing them was impractical from a management standpoint (Nickerson et al, 1997). In a subsequent trial, the daily dietary supplementation of an insect growth regulator helped to suppress fly populations but not enough to prevent new cases of mastitis in dairy heifers (Owens et al., 2000). However, the use of an insecticidal pour-on every 2 weeks for 6 weeks followed by treatment with insecticidal ear tags reduced fly populations and decreased the incidence of new Staph. aureus by 83% during a 6-mo trial in heifers during the warm season in Louisiana (Owens et al., 2002). More recently, an ongoing trial at UGA has found that the use of a pour-on every 2 to 4 weeks drastically reduced fly populations, allowing teats to heal, and reducing two important sources of S. aureus: flies and teat lesions.

29

These studies demonstrate that, during the warm and humid months of the year, horn flies do serve as vectors in the transmission of heifer mastitis, which is associated with elevated SCC in these young dairy animals. Although research has not been conducted to show this same association in lactating and dry adult cows, it is assumed that fly populations play some role in the elevation of mastitis and SCC observed in the hot summer months. And, with the proposed reduction in the SCC legal limit to 400,000/ml in the USA, and in light of the fact that milk buyers are imposing their own limits, some as low as 250,000/ml, it is imperative that dairymen utilize all possible means to prevent new cases of mastitis and their associated SCC. A simple fly control program can serve as an important adjunct to an overall herd plan of mastitis control and assist dairymen in lowering their bulk tank SCC and earning quality premiums for their products. Influence of Dietary Supplementation on Mammary Health Another management tool to reduce the level of infection and SCC when heifers calve, as well as throughout lactation, is through dietary supplementation. Diet plays a role in udder resistance to infection because certain nutrients affect various mammary resistance mechanisms, namely: (1) leukocyte function, (2) antibody transport, and (3) mammary tissue integrity. In one study, heifers received selenium (0.3 ppm/day) and vitamin E (50 to 100 ppm/day) supplementation starting 60 days prepartum and throughout lactation, and a selenium booster injection (50 mg) was administered 21 days prior to freshening (Hogan et al., 1993). This protocol reduced staphylococcal and coliform infections at calving by 42%, and duration of infection was reduced 40 to 50% in supplemented heifers. Clinical mastitis in supplemented heifers was reduced 57% in early lactation and 3.2% throughout lactation, and the mean SCC was lower. Thus, vitamin E and selenium improved udder health of heifers, and the effect of dietary supplementation was most evident at calving and in early lactation. In a more recent study (Eubanks et al., 2012), dairy heifers (n = 40) were fed a daily supplement beginning at 5 months of age containing an immune modulator (OmniGen-AF), which included a proprietary mixture of B-complex vitamins and yeast extract. Blood profile data collected during gestation showed that compared with unsupplemented control animals (n = 40), those supplemented with the immune modulator exhibited greater leukocyte expression of L-selectin and interleukin-8 cell surface receptors, as well as greater leukocyte phagocytic activity and production of reactive oxygen species, suggesting the capability for a greater immune response to bacterial infection. To date, additional data has been collected on 24 heifers that have calved. On a mammary quarter basis, prevalence of mastitis among quarters at 30-60 d prepartum was similar between treatment groups (58.3% vs. 52.5%); however, prevalence among quarters at 3 d postpartum was 1.8-fold lower in OmniGen-AF-supplemented animals (2.8%) compared to controls (5.1%), and by 10 d postpartum, there was no change in mastitis prevalence among quarters in supplemented animals (2.8%), but prevalence was 2.7-fold higher in quarters of unsupplemented controls (7.5%) (Figure 9).

30

Figure 9. Pre- and postpartum prevalence (%) of mastitis among mammary quarters of OmniGen-AF-supplemented and control heifers.

Supplemented 100 50 0
58.3 52.5
2.8 5.1

Control

2.8 7.5

30-60d 3d 10d Prepartum Postpartum Postpartum

Similarly, SCC remained lower in supplemented vs. control heifers (Figure 10). At 3 d postpartum, average SCC for OmniGen-AF-supplemented heifers was 221,000/ml vs. 535,000/ml for controls. By 10d postpartum, SCC in supplemented heifers had fallen to 183,000/ml, but SCC remained elevated 2.4-fold in controls (441,000/ml). Figure 10. Three- and 10-d postpartum SCC among mammary quarters of OmniGen-AFsupplemented and control heifers.

Supplemented
535

Control
441 183

600 400 200 0

221

3d Postpartum

10d Postpartum

31

Interestingly, both supplemented and control heifers entered lactation producing a similar milk yield, although supplemented animal yield (24 lb) was slightly greater than controls (23 lb) (Wk 0, Figure 11); however, during the first few weeks of lactation (the most susceptible time period for development of new IMI), OmniGen-AF-supplemented heifers appeared to remain healthier (less mastitis, lower SCC), and progressively produced a greater daily milk yield than unsupplemented controls through wk 5 postpartum. For example, by wk 1, supplemented heifers were producing an average of 2.4 lb per day more milk than controls, by wk 3, this difference was 7.7 lb, and at wk 5, the difference between OmniGen-AF-supplemented and control animals was still 7 lb. Figure 11. Milk production over the first 5 wk of lactation in OmniGen-AF-supplemented and control heifers.

80 70 60 50 40 30 20 10 0 Week 0 24

Supplemented

Control 63.8

68.9

68.4

56.5 51.7 56.1 53.1 49.3 59.2 61.4

23

Week 1

Week 2

Week 3

Week 4

Week 5

Overall, postpartum results showed that animals supplemented with OmniGen-AF exhibited increased milk production, decreased prevalence of mastitis, and decreased SCC compared with control heifers, indicating a positive effect of feeding the feed supplement. While differences in mastitis prevalence and SCC were not observed prepartum, the postpartum differences observed between OmniGen-AF-supplemented and control cows suggest a positive effect on the immune system after feeding the dietary supplement as heifers, which was manifested during the periparturient period (at time of stress), or more specifically, during the first few days postpartum when animals are subjected to increased risk of IMI, elevated SCC, and decreased production. 32

REFERENCES Akers, R.M. and S.C. Nickerson. 2011. Invited review. Mastitis and its impact on structure and function in the ruminant mammary gland. Journal of Mammary Gland Biology and Neoplasia. 16:275-289. Boddie, R.L., S.C. Nickerson, W.E. Owens and J.L.Watts. 1987. Udder microflora in nonlactating heifers. Agri-Practice, Vol.8, pp. 22-25. Eubanks V.J., D.J. Hurley, L.O. Ely, F.M. Kautz, S.C. Nickerson, N.E. Forsberg, Y.Q. Wang, K. Zanzalari and J. Chapman. 2012. Pre- and postpartum immunomodulatory effects of a dietary supplement on the immune system of dairy heifers. J. Anim. Sci. Vol. 90, Suppl. 3/J. Dairy Sci. Vol. 95, Suppl. 2. Abstr #220, pg. 222. Hogan, J.S., W.P. Weiss and K.L. Smith. 1993. Role of vitamin E and selenium in the host defense responses to mastitis. Journal of Dairy Science, Vol.76, pp. 2795-2802. Nickerson, S.C., W.E. Owens and R.L. Boddie. 1995. Mastitis in dairy heifers: initial studies on prevalence and control. Journal of Dairy Science, Vol.78, pp. 1607-1618. Nickerson, S.C., W.E. Owens, S.M. DeRouen, R.L. Boddie and G.M. Tomita. 1997. Use of insecticide impregnated tail tags may reduce incidence of mastitis in beef cows. The Louisiana Cattleman. August, Pages 11-14. Nickerson, S.C., W.E. Owens, G.M. Tomita and P.W. Widel. 1999. Vaccinating dairy heifers with a Staphylococcus aureus bacterin reduces mastitis at calving. Large Animal Practice, Vol.20, pp. 16-20. Nickerson, S.C., L.O. Ely, E.P. Hovingh and P.W. Widel. 2009. Immunizing dairy heifers can reduce prevalence of Staphylococcus aureus and reduce herd somatic cell counts. In: Dairy Cattle Mastitis and Milking Management, DAIReXNET. <http://www.extension.org/pages/Dairy_Cattle_Mastitis_and_Milking_Managemnt>. Oliver, S.P., M.J. Lewis, B.E. Gillespie, H.H. Dowlen, E.C. Janicke and R.K. Roberts. 2003. Milk production, milk quality and economic benefit associated with prepartum Antibiotic treatment of heifers. Journal of Dairy Science, Vol.86, pp. 1187-1193. Oliver, S.P., S.J. Ivey, B.E. Gillespie, M.J. Lewis, D.L Johnson, K.C Lamar, H. Moorehead, H.H. Dowlen, S.T. Chester and J.W. Hallberg. 2004. Influence of prepartum intramammary infusion of pirlimycin hydrochloride or penicillin-novobiocin on mastitis in heifers during early lactation. Journal of Dairy Science, Vol.87, pp. 17271731. Owens, W.E., S.C. Nickerson and R.L. Boddie. 2000. The effect of methoprene on horn fly numbers and mastitis in dairy heifers. Large Animal Practice, Vol.21, pp. 26-28. Owens, W.E., S.C. Nickerson, R.L. Boddie, G.M. Tomita and C.H. Ray. 2001. Prevalence of mastitis in dairy heifers and effectiveness of antibiotic therapy. Journal of Dairy Science, Vol.84, pp. 814-817. Owens, W.E., S.C. Nickerson, P.J. Washburn and C.H. Ray. 1991. Efficacy of a cephapirin dry cow product for treatment of experimentally induced Staphylococcus aureus mastitis in heifers. Journal of Dairy Science, Vol.74, pp. 3376-3382. Owens, W.E., S.C. Nickerson, P.J. Washburn and C.H. Ray. 1994. Prepartum antibiotic therapy with a cephapirin dry cow product against naturally occurring intramammary infections in heifers. Veterinary Medicine Series B, Vol.41, pp. 90-100. Owens, W.E., S.P. Oliver, B.E. Gillespie, C.H. Ray and S.C. Nickerson. 1998. Role of horn Flies (Haematobia irritans) in Staphylococcus aureus-induced mastitis in dairy 33

heifers. American Journal of Veterinary Research, Vol.59, pp.1122-1124. Owens, W.E., S.C. Nickerson and C.H. Ray. 2002. Effect of a pour-on and fly tag insecticide combination in controlling horn flies and Staphylococcus aureus mastitis in dairy heifers. 2002 Louisiana Dairy Report. Baton Rouge, Louisiana, USA, pp. 39-42. Parker, K.I., C. Compton, F.M. Anniss, A. Weir, C. Heuer and S. McDougall. 2007. Subclinical and clinical mastitis in heifers following the use of a teat sealant precalving. J. Dairy Science, 90: 207-218. Parker, K.I., C. Compton, F.M. Anniss, C. Heuer and S. McDougall. 2008. Quarter level analysis of subclinical and clinical mastitis in primiparous heifers following the use of a teat sealant or an injectable antibiotic, or both, precalving. J. Dairy Science, 91: 169-181. Trinidad, P., S.C. Nickerson and R.W. Adkinson. 1990a. Histopathology of staphylococcal mastitis in unbred dairy heifers. Journal of Dairy Science, Vol.73, pp. 639-647. Trinidad, P., S.C. Nickerson, T.K. Alley and R.W. Adkinson. 1990b. Efficacy of intramammary treatment in unbred and primigravid dairy heifers. Journal of the American Veterinary Medical Association, Vol.197, pp. 465-470.

34

The Physiology of Stress and Effects on Immune Health in Ruminants

J.A. Carroll*, and N.C. Burdick Sanchez Livestock Issues Research Unit USDA-ARS Corresponding author: jeff.carroll@ars.usda.gov SUMMARY As researchers have continued to explore the complex interactions among stress and production parameters such as growth, feed efficiency, and health, multidisciplinary efforts have emerged leading to a greater understanding of homeostatic regulation. The immune system can be regulated by several different endocrine secretions, with the most prominent being those secreted in response to stress. Ultimately, within the animal, the immune system response to stress is dependent upon the type of stress encountered (i.e., acute versus chronic). Given that the innate immune system provides the first line of defense, understanding the effects of stress hormones on innate immunity holds a great deal of potential with regard to improving cattle health, and ultimately productivity. INTRODUCTION Livestock are exposed to stress at various points during production. Examples include weaning, the transportation process, and castration and other less appreciated stressors such as extreme fluctuations in temperature, poor nutrition, and mixing of unfamiliar animals. It has been well established that stress can influence how an animal responds and recovers from exposure to pathogens, and therefore, it is essential that animals have a well-developed and properly functioning immune system. While chronic stress negatively impacts immunity, literature has demonstrated that acute stress can potentially prime the immune system (Dhabhar, 2000; Dhabhar, 2009). Animals that possess an adequate level of immunological protection have been demonstrated to exhibit greater reproductive capabilities, enhanced growth, and increased feed efficiency (Galyean et al., 1999).

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. The U.S. Department of Agriculture (USDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, age, disability, and where applicable, sex, marital status, familial status, parental status, religion, sexual orientation, genetic information, political beliefs, reprisal, or because all or part of an individual's income is derived from any public assistance program. (Not all prohibited bases apply to all programs.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA's TARGET Center at (202) 720-2600 (voice and TDD). To file a complaint of discrimination, write to USDA, Director, Office of Civil Rights, 1400 Independence Avenue, S.W., Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or (202) 720-6382 (TDD). USDA is an equal opportunity provider and employer. 35

As researchers have continued to explore the complex interactions among stress and production parameters such as growth, feed efficiency, and health, multidisciplinary efforts have emerged leading to a greater understanding of homeostatic regulation. Additionally, there has been an increased effort to elucidate the interactions between stress responsiveness and immunological parameters in animals that may be either predisposed to or resistant to the detrimental effects of stress due to factors such as genetic programming, management practices and (or) prior experiences. Today, the scientific community and producers alike acknowledge the fact that stress can potentially have detrimental effects on animal productivity, and overall health and well-being. Even though the debate among animal scientists concerning the definition and quantification of stress is ongoing, an increased understanding and appreciation with regard to the effects of stress on livestock production now exists both within the scientific community and with livestock producers. While the physiological consequences of stress on the body have been of scientific interest for many years, scientists have yet to fully elucidate the complex interactions among stress hormones and the immune system. However, there is substantial literature available documenting the detrimental effects of prolonged stress on the immune system and overall health of livestock. STRESS Stress, as it relates to bodily functions, has been defined as the sum of all biological reactions to physical, emotional, or mental stimuli that disturb an individuals homeostasis ( Pack and Palkovits, 2001). Therefore, a stressor can be defined as any internal or external stimuli or threat that disrupts homeostasis of the body, and elicits a coordinated physiological response (stress response) in an attempt to reestablish homeostasis. Maintaining a state of homeostasis requires proper functioning of all physiological processes including the stress and immune systems which are influenced by numerous factors including environmental conditions, pathogen exposure, genetic makeup, animal temperament, and nutrient availability. Research related to stress in domestic animals continues to evolve and expand, with emergent multidisciplinary efforts leading to a greater understanding of homeostatic regulation. Not only has the definition of stress been refined and updated based upon continued scientific discoveries, but the perception of stress in domestic animals has evolved as well. Stress, as we now know, includes indices such as environmental stress, nutritional stress, social stress, and even prenatal stress. Animal stress is now identified as a unique event that elicits a specific behavioral, physiological, neuroendocrine, endocrine, and/or immune response that may be as unique as the stressful event itself. The stress response is a complex and coordinated series of events initiated when stress sensors in the brain stimulate the release of two neurohormones, corticotropin-releasing hormone (CRH) and vasopressin (VP) in response to a stimulus. The parvocelular neurons within the paraventricular nucleus (PVN) of the hypothalamus produce CRH, while magnocellular neurons of the paraventricular and supraoptic nuclei are responsible for production of VP (Carrasco and Van de Kar, 2003). Studies have indicated that some parvocellular neurons are capable of producing both CRH and VP, suggesting a biological interaction between these two neuropeptides. After traversing the blood vessels in the median eminence, CRH and VP can 36

independently stimulate the production of adrenocorticotropic hormone (ACTH) from the anterior pituitary (Aguilera, 1998; Carrasco and Van de Kar, 2003; Webster Marketon and Galser, 2008). Subsequently, ACTH stimulates the production of glucocorticoids from the cortex of the adrenal gland (Markara et al., 1981; Carrasco and Van de Kar, 2003). This systematic cascade is often referred to as the hypothalamic-pituitary-adrenal (HPS) axis. In mammals the primary glucocorticoid is cortisol. However, rodents lack the enzyme P450c17, which is responsible for producing cortisol in the adrenal, and therefore their primary glucocorticoid is corticosterone (Ashwell et al., 2000). When released into the blood stream, cortisol can elicit a plethora of biological effects on the body including changes in metabolism of carbohydrates and protein, alterations in the growth and reproductive axes, regulation of the stress response, and influencing overall immune function (Figure 1). Cortisol plays an important role in gluconeogenesis, the generation of glucose from other organic molecules like pyruvate, lactate, glycerol, and amino acids, during the fight or flight response. Cortisol increases blood glucose concentrations by stimulating the liver to convert fat and protein to these intermediate metabolites that are ultimately converted to glucose for energy (Long et al., 1940; McGuinness et al., 2005). Cortisol also supports the primary defense response by enhancing the synthesis and secretion of catecholamines, other stress hormones produced by the adrenal glands, which control physiological processes such as heart rate, pupil dilation, vasoconstriction in the skin and gut, vasodilation in leg muscles, and increased glucose production by the liver, all of which are essential pr ocesses during the fight or flight response (Charmandari et al., 2005). Figure 1. When an animal perceives either an internal or external threat, neurotransmitters are released in the brain that cause the release of corticotrophin-releasing hormone (CRH) and vasopressin (VP) which stimulate the release of adrenocorticotrophin (ACTH). ACTH in turn stimulates the release of cortisol, epinephrine (Epi) and norepinephrine (NE), each of which affects various target tissues in the body including the immune system.

STRESS
Hypothalamus

CRH/VP
Pituitary

Visceral Adiposity
Adipose

Muscle

Bone

ACTH
Cortex Medulla

Adrenals

Mass

Cortisol
NE/Epi
Immune cells

37

The sympathetic nervous system is activated in response to many stressors in parallel to and often prior to the stimulation of the HPA axis. Upon stimulation, noradrenergic neurons in the brain and postganglionic sympathetic neurons innervating peripheral organs (e.g., heart, vasculature, kidneys, gut, and adipose) secrete norepinephrine into circulation, resulting in increased blood pressure, heart rate, and respiration rate. Additionally, nerve impulses in high cortical centers within the brain relay messages through the limbic system resulting in the release of norepinephrine, serotonin, and acetylcholine, which activate the PVN (Black, 2002). In conjunction with these actions, preganglionic sympathetic fibers innervating the adrenal medulla stimulate the production and secretion of epinephrine and norepinephrine via acetylcholine (Butcher and Lord, 2004). The sympathetic nervous system regulates many functions in the body including cardiovascular, gastrointestinal, respiratory, and renal systems, all of which can be modulated in response to sympathomedulary system (SMS) activation (Charmandari et al., 2005). An increase in epinephrine concentrations in the brain serves as an alarm system, resulting in a decrease in neurovegetative activities (e.g., eating and sleeping) and the activation of the stress response (HPA axis activation; Tsigos and Chrousos, 2002). The secretion of norepinephrine within the brain also activates the fear behaviors and enhances long term memory and storage of adversely charged emotions in the hippocampus (Salpolsky et al., 2000; Tsigos and Chrousos, 2002). Additionally, the responses of the HPA axis and the SMS to stress are highly concordant. In response to most stressors both systems are activated and have the ability to activate each other. THE IMMUNE SYSTEM The immune system is not a single entity, but rather a complex, integrated system regulated by a multitude of specialized cells and chemical messengers. In general, however, the immune system can be separated into three broad components; natural immunity, innate immunity, and acquired immunity, all of which must be fully developed and functioning properly to provide adequate immunological protection. Natural and innate immunity are typically grouped together under the category of innate immunity. Therefore, when discussing innate immunity, it is typically assumed that one is including natural immunity as well. Innate immunity is considered to be the first line of defense against pathogens; whether bacterial, viral, protozoal or fungal. It includes physical barriers such as the skin, mucosal secretions, tears, urine, and stomach acid, as well as complement and antigen-nonspecific cellular components and is designed to elicit an immediate or acute response (0 to 4 h) following exposure to an antigenic agent (Mnnel, 2007; Barton, 2008). Until recently, the innate immune system was thought to represent the antigen-nonspecific aspect of the immune system. However, recent evidence suggests that the innate response may be specific to the pathogenic agent encountered. While it is often assumed that this aspect of the immune system becomes a constant entity once developed by the animal, this is certainly not the case. The innate immune system, while always present to some degree, can be modulated in either a beneficial or detrimental manner by a number of factors including wounds, dehydration, nutritional status, genetics, stress, and various peptide hormones (Figure 2). In response to inflammation, tissue damage, and infection, the body initiates an acute phase response (APR). The APR is a set of reactions that promote tissue damage repair, control of 38

invading organisms, would healing, and (or) recruitment of host defense mechanisms (Black, 2002). This includes the production of acute phase proteins from the liver, and production of cytokines, catecholamines, and to a lesser extent, glucocorticoids. The APR is also characterized by physiological responses within the animal including fever and sickness behavior (Black, 2002). This response usually subsides within 24 to 48 hours following stimulation. Figure 2. Factors that can have a significant influence on the function of the innate immune system of cattle. Naturally occurring deviations in the innate immune system as well as the influence of various management practices on the immune system are often overlooked in production systems.

Genetic/Natural Deviations
Breed Effects Sexual Dimorphic Effects Animal Temperament Effects

Management Influences
Diet Housing Conditions Early vs Normal Weaning

Variations in Innate Immune Function

Stress Hormones

STRESS EFFECTS ON IMMUNE FUNCTION The immune system can be regulated by several different endocrine secretions, with the most prominent being those secreted in response to stress. There has been an increased effort to elucidate the interactions between stress responsiveness and immunological parameters in cattle that may be either predisposed to or resistant to the detrimental effects of stress due to genetic programming and/or prior experiences. Interestingly, there are cattle that demonstrate differential stress and immunological responses due to previous exposure to various managerial, environmental, nutritional, or pathogenic stressors or due to varying temperaments within a genetically similar group of animals. As researchers have continued to explore the complex interactions between stress and production parameters such as growth, reproduction, and health, multidisciplinary efforts have emerged that have led to a greater understanding of homeostatic regulation. Based upon these efforts, our knowledge has extended beyond the "all or none" biological activity strictly associated with the "fight or flight" response. For instance, researchers have demonstrated that the combined immunological effects of cortisol and catecholamines result in a well-orchestrated biological event designed to prevent over-stimulation of innate immunity and the production of pro-inflammatory cytokines while simultaneously priming the humoral immune response in an effort to provide adequate immunological protection (Sorrells and Salpolsky, 2007). 39

Specifically, cortisol suppresses the release of various cytokines produced by cells of the immune system which can cause systemic disease. What is the purpose for inhibiting immune function in response to stress? First, it minimizes potential autoimmune damage stemming from novel self-antigens that arise from catabolic activity of glucocorticoids on immune cells and other tissues (Rberg et al., 1998). Secondly, it redirects resources to processes essential for survival such as heart rate, lung ventilation, and other processes needed for escape or aggression (Sapolsky et al., 2000). In addition to suppression of immune activity, there is catabolism of immune cells and other tissues in order to increase availability of glucose and proteins for other body systems. However, suppressing the immune system during acute stress would be counter intuitive for survival, and for a significant energy savings to occur would require longer immune suppression (Martin, 2009). Today, our knowledge base has expanded, and a greater appreciation and understanding has emerged regarding the plethora of immune system activities that are influenced by cortisol such as stimulation of immune system chemical messengers, and stimulation of immune cell growth and function. In addition to these stimulatory actions, long-term exposure to cortisol is known to inhibit aspects of immune function. Ultimately, within the animal, the immune system response to stress is dependent upon the type of stress encountered (i.e., acute versus chronic). In some instances of acute stress, such as that resulting from bites, punctures, scrapes or other challenges to the integrity of the body, stress hormones are associated with priming the immune system in a manner to prepare for potentially invading pathogens and subsequent infection. However, when an animal experiences prolonged or chronic stress, the effect of stress hormones on the immune system shifts from a preparatory event to a series of suppressive events first at the cellular level and then eventually across the entire immune system spectrum. Several factors influence the stress-immune interaction, including age, breed/species, environment, gender, health status, previous exposure, and personality/temperament. Acute Stress While is has been known for decades that stress can have detrimental effects on the immune system, it was only recently that the divergent effects of acute stress compared to long -term or chronic stress were revealed. As the scope of scientific exploration beyond traditionally defined pathways of neuroendocrinology, endocrinology, and immunology, multidisciplinary efforts emerged, cross-communication pathways among the stress and immune systems have emerged, and have lead to a better understanding of homeostatic regulation within the animal. No longer is stress considered strictly immunosuppressive. Indeed, stress may elicit bi directional effects on immune function such that acute stress may be immunoenhancing, while chronic stress may be immunosuppressive. Acute stress is stress that occurs for a short period of time typically seconds or minutes. While chronic stress, discussed below, has typically been associated with negative effects on immune function, acute stress has been demonstrated to have a priming effect on the immune system. Specifically, studies have demonstrated an increase in maturation and trafficking of immune cells (i.e., neutrophils and natural killer cells) to the periphery and target tissues, up-regulation of several cytokines and chemokines, enhanced response to vaccination and an enhanced response to a second pathogen exposure (Dhabhar, 2000; Dhabhar, 2002; Dhabhar et al., 2009). 40

Stress hormones, particularly glucocorticoids produced by the adrenal gland, are necessary for the development of the thymus, as adrenal insufficiency or adrenalectomy results in hypertrophy of the thymus that is not reversed by epinephrine administration (Ashwell et al., 2000). Additionally, the ability to respond to glucocorticoids though the glucocorticoid receptor (GR) is necessary for the development of T cells in the thymus, as inhibition of the GR limits the production of mature T cells (Bellinger et al., 2008). Stress hormones are responsible for increases in cholesterol, lipoproteins, triglycerides and free fatty acids, which may be beneficial during the stages of early infections as lipoproteins can bind and thereby neutralize lipopolysaccharide (LPS; component of the cell wall of gram negative bacteria; Black, 2002). Studies have also demonstrated that cortisol administered 144 hour or less prior to an endotoxin (LPS) challenge can enhance the cytokine response, perhaps by priming the immune system (Besedovsky et al., 1996; Sapolsky et al., 2000; Sorrells and Sapolsky, 2007). In contrast, glucocorticoid administered at the same time or after the administration of endotoxin suppresses these responses (Sapolsky et al., 2000). Additionally, in a review by Sorrells and Sapolsky (2007), the authors stated that the early response to invading pathogens is characterized by low concentrations of glucocorticoids that are permissive; later responses to pathogens, characterized by high concentrations of glucocorticoids, results in the negative effects on immune function. Therefore, the timing relative to immune system activation is extremely important in determining whether stress hormones will have stimulatory and inhibitory effects on the immune system. A study in which ACTH was administered to Brahman heifers reported an increase in the expression of interleukin-10 (IL-10), interferon- (IFN-), IL-4, tumor necrosis factor- (TNF), and the GR in isolated peripheral blood mononuclear cells (PBMCs; Burdick et al., 2010). This suggested that acute ACTH-induced increases in cortisol can modulate cytokine and GR gene expression, potentially resulting in a priming effect on the immune system. Administration of short-acting dexamethasone to Hereford steers, mimicking in vivo increases in cortisol, resulted in an increase in many immune cells and immune cell function parameters, which also supports the conclusion that acute stress can prime the immune system for subsequent responses (Anderson et al., 1999). Acute stress induced by weaning has also been reported to influence immune cell numbers as well as cytokine gene expression in calves. Specifically, expression of IL-, toll-like receptor 4 (TLR4), and GR by leukocytes were increased in response to weaning (OLoughlin et al., 2011). However, while weaning alone may result in an acute stressor, weaning is typically accompanied by transportation and the addition of novel management practices and handling, which may heighten the exposure to stress resulting in chronic stressor exposure. Chronic Stress In contrast to acute stress, chronic stress is stress that is either repeated or exerted for an extended period of time typically hours to days, weeks, months, or longer. Chronic stress has been associated with delayed wound healing, enhanced immunosuppressive actions, increased changes for development of autoimmune and inflammatory diseases and cancer, reduced cytokine responsiveness of natural killer cells, reduced mucosal immunity, and decreased ability of lymphocytes to proliferate, respond to pathogens, and release immunoglobulins (McEwen et al., 1997; Ashwell et al., 2000; Sapolsky et al., 2000; Silberman et al., 2003). 41

Chronic exposure to high concentrations of cortisol can cause severe physiological and psychological problems such as excessive protein catabolism, hyperglycemia, immunosuppression, and depression (McEwen et al., 1997). In domestic livestock, excessive concentrations of cortisol have been linked to reduced rates of reproduction, suboptimal growth, suppressed milk production, and suppression of immune function that could increase susceptibility to disease (Moberg, 1987; Dobson et al., 2001; Silberman et al., 2003; Zhao et al., 2008). Additionally, glucocorticoids can modulate the expression of cytokines and their receptors, including pro-inflammatory TNF- (Ashwell et al., 2000). Corticosteroids have been noted to decrease the secretion of IL-1, IL-2, IL-6 and IFN- and increase receptors for IFN-, IL-1, IL-6, VP, CRH, serotonin, and insulin (Black, 2002). In livestock, administration of dexamethasone to Holstein steers induced neutrophilia, or an increase in circulating neutrophils, while reducing the expression of L-selectin, an adhesion molecule that allows neutrophils to extravasate into tissues (Weber et al., 2004). Glucocorticoids also increase the half-life of neutrophils. This can result in excessive damage to healthy tissue due to their longer life and the extended time in which neutrophils can produce cytotoxic granules (McEwen et al., 1997). Interestingly, neutrophils can release specific proteases that cleave glucocorticoids from cortisol binding globulin, therefore increasing the amount of free cortisol within tissues (McEwen et al., 1997). There is also evidence that stress hormones can affect the maintenance of the memory cell pool (Burns et al., 2003). In neonatal Holstein bull calves administered dexamethasone twice daily from 3 to 56 days of age, expression of TLR2, TLR4 and IL-1 were reduced in blood leukocytes, suggesting chronic administration of dexamethasone inhibited the recognition of pathogens in young calves (Eicher et al., 2004). Additionally, a study in Angus and Romosinuano heifers exposed to heat stress for 2 weeks reported a diminished cortisol response and an altered immune response to subsequent LPS challenge, suggesting that chronic heat stress down-regulated immune responsiveness (Burdick et al., 2012). Therefore, chronic stress may prevent cattle from recognizing and eliciting an adequate immune response to an invading pathogen. CONCLUSION Ultimately, the combined immunological effects of cortisol and catecholamines result in a wellorchestrated event designed to prevent over-stimulation of innate immunity while simultaneously priming the acquired immune response. Therefore, the final type of immune response that prevails within an animal is dependent upon the overall type and duration of the stress response in the animal. Given that the innate immune system provides the first line of defense, understanding the effects of stress hormones on innate immunity holds a great deal of potential with regard to improving cattle health, and ultimately productivity. Continued research efforts into these complex interactions may allow the implementation of alternative management practices, improved selection programs, and/or implementation of various nutritional strategies to prevent or overcome significant production losses and animal health care costs for livestock producers.

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REFERENCES Aguilera, G. 1998. Corticotropin releasing hormone, receptor regulation and the stress response. Trends Endocrinol. Metab. 9:329-36. Anderson, B.H., D.L. Watson, and I.G. Colditz. 1999. The effect of dexamethasone on some immunological parameters in cattle. Vet. Res. Commun. 23:399-413. Ashwell, J.D., F.W.M. Lu, and M.S. Vacchio. 2000. Glucocorticoids in T-cell development and function. Barton, G.M. 2008. A calculated response: control of inflammation by the innate immune system. J. Clin. Invest. 118:413-20. Bellinger, D.L., C. Lubahn, and D. Lorton. 2008. Maternal and early life stress effects on immune function: relevance to immunotoxicology. J. Immunotoxicol. 5:419-444. Besedovsky, H.O., and A. del Rey. 1996. Immune-neuro-endocrine interactions: facts and hypotheses. Endocr. Rev. 17:64-102. Black, P.H. 2002. Stress and the inflammatory response: a review of neurogenic inflammation. Brain Behav. Immun. 16:622-653. Burdick, N.C., B.J. Agado, R.D. Randel, D.A. Neuendorff, J.A. Carroll, R.C. Vann, C.G. Chitko-McKown, S.D. Lawhon, and T.H. Welsh, Jr. 2010. Endogenous cortisol: acute modulation of cytokine gene expression in bovine PBMCs. J. Anim. Sci. 88(E-Suppl 3):20 (Abstract # 61). Burdick, N.C., R. Chaffin, J.A. Carroll, C.C. Chase, Jr., S.W. Coleman, and D.E. Spiers. 2012. Influence of heat stress on the immune response of Angus and Romosinuano heifers to an LPS challenge. J. Anim. Sci. 90(E-Suppl. 2):22 (Abstract # 67). Burns, V.E., D. Carroll, C. Ring, and M. Drayson. 2003. Antibody response to vaccination and psychosocial stress in humans: relationships and mechanisms. Vaccine. 21:2523-2534. Butcher, S.K. and J.M. Lord. 2004. Stress responses and innate immunity: aging as a contributing factor. Aging Cell. 3:151-160. Carrasco, G.A., and L.D. Van de Kar. 2003. Neuroendocrine pharmacology of stress. Eur. J. Pharmacol. 463:235-272. Charmandari, E., C. Tsigos, and G. Chrousos. 2005. Endocrinology of the stress response. Ann. Rev. Physiol. 67:259-284. Dhabhar, F.S. 2000. Acute stress enhances while chronic stress suppresses skin immunity. The role of stress hormones and leukocyte trafficking. Ann. N.Y. Acad. Sci. 917:876-893. Dhabhar, F.S. 2002. Stress-induced augmentation of immune function the role of stress hormones, leukocyte trafficking, and cytokines. Brain Behav. Immun. 16:785-798. Dhabhar, F.S. 2009. Enhancing versus suppressive effects of stress on immune function: implications for immunoprotection and immunopathology. Neuroimmunomodulation 16:300-317. Dobson, H., J.E. Tebble, R.F. Smith, and W.R. Ward. 2001. Is stress really all that important? Theriogenology. 55:65-73. Eicher, S.D., K.A. McMunn, H.M. Hammon, and S.S. Donkin. 2004. Toll-like receptors 2 and 4, and acute phase cytokine gene expression in dexamethasone and growth hormone treated dairy calves. Vet. Immunol. Immunopathol. 98:115-125. Galyean, M.L., L.J. Perino, and G.C. Duff. 1999. Interaction of cattle health/immunity and nutrition. J. Anim. Sci. 77:1120-1134. Long, C.N.H., B. Katzin, and E.G. Fry. 1940. The adrenal cortex and carbohydrate metabolism. Endocrinology. 26:309-344. 43

Makara, G.B., E. Stark, M. Karteszi, M. Paklovits, and G. Rappay. 1981. Effects of paraventricular lesions on stimulated ACTH release and CRF in stalk-median eminence of the rat. Am. J. Physiol. 240:E441-E446. Mnnel, D.N. 2007. Advances in sepsis research derived from animal models. Int. J. Med. Microbiol. 297:393-400. Martin, L.B. 2009. Stress and immunity in wild vertebrates: timing is everything. Gen. Comp. Endocrinol. 163:70-76. McEwen, B.S., C.A. Biron, K.W. Brunson, K. Bulloch, W.H. Chambers, F.S. Dhabhar, R.H. Goldfarb, R.P. Kitson, A.H. Miller, R.L. Spencer, and J.M. Weiss. 1997. The role of adrenocorticoids as modulators of immune function in health and disease: neural, endocrine and immune interactions. Brain Res. Rev. 23:79-133. McGuinness, O.P. 2005. Defective glucose homeostasis during infection. Annu. Rev. Nutr. 25:9-25. Moberg, G.P. 1987. A model for assessing the impact of behavioral stress on domestic animals. J. Anim. Sci. 65:1228-1235. OLoughlin, A., M. McGee, S.M. Waters, S. Doyle, and B. Early. 2011. Examination of the bovine leukocyte environment using immunogenetic biomarkers to assess immunocompetence following exposure to weaning stress. BMC Vet Res. 7:45. Pack, K., and M. Palkovits. 2001. Stressor specificity of central neuroendocrine responses: implications for stress-related disorders. Endocr. Rev. 22:502-548. Rberg, L., M. Grahn, D. Hasselquist, and E. Svensson. 1998. On the adaptive significance of stress-induced immunosuppression. Proc. R. Soc. Lond. B. 265:1637-1641. Sapolksy, R.M., LM. Romero, and A.U. Munck. 2000. How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr. Rev. 21:55-89. Silberman, D.M., M.R. Wald, and A.M. Genaro. 2003. Acute and chronic stress exert opposing effects on antibody responses associated with changes in stress hormone regulation of T-lymphocyte reactivity. J. Neuroimmunol. 144:53-60. Sorrells, S.F., and R.M. Sapolsky. 2007. An inflammatory review of glucocorticoid actions in the CNS. Brain Behav. Immun. 21:259-72. Tsigos, C., and G.P. Chrousos. 2002. Hypothalamic-pituitary-adrenal axis, neuroendocrine factors and stress. J. Psychosomatic Res. 58:865-871. Weber, P.S., T. Toelboell, L.C. Chang, J.D. Tirrell, P.M. Saama, G.W., Smith, and J.L. Burton. 2004. Mechanisms of glucocorticoid-induced down-regulation of neutrophil L-selectin in cattle: evidence for effects at the gene-expression level and primarily on blood neutrophils. J. Leukoc. Biol. 75:815-827. Webster Marketon, J.I. and R. Glaser. 2008. Stress hormones and immune function. Cell. Immunol. 252:16-26. Zhao, J., K.D. Kim, X. Yang, S. Auh, Y.X. Fu, and H. Tang. 2008. Hyper innate responses in neonates lead to increased morbidity and mortality after infection. Proc. Natl. Acad. Sci. U.S.A. 105:7528-7533.

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Predicting Transition Cow Health and Performance Use of Blood and Fecal Biomarkers for Herd-Level Evaluation and Diagnostics
J. M. Huzzey1,, D. V. Nydam2, P. A. Ospina1, and T. R. Overton1 1 Department of Animal Science 2 Department of Population Medicine and Diagnostic Sciences Cornell University, Ithaca NY Corresponding author: tro2@cornell.edu SUMMARY Nonesterified fatty acids and beta-hydroxybutyrate in plasma are indices of energy metabolism in transition cows and can be used to assess opportunities for improved health, performance, and reproduction Plasma haptoglobin is a nonspecific indicator of inflammation and acute phase response and is associated with decreased subsequent milk production and reproductive performance Fecal cortisol metabolites can also be used as a nonspecific biomarker of stress in transition dairy cows, and its most meaningful associations are with milk yield and reproductive performance rather than clinical disease INTRODUCTION Blood metabolites have been used in herd-level diagnostics of transition cow management for a number of years (Ingraham and Kappel, 1988; Herdt, 2000; Oetzel, 2004), mostly with focus on identifying opportunities to decrease the incidence of metabolic disorders related to energy metabolism. Recently, there has been a resurgence of interest in the use of blood metabolites, primarily nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA) for evaluation of transition cow programs. This resurgence is largely based on recent findings that both NEFA and BHBA are associated with economically important herd parameters beyond metabolic disease incidence, namely milk production and reproductive performance. In addition, the increased availability of accurate cow-side tests for BHBA in blood (Precision Xtra, Abbott Laboratories) and milk (KetoTest, Elanco Animal Health) has made routine evaluation of BHBA in herds simple, fast, and low cost. In light of these findings, new research has begun to evaluate the relationships of biomarkers related to stress and inflammation [e.g. plasma haptoglobin (Hp) and fecal cortisol metabolites (FCORT)] with metabolic disease, milk production, and reproduction in order to identify other useful and physiologically relevant markers for herd-level diagnostics and evaluation of transition cow programs. This paper reviews our recent findings in these areas and provides recommendations on how these markers should be interpreted for evaluating transition cow programs. USE OF ANALYTES RELATED TO ENERGY METABOLISM NEFA & BHBA Oetzel (2004) characterized well the typical use of blood analytes related to energy metabolism in transition management diagnostics NEFA during the prepartum period to assess precalving energy status and BHBA during the postpartum period to assess incidence of subclinical (and

Current affiliation: Animal Welfare Program, University of British Columbia, Vancouver BC 45

clinical) ketosis. This approach was supported in part by work conducted in Michigan (Cameron et al., 1998) that associated increased prepartum concentrations of NEFA, reflective of negative energy balance, with a greater incidence of displaced abomasum. Duffield et al. (1998) defined and characterized subclinical ketosis in herds in Ontario during the postpartum period and demonstrated that administration of monensin in a controlled-release capsule would decrease the incidence of subclinical ketosis in dairy cows during early lactation. Recently, our group conducted a large-scale evaluation of the associations of prepartum NEFA and postpartum NEFA and BHBA with postpartum health, milk production, and reproductive performance in dairy herds in the northeastern US (Ospina et al., 2010a, 2010b, 2010c). In order to have been included in the study a herd must have: 1) had greater than 250 milking cows, 2) housed cows in free-stalls, 3) fed a total mixed ration (TMR), and 4) participated in DHIA and/or use Dairy Comp 305 (Valley Ag. Software, Tulare CA). Farms were visited once and during the farm visit two cohorts of animals were selected: those 14 to 2 days prepartum and those 3 to 14 days postpartum. Within each cohort, convenience samples of 15 apparently healthy animals were evaluated. Briefly, 10 mL of blood was collected from the coccygeal vein or artery into a red-top tube. The sera from the prepartum cohort were analyzed for NEFA and the sera from animals sampled after calving were analyzed for NEFA, BHBA. For all animals sampled, the incidence of the diseases of interest [displaced abomasum (DA), clinical ketosis (CK), and metritis (MET) and/or retained placenta (RP)] within 30 days in milk, time to pregnancy within 70 days post voluntary waiting period and Mature Equivalent 305 (ME 305) milk at 120 days in milk were recorded. The final dataset included 100 herds with an average herd size of 840 cows. A total of 2758 cows were sampled within these herds (1440 animals sampled prepartum and 1318 sampled postpartum) with an approximate distribution of 35% primiparous (entering first lactation) and 65% multiparous (entering second or greater lactation) cows. Critical threshold values for prepartum NEFA and postpartum NEFA and BHBA and the associated risk ratios for disease are presented in Table 1. If animals had prepartum serum NEFA concentrations greater than about 0.30 mEq/L, they were twice as likely to develop one or more of the diseases of interest. Animals with postpartum serum NEFA and BHBA concentrations greater than about 0.60 mEq/L and 10 mg/dL, respectively, were four times as likely to develop one of more of the diseases of interest than animals with lower concentrations of these metabolites. The risk ratio for individual disorders varied widely within these groups. These results are consistent with prior work and support the importance of maintaining adequate energy intake prepartum and controlling body condition score loss and overall energy status during the postpartum period with respect to disease.

46

Table 1. Receiver operator characteristic (ROC) curve determination of critical NEFA (mEq/L) and BHBA (mg/dL) thresholds as predictors of disease and risk ratios of disease based upon these critical thresholds (Ospina et al., 2010b). Prepartum cohort (2 to 14 days prepartum) Disease DA CK Met and/or RP Any of the three Disease DA CK Met Any of the three Disease DA CK Met Any of the three
1 2

Critical NEFA1 0.27 0.26 0.37 0.29 Critical NEFA1 0.72 0.57 0.36 0.57

prepartum

Risk Ratio 2.0 1.8 2.2 1.8

95 % CI2 1.1 3.7 1.2 2.5 1.6 3.0 1.4 2.2 95 % CI2 4.2 22 2.3 11 2.0 134 2.6 7.3 95 % CI2 3.7 12.9 3.2 7.3 1.1 5.1 3.1 6.3

P-value 0.03 0.001 < 0.0001 < 0.0001 P-value <0.0001 <0.0001 0.008 < 0.0001 P-value <0.0001 <0.0001 0.037 <0.0001

Postpartum cohort (3 to 14 days postcalving) postpartum Risk Ratio 9.7 5.0 17 4.4 Risk Ratio 6.9 4.9 2.3 4.4

Critical BHBA1 10 10 7 10

Highest combination of specificity and sensitivity based upon ROC analysis Risk ratio confidence interval The relationships of prepartum NEFA and postpartum NEFA and BHBA with reproductive performance for the first 70 days after voluntary waiting period are described in Table 2. Animals with prepartum NEFA greater than about 0.3 mEq/L were nearly 20% less likely to become pregnant than animals with lower concentrations. Animals with greater than about 0.70 mEq/L of NEFA (while controlling for BHBA) and/or greater than 10 mg/dL of BHBA were 13 to 16% less likely to become pregnant than animals with lower concentrations. In all models, multiparous cows were less likely than primiparous cows to become pregnant in the first 70 days following the voluntary waiting period.

47

Table 2. Cox proportional hazard model of the effect of NEFA (mEq/L) and/or BHBA (mg/dL), covariates, and animals clustered within herds on days to conception after voluntary waiting period (Ospina et al., 2010a). Sampled population Prepartum cohort Postpartum cohort Variable NEFA 0.27 Parity NEFA 0.72 BHBA 10 Parity BHBA 10 Parity Hazard 0.81 0.73 0.84 0.93 0.81 0.87 0.80 P-value 0.01 0.001 0.05 0.4 0.01 0.1 0.01

Postpartum cohort

Associations of analytes related to energy metabolism with subsequent milk production (assessed as mature-equivalent 305-day lactational milk, predicted at approximately 120 DIM) are depicted in Table 3. Regardless of parity, animals with greater than about 0.3 mEq/L of NEFA during the prepartum period had nearly 700 kg less ME305 projected milk than animals with lower concentrations. During the postpartum period, there were interesting differences in associations of energy-related analytes with milk production depending upon parity. In primiparous cows (heifers), postpartum NEFA concentrations greater than about 0.6 mEq/L and BHBA concentrations over about 9 mg/dL were associated with increased milk yield. In multiparous cows, postpartum NEFA concentrations greater than about 0.7 mEq/L and BHBA concentrations greater than about 10 mg/dL were associated with lower predicted milk yield. Table 3. Mixed models for the effect of NEFA (mEq/L) and/or BHBA (mg/dL), covariates, and herd as a random effect on milk production assessed as ME305 milk at 120 days in milk (Ospina et al., 2010a). Sampled Population Prepartum Postpartum -- heifers Postpartum -- heifers Postpartum -- cows Post-partum -- cows Variable NEFA 0.33 Parity NEFA 0.57 BHBA 10 BHBA 9 NEFA 0.72 BHBA 10 BHBA 10 Difference in ME milk yield (kg) -683 -556 +488 -143 + 403 -647 -165 -393 P-value 0.001 0.01 0.02 0.5 0.04 0.001 0.4 0.04

Among animals sampled during the prepartum period (2 to 14 days before calving), 45% of primiparous animals and 26% of multiparous cows had NEFA concentrations at or above 0.3 mEq/L. Among animals sampled during the postpartum period (3 to 14 days after calving), 25% of primiparous animals and 33% of multiparous cows had NEFA concentrations at or above 0.7 mEq/L. Furthermore, 15% of primiparous animals and 27% of multiparous cows had BHBA concentrations at or above 10 mg/dL. In the vast majority of participating farms, primiparous and multiparous animals would have been commingled during the period before calving these results suggest that heifers in particular may be compromised from the standpoint of energy 48

intake relative to requirements in these systems. Furthermore, these energy-related analytes appear more likely to be elevated in multiparous cows than primiparous cows during the period after calving. Ospina et al. (2010c) also used this dataset to compare herds with greater than 15% of animals over the critical thresholds for the analytes during the prepartum and postpartum periods with those with less than 15% of animals over the thresholds during each period and results from this analysis are presented in Table 4. It should be noted that the numbers in this table reflect the associations among all animals in the herd, not just sampled animals in the study. As suggested by the results in the table, those herds with more than 15% of animals with prepartum NEFA and/or postpartum NEFA and BHBA over the critical thresholds had slightly greater disease incidence, poorer reproductive performance, and lower ME305 projected milk yield in both primiparous and multiparous cows. In the U.S. system, the associations of these analytes at the herd-level with decreased milk yield and poorer reproductive performance would be much more economically meaningful than those with disease incidence. Table 4. Herd-level impacts of elevated prepartum and postpartum nonesterified fatty acids (NEFA) and postpartum beta-hydroxybutyrate (BHBA) in commercial dairy farms (Ospina et al., 2010c) Metabolite level Prepartum NEFA (14 to 2 d prepartum) > 0.3 mEq/L Herd alarm > 15% Herd-level impact - 1.2% 21-d pregnancy rate + 3.6% disease incidence - 282 kg ME305 milk - 0.9% 21-d pregnancy rate + 1.7% disease incidence Heifers: - 288 kg ME305 milk Cows: -593 kg ME 305 milk - 0.8% 21-d pregnancy rate + 1.8% disease incidence *Heifers: -534 kg ME305 milk Cows: -358 kg ME 305 milk

Postpartum NEFA (3 to 14 d postpartum) > 0.6 (heifers) 0.7 (cows) mEq/L

> 15%

Postpartum BHBA (3 to 14 d postpartum) > 10 (cows) 12 (heifers) mg/dL

> 15% > 20%*

15% of 15 animals sampled = 2 to 3 animals over threshold; 90% confidence interval that it sample represents herd prevalence In terms of practical application of this information, we believe that measurement of energyrelated analytes is a useful tool for monitoring herds, evaluation of potential opportunities for improved transition cow management, or diagnostics. In terms of the target windows, we recommend sampling 12 to 15 cows per group within the windows of interest described above prepartum samples should be analyzed for NEFA and postpartum samples can be analyzed for NEFA and/or BHBA. The cowside blood or milk tests for BHBA described above are very accurate and represent an excellent first step or front line analysis because of convenience and cost. Because the incidence of herds with high postpartum NEFA in our dataset was much greater than that with high postpartum BHBA, we would encourage practitioners and 49

consultants to take the extra step and consider analysis for postpartum NEFA in situations where they believe that early lactation milk production and reproductive performance are compromised yet the BHBA data are unrevealing. Finally, prepartum NEFA continue to be useful in helping to identify situations in which larger than desired proportions of prepartum cows have compromised energy status. Table 5 describes three possible outcomes and potential interpretations for a herd to consider after NEFA and/or BHBA evaluation in prepartum and postpartum groups. If NEFA is elevated in prepartum cows, it is generally a good signal that either energy intake as a whole is inadequate or facility/management issues exist and are causing significant cow to cow variation in DMI and hence NEFA concentration. Independent of postpartum analyte values, we associate elevated prepartum NEFA with negative disease, reproductive, and production outcomes at the herd level (Table 4). The most likely analyte pattern for a herd that is overfeeding energy either far-off or close-up is low NEFA values prepartum but high NEFA and/or BHB values postpartum. Herds and consultants should remember, however, that a number of factors specific to either nutritional management or facility/grouping management also can elevate postpartum concentrations of NEFA and/or BHB independent of prepartum values. Typically, when herds are overfed either far-off or close-up, we see a subsequent rapid and marked loss of BCS among fresh cows NEFA testing of the fresh cows can help to confirm this. Table 5. Interpretation of energy-related metabolites [nonesterified fatty acids (NEFA) and beta-hydroxybutyrate (BHBA)] to assess herd-level opportunities. Scenario High prepartum NEFA High postpartum NEFA and/or BHBA Likely cause and possibilities Likely starting with low DMI in close-up cows Too low energy in prefresh diet, facility and/or management issues (grouping, stocking density, heat stress?) Low DMI in close-up cows Sampling the survivors in the fresh pen? Is herd outmanaging or putting band-aids on fresh cow issues? Is herd overfeeding energy either far-off or close-up? Diet or facility/management issues specific to maternity/fresh group

High prepartum NEFA Low postpartum NEFA and/or BHBA

Low prepartum NEFA High postpartum NEFA and/or BHBA

POTENTIAL FIELD-BASED MARKERS FOR INFLAMMATION AND STRESS IN TRANSITION COWS An emerging area of focus within our research group is in understanding the relationship of inflammation and stress with transition cow health and performance (Huzzey et al., 2011; Huzzey and Overton, 2010). We envision that biomarkers related to inflammation [e.g. Haptoglobin (hp)] or stress [fecal cortisol metabolites (FCORT)] could also be useful for evaluating the effects of non-nutritional management factors, such as overstocking or commingling of cows and heifers, on physiology. Haptoglobin is an acute phase protein that is synthesized and released by liver as part of the inflammatory response and has been shown to be elevated in cows with metritis (Huzzey et al., 2009). Although there are many acute phase 50

proteins, Hp has been of particular interest for the detection of sick animals due to its very low concentrations in the blood of healthy animals (Eckersall, 2000). Fecal cortisol metabolites are reflective of circulating concentrations of cortisol approximately 10 to 12 hours prior to the collection of the fresh fecal sample. Concentrations of FCORT have been suggested to be a better indictor of physiological stress responses than direct measurements of plasma cortisol due to the feedback-free nature of the sampling method (Palme et al., 1999). Restraint and handling, which are required during blood sampling, can cause a physiological stress response and raise circulating cortisol concentrations quickly (Cook et al., 2000). Further, the release of cortisol from the adrenal gland during the day is pulsatile and has a diurnal cycle that is subject to substantial individual variation. Because FCORT does not have the same limitations relative to sampling, as does serum or plasma cortisol, it offers a potential way to study activation of the hypothalamic-pituitary-axis in response to exposure to stressors. To evaluate whether prepartum physiological indicators of stress and inflammation were associated with the occurrence of health disorders after calving and milk yield, data were collected from 412 cows on two commercial dairies in New York State. Farms were visited weekly to collect blood, fecal samples, and BCS. Sampling began approximately 4 wk prior to each cows expected calving date. One blood and fecal sample per cow was collected between d -21 to -15 relative to the actual calving date to represent wk -3, d -14 to -8 (wk -2), and d -7 to 2 (wk -1). Prepartum plasma was analyzed for NEFA, Hp, and cortisol and FCORT concentrations (11,17-dioxoandrostanes) were determined from fecal samples. Health events occurring within 30 DIM, including retained placenta ( RP), displaced abomasum (DA), and death (not including voluntary culls) were collected from DairyCOMP 305. A postpartum blood sample was collected within 3 to 10 d after calving. Based on this postpartum blood sample, sub-clinical ketosis (SCK) was diagnosed when plasma BHBA was 10 mg/dl (Ospina et al., 2010a) and High Haptoglobin ( HiHp, suggestive of an infection such as metritis) was diagnosed when plasma Hp was 1 g/L (Huzzey et al., 2009). Cows were divided into 3 health categories for statistical analysis: 1) No disorder of interest ( NDI); 2) One disorder (RP, DA, SCK, or HiHp); or 3) More than one disorder (RP, DA, SCK, HiHp) or death. As expected prepartum plasma NEFA was a strong predictor of postpartum health; however, this relationship was dependent on the degree of illness after calving. Cows that developed multiple disorders after calving or that died had the greatest concentrations of NEFA, relative to the other two health categories, particularly during the 2-week period before calving. There were no associations between prepartum Hp or FCORT concentration and the occurrence of one disorder (RP, DA, SCK or HiHP) by 30 DIM. Hp concentration tended to be greater during wk 2 and -1 and FCORT tended to be greater during wk -3 and -2 for cows that developed more than one disorder or that died by 30 DIM relative to cows the NDI category; however, neither of these analytes could predict which cows would go on to develop health complications as well as prepartum NEFA concentration (Huzzey et al., 2011). In order to evaluate the relationships of these analytes with subsequent milk yield, herd DC305 records were used to collect information on each cows predicted 305ME from the 2 nd test day (approximately 62 DIM). A range of metabolic cutpoints were evaluated for each period and the effect of being above or below the cutpoint on predicted 305ME was then evaluated (Huzzey and Overton, 2010). 51

Projected 305ME milk yield tended to be 2328 lbs lower in heifers with haptoglobin concentrations > 1.1 g/L during weeks -3 and -2, relative to heifer below this cutpoint. Multiparous cows with haptoglobin > 1.1 g/L during weeks -2, -1 or +1 had on average 3315 lbs lower projected 305ME milk yield (Figure 1), relative to multiparous cows below this cutpoint. Figure 1. Difference in predicted 305ME milk yield for cows above the indicated Haptoglobin (Hp) cutpoints relative to cows that are below the cutpoints
wk -3
Difference* in 305ME Milk Yield (lbs)

wk -2

wk -1

wk +1

0 -1000 -2000 -3000 -4000 -5000

-5130 -2654 -1634 -2002 -2224 -721

-2315

-2500

***

primiparous multiparous
** Hp Cutpoint: > 1.1 g/L
* Dif f erence = (305MP cows abov e cutpoint) - (305ME cows below cutpoint)

-6000

There was no association between plasma cortisol and milk yield at any period relative to calving for either multiparous or primiparous cows (data not shown). Concentrations of FCORT were not significantly associated with 305ME milk yield in primiparous cows; however, multiparous cows with FCORT > 2500 ng/g fecal DM during weeks -3 or -2 relative to calving had on average 2429 lbs lower 305ME milk yield relative to cows below this cutpoint. Projected 305ME milk yield was 2926 lbs lower among MP cows with fecal cortisol metabolites > 700 ng/g fecal DM during week +1 (Figure 2). Figure 2. Difference in predicted 305ME milk yield for cows above the indicated fecal cortisol metabolite (FCORT) cutpoints relative to cows that are below the cutpoints
1500
Difference* in 305ME Milk Yield (lbs)
1176

1000 500 0 -500 -1000 -1500 -2000 -2500 -3000 -3500

FCORT Cutpoints: > 2500 ng/g fecal 52DM
-2275

primiparous multiparous
wk -2
-257 -863 -999

wk -3

wk -1

wk +1

-1041

-2583

**

-2926

*** > 700 ng/g fecal DM

* Dif f erence = (305MP cows abov e cutpoint) - (305ME cows below cutpoint)

In summary, cows with higher concentrations of NEFA (data not shown; with the exception of heifers during week +1), haptoglobin, or fecal cortisol metabolites around calving have lower projected 305ME at the 2nd test day and these associations are apparent up to 3 weeks prior to the onset of lactation. While higher concentrations of NEFA, Hp, and FCORT are associated with lower predicted milk yield, the significance of these relationships was stronger for Hp and FCORT relative to NEFA, particularly when these analytes are measured during the week after calving. If we focus on the postpartum period it is also appears that both Hp and FCORT are more sensitive to 305ME milk yield projections than NEFA; the magnitude of the difference in projected 305ME between those animals that were above versus below the indicated cutpoints were much greater when using Hp or FCORT to predict these differences compared to NEFA. Finally, a greater proportion of animals were above the Hp and FCORT cutpoints during week +1 then were above the NEFA cutpoint. These results suggest that haptoglobin or fecal cortisol metabolites may be alternative and perhaps more effective analytes for detecting cows at risk for reduced milk yield, than NEFA. Associations of postpartum concentrations of NEFA and Hp with reproductive performance as assessed using Kaplan-Meier analysis are shown in Figure 3. Consistent with previous work, cows with elevated concentrations of NEFA postcalving had longer times to pregnancy. Similarly, cows with elevated concentrations of Hp had longer times to pregnancy. Collectively, these results suggest that both compromised energy balance and inflammation have strongly negative associations with subsequent reproductive performance.

53

Figure 3. Graph of Kaplan-Meier estimator of days to conception for primiparous cows with postpartum (3 to 10 DIM) nonesterified fatty acid (NEFA; A) concentrations > or 0.45 mEq/L (P = 0.02) and postpartum haptoglobin (Hp; B) concentrations > or 1.3 g/L ( P = 0.02).
100 90 80 70 60 50 40 30 20 10 0 0

NEFA < 0.45 mEq/L Postpartum NEFA > 0.45 mEq/L Postpartum

Primiparous Cows not Pregnant (%)

30

60

90

120

150

100 90 80 70 60 50 40 30 20 10 0 0

Hp < 1.3 g/L Postpartum Hp > 1.3 g/L Postpartum

30

60

90

120

150

Days in Milk
SUMMARY AND CONCLUSIONS Circulating concentrations of energy-related metabolites (prepartum NEFA and postpartum NEFA and/or BHBA) are highly associated with postpartum outcomes relative to disease, milk production, and reproductive performance in dairy cattle. As such, they can be an important component of evaluation of transition cow programs. Our recent data suggest that associations of elevated concentrations of these metabolites during the prepartum and postpartum period with subsequent milk yield and reproductive performance may be more meaningful at the farm level than their associations with metabolic disease. Because of convenience and cost, evaluation of 54

postpartum BHBA in milk or blood at the farm level is a preferred first-line monitoring tool, although prepartum and postpartum NEFA concentrations in serum or plasma can provide additional insight into transition management opportunities. New research from our group suggests that elevated concentrations of biomarkers related to inflammation and stress (plasma haptoglobin and fecal cortisol metabolites) during the transition period also are associated with decreased milk yield and reproductive performance in early lactation we will be conducting further research to develop these as potential tools to help identify opportunities for improved transition cow management. REFERENCES Cameron, R. E., P. B. Dyk, T. H. Herdt, J. B. Kaneene, R. Miller, H. F. Bucholtz, J. S. Liesman, M. J. VandeHaar, and R. S. Emery. 1998. Dry cow diet, management, and energy balance as risk factors for displaced abomasums in high producing dairy herds. J. Dairy Sci. 81:132139. Cook, C.J., D. J. Mellor, P. J. Harris, J. R. Ingram, and L. R. Matthews. 2000. Hands-on and hands-off measurement of stress. In: Moberg G.P. and J. A. Mench, ed. The Biology of Animal Stress. CABI Publishing, p.123-46. Duffield, T. F., D. Sandals, K. E. Leslie, K. Lissemore, B. W. McBride, J. H. Lumsden, P. Dick, and R. Bagg. 1998. Efficacy of monensin for the prevention of subclinical ketosis in lactating dairy cows. J. Dairy Sci. 81:2866-2873. Eckersall, P. D. 2000. Recent advances and future prospects for the use of acute phase proteins as markers of disease in animals. Rev. Med. Vet. 151:577-584. Herdt, T. H. 2000. Variability characteristics and test selection in herd-level nutritional and metabolic profile testing. Vet. Clin. North Am. Food Anim. Pract. 16:387-403. Huzzey, J. M., T. F. Duffield, S. J. LeBlanc, D. M. Veira, D. M. Weary, and M. A. von Keyserlingk. 2009. Short communication: Haptoglobin as an early indicator of metritis. J. Dairy Sci. 92:621-625. Huzzey, J. M., and T. R. Overton. 2010. Measuring the effect of stress during the transition period on subsequent health and performance of dairy cattle. Proceedings, Cornell Nutrition Conference for Feed Manufacturers, Syracuse, NY, pp. 76-86. Accessed on 6/1/2011 at http://www.ansci.cornell.edu/cnconf/2010proceedings/index.html Huzzey J. M., D. V. Nydam, R. J. Grant, and T. R. Overton. 2011. Associations of prepartum plasma cortisol, haptoglobin, fecal cortisol metabolites, and nonesterified fatty acids with postpartum health status in Holstein dairy cows. J. Dairy Sci. 94 :58785889. Ingraham, R. H., and L. C. Kappel. 1988. Metabolic profile testing. Vet. Clin. North Am. Food Anim. Pract. 4:391-411. Oetzel, G. R. 2004. Monitoring and testing dairy herds for metabolic disease. Vet. Clin. North Am. Food Anim. Pract. 20:651-674. Ospina, P. A., D. V. Nydam, T. Stokol, and T. R. Overton. 2010a. Associations of elevated nonesterified fatty acids and beta-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States. J. Dairy Sci. 93:1596-1603. Ospina, P. A., D. V. Nydam, T. Stokol, and T. R. Overton. 2010b. Evaluation of nonesterified fatty acids and beta-hydroxybutyrate in transition dairy cattle in the northeastern United States: Critical thresholds for prediction of clinical diseases. J. Dairy Sci. 93:546-54.

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Ospina, P. A., D. V. Nydam, T. Stokol, and T. R. Overton. 2010c. Association between the proportion of sampled transition cows with increased nonesterified fatty acids and hydroxybutyrate and disease incidence, pregnancy rate, and milk production at the herd level. J. Dairy Sci. 93:3595-3601. Palme, R., C. Robia, S. Messmann, J. Hofer, and E. Mstl. 1999. Measurement of faecal cortisol metabolites in ruminants: a non-invasive parameter of adrenocortical function. Wien. Tierrztl. Mschr. 86:237-241.

56

Economics of Feeds in Dairy Rations

N. R. St-Pierre and W. P. Weiss Department of Animal Sciences The Ohio State University, Columbus, OH-43210 Corresponding author: st-pierre.8@osu.edu SUMMARY The economic value of a feed rests in the value of its nutrients. In dairy, the economically important nutrients are net energy lactation (NEL), metabolizable protein (MP), effective NDF (eNDF), and non-effective NDF (neNDF). Unit prices of the important nutrients can be calculated using the composition and prices of all feeds being traded in a given market. This method is available in a Windows-based software. Alternatively, estimates of unit prices are being published on a monthly basis in a national dairy magazine for all major dairy regions. Forage composition for NEL, MP, eNDF and neNDF can easily be determined using 11 compositional inputs and equations provided in the appendix of this paper. Using data from our experimental research station, we do not find a strong association between in vivo total tract NDF digestibility and in vitro NDF digestibility (NDFd). At this point we are still recommending the use of protein-free NDF and lignin to estimate NDF digestibility. For most feeds, the economic value is calculated as the simple sum of the values of their nutrients. With forages, however, one must introduce a correction associated with quality because forages are not entirely substitutable. Dairy cows exhibit a small, but significant response in milk yield when high quality forages are substituted for low quality forage in otherwise equally balanced diets. The values of three levels of quality within alfalfa and grass hay are calculated over the period of January through March 2012. In alfalfa, nearly 65% of the total value is associated with the NEL content, whereas in grass NEL content accounts for nearly 75% of forage values. Of the 11 compositional inputs required in the calculation of hay and silage values, NDF, lignin and ash appear to have the greatest importance. INTRODUCTION What Are Feeds Used For? Animals do not require feeds; animals require nutrients. Feeds are nothing else than containers, packages of nutrients. The sole value of a feed is in the value of the nutrients that it contains. A feed containing no nutrient has no economic value. No economic value means that it is worthless - ZERO. What are the Nutrients of Economic Value? The answer to that question depends on two things. First, the nutrients of economic value are dependent on the class of animals under consideration. For example, the nutrients of economic importance are not the same for beef cattle, lactating dairy cows, dry cows, and replacement 57

animals. Hence a given feed has a different economic value to lactating dairy cows and replacement heifers. Second, the nutrients of economic value depend whether one is interested in the strategic value of a feed versus its tactical value. This begs further explanations. A dairy producer feeding 50 lbs/day of a finely chopped corn silage is looking for attributes in purchased hay that are very specific to the narrow conditions in which it is to be fed. The value of a given lot of hay to this producer would be entirely tactical i.e., determined by how well it fits as a complement to other feed ingredients that are essentially pre-determined. On the other hand, a dairy producer who considers all feed components of his dairy rations to be exchangeable (tradable) would look at a given lot of hay with a strategic view. The hay would no longer be looked at as a complement to other pre-determined feeds, but as a component of the whole diet. To put it differently, tactical is when you have painted yourself in a corner; strategic is when you look at the floor configuration before you start painting. The economic values calculated in this paper are (1) exclusively for lactating dairy cows, and (2) entirely strategic. Of the large set of nutrients required by dairy cows, some have large economic values while others have small economic values. The calcium content of feeds is a good example of a nutrient with a small economic value. Calcium can be supplemented very inexpensively in any dairy diets. This is not to say that calcium is not important to dairy cows or that ration balancing should ignore calcium. It just says that the economics of feeding cows have little to do with calcium. We have extensively studied the major dairy feed markets in the U.S. over a period of 30 years. Across all 3 major markets (Midwest, Northeast, West), two sets of nutrients explain over 98% of the variation in feed prices. These are: 1. Net energy for lactation (NEL), rumen degradable protein (RDP), digestible rumen undegradable protein (dRUP), effective neutral detergent fiber (eNDF), and non-effective neutral detergent fiber (neNDF), or NEL, metabolizable protein (MP), eNDF, and neNDF.

2.

The two sets are entirely interchangeable and give very similar results. The protein requirements of dairy cows, however, are best expressed as metabolizable protein. Therefore, nutrient set #2 will be exclusively used in the balance of this paper. UNIT PRICES OF IMPORTANT NUTRIENTS Calculating Nutrient Unit Prices Feed markets do exist: there are people selling and buying feeds in all major dairy regions. But there is no market for nutrients. Or is there? Can we calculate the implicit nutrient prices from the market prices of feedstuffs?

58

The problem of determining the implicit price of attributes (the nutrients) embedded in various products (feedstuffs) is no at all unique to the feed/nutrient complex. Economists have found an elegant way to solve this problem using a method called hedonic pricing. We will not review the details of how this work in this paper. Interested reader can consult St-Pierre and Cobanov (2000) for further details. In short, prices and nutritional composition of all ingredients traded in a given market are used to back-calculate, using statistical methods, what the markets are implicitly pricing the nutrients contained in feeds. Market prices of nutrients in the Midwest for the 86 months between January 2005 and March 2012 are shown in Figure 1. Southwest prices would be a bit different, but the curves would largely parallel those for the Midwest. Figure 1. Price of nutrients in the Midwest from January 2005 through March 2012. NEl$ = Net energy for lactation ($/Mcal), MP$ = metabolizable protein ($/lb), and e-NDF$ = effective NDF ($/lb).

During this period, the cost per unit of NE L has more than tripled, the cost per unit of MP has doubled, while the cost per unit of eNDF has surged in the first half of 2011. The U.S. has experienced drastic changes in renewable energy policies during the last decade, some of which have had a substantial effect on feed prices. However, large variation in nutrient unit prices are still evident even over a much shorter period of time such as what has occurred since January 2011 (Table 1).

59

Table 1. Nutrient unit prices in the Southwest between January 2011 and March 2012. Nutrients NEL (/Mcal) MP (/lb) eNDF (/lb) neNDF (/lb) Average 16.5 23.6 3.6 -8.7 S.D. 2.7 9.6 3.5 3.7 Min 11.1 9.7 0.0 -13.5 Max 20.8 40.6 11.9 -3.2

What Affects Nutrient Unit Prices? Market prices of all feeds, not just the prices of corn, soybean meal and alfalfa hay, affect nutrient unit prices. Therefore, nutrient unit prices change through time and location. We have already shown how nutrients can quickly change through time in Table 1. Because of regional differences in feed prices and availability, nutrient unit prices also show regional differences (Table 2). In this paper we will be using southwest nutrient unit prices whenever this information was available. When examining the effect of time, we will use January 2011 to March 2012 prices. Table 2. Nutrient unit prices across 3 regions1, December 2012. Nutrients NEL (/Mcal) MP (/lb) eNDF (/lb) neNDF (/lb)
1

Midwest 15.8 38.9 4.0 -3.2

Southwest 13.7 49.7 5.6 -1.5

West 15.9 35.3 6.9 0.0

Midwest prices are for WI and eastern MN; Southwest prices are for northwest Texas; West prices are for the San Joaquin Valley of CA.

Where Do I Find the Nutrient Unit Prices? You can purchase a Windows-based software that we wrote (Sesame) for $10 at www.sesamesoft.com. Beware that it is NOT the easiest software to use. Alternatively, we publish a regular column in Progressive Dairyman where we publish the nutrient unit prices for the major dairy regions of the U.S. Some nutrition consultants also provide this information to their clients. FORAGE COMPOSITION Nutrient Composition Used in This Paper. Each lot of forage has a unique nutrient composition that affects its value. The nutrient composition of forages used as examples in this paper is reported in Table 3. We used 3 levels of quality for legume hay and grass hay.

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Table 3. Nutrient composition of the reference alfalfa hay, reference grass hay, and low and high quality hays used as examples.
Nutrients1 Dry matter Crude protein NDICP ADICP Ether extracts NDF ADF Lignin Ash RUP RUPd Effective NDF TDN from NFC TDN from NDF TDN from CP TDN from EE TDN at 3X NEL at 3X MP at 3X NEL MP eNDF neNDF
1

Units % % % % % % % % % %CP % RUP % NDF % % % % % Mcal/cwt % Mcal lbs lbs lbs

Reference 88 20 2.5 1.5 2.0 40 30 7.0 10 25 70 92 29.9 15.4

Alfalfa Low 88 16 2.5 1.5 2.0 44 34 8.8 10 25 70 92 29.9 15.8

High 88 24 2.5 1.5 2.0 36 26 5.4 10 25 70 92 29.9 14.8

Reference 88 12 4.0 1.0 2.5 60 40 6.5 7 30 65 98 22.1 28.3

Grass Low 88 8 4.0 1.0 2.5 68 48 8.5 7 30 65 98 18.1 30.8

High 88 16 4.0 1.0 2.5 52 32 4.5 7 30 65 98 26.0 25.9

18.3 14.3 22.3 10.9 6.9 14.8 2.3 2.3 2.3 3.4 3.4 3.4 54.0 50.7 57.1 52.9 47.9 57.9 57.6 51.5 63.5 53.0 44.6 61.5 7.99 7.02 8.95 6.73 5.55 7.94 ------------------------------ Units per Ton ----------------------------1014.2 906.7 1118.1 932.6 785.5 1082.3 140.7 123.5 157.6 118.6 97.6 139.7 647.7 712.4 582.9 1034.9 1172.9 896.9 56.3 62.0 50.7 21.1 23.9 18.3

NDICP = NDF insoluble crude protein; ADICP = ADF insoluble crude protein; NDF = neutral detergent fiber; ADF = acid detergent fiber; RUP = rumen undegradable protein; RUPd = RUP digestibility; TDN = total digestible nutrients; NEL at 3X = net energy for lactation calculated at an intake of 3 times maintenance; MP at 3X = metabolizable protein calculated at 3 times maintenance.

A few things are worth mentioning here. First, notice that the quality of alfalfa (and grass) has a much smaller effect on its metabolizable protein than on its crude protein. Second, observe that in alfalfa the non-fiber carbohydrates (NFC) contribute 2 times more to its energy content (i.e., TDN) than the NDF. In grass, the situation is reversed, with NDF contributing significantly more to the energy content than NFC. Total tract NDF digestibility is greater in grass (~47%) than in alfalfa (~39%) of equivalent quality. How are NEL and MP Calculated? Equations used in the calculation of NE L and MP according to NRC (2001) are reported in the appendix. Although these equations may seem intimidating at first, they can easily be programmed in a computer spreadsheet. While 9 chemical entries are required for the 61

calculation of NEL only 5 measurements will have much effect on NEL in practice: DM, CP, NDF, lignin, and ash. Other entries can simply be taken from standard feed composition tables. The calculation of MP requires a measurement of CP, rumen degradability of protein (RUP_CP), and post-ruminal digestibility of RUP (RUPd). Some feed laboratories provide estimates of RUP_CP, but the variation within a type of hay is relatively small and largely inconsequential to the value of the forage. Likewise, table values for RUPd should be used. What NDF Digestibility Should be Used? The ratio of lignin to protein-free NDF is used to estimate in vivo total tract NDF digestibility (TT-NDFd) in the equation for TDN_NDF reported in the Appendix. The 2/3 exponent is used to convert mass to surface area, hence representing the decrease in NDF digestibility due to the surface interaction (i.e., coating) of cell walls by lignin. This conceptual interaction is necessarily a simplification of the complex anatomy and chemistry of plant cell walls. Some have advocated the use of in vitro NDF digestibility (NDFd) as a proxy for the calculated TTNDFd used when calculating TDN_NDF. Although this approach is appealing, much doubt remains regarding the relationship between NDFd and TT-NDFd. For example, we have summarized the relationship between TT-NDFd and NDFd for 23 diets where TT-NDFd was measured using total fecal collection in trials conducted at our experimental research station (Figure 2). Figure 2. Relationship between whole diet total tract in vivo NDF digestibility (TT-NDFd) expressed as deviation from a control diet and in vitro NDF digestibility (NDFd).

In this figure, it is apparent that NDFd overestimates differences between treatments and that the magnitude of the difference in NDFd is not related to the magnitude of the difference in TTNDFd. The ranking within experiment was often OK with NDFd, raising the possibility of using NDFd for energy calculation. It is clear, however, that NDFd cannot be directly substituted for TT-NDFd when calculating the energy of a feed. Much work is needed in this 62

area. Meanwhile, we still recommend using the equation with the ratio of lignin to protein-free NDF for estimating the energy contribution of NDF. CALCULATING THE VALUE OF A FORAGE The Value of the Nutrients So far, we have shown that nutrient unit prices can be calculated from market information from all feedstuffs traded in an area. We also explained how the nutrient composition of forages for the economically important nutrients is calculated. Determining the value of the nutrients in a given forage involves a series of simple arithmetical operations. These are illustrated for the reference alfalfa in Table 4 using the average price of nutrients for the Southwest from January 2011 to March 2012. Table 4. Calculation of the value of the nutrients in one ton of the reference alfalfa hay using average nutrient unit prices from January 2011 through March 2012. Composition NEL (Mcal/cwt) MP (% DM) eNDF (% DM) neNDF (% DM) Total 57.6 7.99 36.8 3.2 DM % 88 88 88 88 Mcal or Pounds per Ton 1014.2 140.7 647.7 56.3 Unit Prices 16.5 23.6 3.6 -8.7 Value $/ton 167.34 33.20 23.31 -4.90 218.95

Correcting for Milk Production Response For most feeds, the sum of the values of the nutrients as calculated in the preceding paragraph is its average economic value; but not for forages. While most feeds are substitutable based on their nutrient content, this is not entirely true for forages. What this means is that two rations balanced for exactly the same nutrient density (NE L, MP, eNDF, neNDF) but using forages of different quality do not result in exactly the same milk production. Cows fed the ration based on a high quality forage respond to forage quality with additional milk production mainly through greater dry matter intake (DMI). Note that this is not the same as the response to feeding forages of different quality, but without any ration re-balancing. Here the rations are identical in their nutritional content, but cows fed rations based on higher quality forages achieve a greater level of milk production. We used results from many research trials to calculate the response to forage quality. Although one could think of a better marker of quality than the total NDF content of forage, the data did not allow the calculation of anything more than NDF. The resulting equations used to calculate the value per ton of forage due to milk production responses are: Alfalfa: Value of Response ($/ton) = [(P-Milk x 0.273 x (44 NDF)] x DM 100 Grass: Value of Response ($/ton) = [(P-Milk x 0.3 x (53 NDF)] x DM 100 63

where P-Milk is the price of milk ($/cwt). It is important to understand that this adjustment to the value of forages means that forage values are dependent on milk prices. The difference in the value of a high quality forage compared to that of a low quality forage is much smaller when milk prices are low (such as in 2009) then when milk prices are high (as in 2011). Effect of Forage Type and Quality on the Historical Value of Forages Table 5 summarizes the values of alfalfa and grass hay for 3 levels of quality for the period of January 2011 to March 2012. On an average, the range in values due to quality is greater in grass ($124/ton) compared to alfalfa ($73/ton). This could be due to the arbitrary range of quality selected for the two types of forages. On an average, alfalfa is worth $50/ton more than grass. Compared to alfalfa, grass hay shows a greater range in value through time: $109 and $126/ton for alfalfa and grass, respectively. More importantly, the value of forages changes considerably through time even over a short time span. Table 5. Value ($/ton) of alfalfa and grass hay of 3 quality levels, Southwest, January 2011 to March 2012. Feeds Alfalfa - Reference - Low - High Grass - Reference - Low - High
1

Average 241 204 277 191 129 253

SD1 37 36 38 46 47 47

Min 193 162 223 145 77 197

Max 302 265 338 271 210 332

SD = standard deviation.

Contribution to Calculated Values The value of a forage is the sum of the values of its important nutrients plus the milk response associated with the forage quality expressed as NDF content. The contribution of each nutrient to the value of a forage is not the same. Table 6 shows the average contribution of NE L, MP, eNDF, neNDF, and milk response for the 15 months from January 2011 to March 2012 for the reference alfalfa and grass hays. On an average, energy (NE L) content accounts for nearly 65% of the value of alfalfa hay and 75% of the value of grass hay. Notice that the protein in alfalfa (20% crude protein) is only worth $7/ton more than the protein in grass (12% crude protein). This is because most of the protein in forage is rumen degradable (RDP), and the unit value of RDP ($/lb) is generally null and often even slightly negative (results not shown). Producing forages of greater protein content is of little value unless the protein increase is associated with an increase in the digestibility (energy) of the resulting feeds.

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Table 6. Average contribution of NEL, MP, eNDF, neNDF, and milk response to the value of our reference alfalfa and grass hays between January 2011 and March 2012. Alfalfa Component NEL MP eNDF neNDF Milk Total $/ton 154.55 43.75 30.44 -4.78 17.29 240.86 % of Total 64.0 18.2 12.6 -2.0 7.2 100.0 $/ton 141.75 36.88 48.64 -1.79 -33.26 192.21 Grass % of Total 73.7 19.2 25.3 -0.9 -17.3 100.0

In hay and most haycrop silages, most of the NDF is effective, resulting in very small neNDF content (Table 2). Therefore, the contribution of neNDF to the value of long hays could be entirely ignored without meaningful losses in the accuracy of estimating their economic values. Marginal Changes from Compositional Values The 4 nutrients used to calculate the value of a forage are calculated from 11 compositional entries. We can calculate the change in the value of our reference alfalfa and grass hay from a one-unit change in each of the 11 entries (Table 7). Table 7. Marginal change in value of forage hay ($/ton) from a one-unit increase in each of the compositional entries between January 2011 and March 2012. Composition entries Dry matter (%) Crude protein (% DM) NDICP (% DM) ADICP (% DM) Ether Extracts (% DM) NDF (% DM) Lignin (% DM) Ash (% DM) RUP (% CP) RUPd (% RUP) NDF Effectiveness (% NDF) Change in forage value ($/ton) Alfalfa - Reference Grass - Reference 2.73 2.19 2.12 2.23 1.28 1.14 -5.17 -5.01 4.72 4.72 -4.96 -5.10 -4.42 -5.38 -3.68 -3.67 0.76 0.43 0.27 0.20 0.93 1.40

One should be extremely careful in the interpretation of these results. First, a one-unit change does not represent the same degree of difficulty across all compositional elements. For example, it is considerably easier to raise the NDF of grass by one unit than to raise its ether extracts also by one unit. Second, and even more importantly, it is very difficult in nature to change a compositional element by one unit without affecting any of the other compositional elements. For example, crude protein in alfalfa is negatively associated with NDF content. On average, raising crude protein lowers the NDF content (i.e., the plant is more immature). Likewise, NDF and lignin content are positively associated, meaning that an increase in NDF content is generally associated with an increase in lignin content. 65

Keeping these reservations in mind, compositional elements can be loosely grouped into 3 categories. 1. 2. 3. Those with a small effect on the value of hay: RUP, RUPd and NDFe, Those with a medium effect on the value of hay: DM, CP, NDICP, and Those with a large effect on the value of hay: ADICP, ether extracts, NDF, lignin and ash.

Ether extracts and ADICP are relatively constant within hay type compared to NDF, lignin and ash. NRC (2001) reports standard deviations of 0.4 (ADICP), 0.5 (ether extracts), 0.9 (lignin), 1.0 (ash), and 6.3 (NDF) percent. Thus, NDF, lignin, and ash are arguably the most influential compositional entries to hay values. Note that the net effect of NDF on hay values incorporates its positive effect on forage value from its positive contribution to eNDF, and its negative effects on forage value from its negative contribution to NE L and MP. CONCLUDING REMARKS The values calculated in this paper are on a farm-gate basis (i.e., delivered) and not FOB. Therefore, forage growers would have to account for delivery costs when estimating the value of a given hay or silage. In addition, the values calculated in this paper are averages and represent what coherent buyers should be willing to pay. Coherent behavior is often an elusive attribute in feed markets. Some buyers consistently shop for supreme quality alfalfa hay because thats what they have been using for years without ever considering whether other quality levels or even other types of feeds make more economic sense. A very thirsty man is much more willing to pay an exorbitant price for a cold beer, especially if he fails to consider the free water available from a nearby fountain. REFERENCES St-Pierre, N. R., and D. Glamocic. 2000. Estimating unit costs of nutrients from market prices of feedstuffs. J. Dairy Sci. 83:1402-1411. APPENDIX Equations used to calculate NEL (NE_3X, Mcal/lb) of forages: TDN_NFC = 0.98 x (100 NDF + NDICP CP EE ASH) TDN_NDF = 0.75 x (NDF NDICP LIG) x (1-(LIG/(NDF-NDICP))0.667) TDN_CP = CP x exp(-1.2 x ADICP/CP) TDN_EE = (EE 1) x 2.25 TDN_1X = TDN_NFC + TDN_NDF + TDN_CP + TDN_EE - 7 DE_1X = (TDN_NFC x 0.042) + (TDN_NDF x 0.042) + (TDN_CP x 0.056) + ((EE-1) x 0.094) - 0.3 TDN_3X = TDN_1X x 0.92 NE_3X = (0.6532 x DE_1X) 0.5064

Equations used to calculate the metabolizable protein (MP, % of DM) of forages: dRUP = CP x RUP_CP x RUPd 10000 dMTP = TDN_3X x 1.3 x 0.64 10 66

MP

dRUP + dMTP

Equations used to calculate effective and non-effective NDF of forages: eNDF neNDF where: ADICP ASH CP dRUP dMTP EE eNDF exp LIG NDF NDFe NDICP NE_3X neNDF RUP_CP RUPd TDN_1X TDN_3X TDN_CP TDN_EE TDN_NDF TDN_NFC = = = = = = = = = = = = = = = = = = = = = = = = NDF x NDFe NDF - eNDF ADF insoluble crude protein (% of DM) Ash (% of DM) Crude protein (% of DM) Digestible RUP (% of DM) Digestible microbial true protein (% of DM) Ether extracts (% of DM) Effective NDF (% of DM) The exponential function (i.e., e exponent the value in parentheses) Lignin (% of DM) Neutral detergent fiber (% of DM) NDF effectiveness (% of NDF) NDF insoluble crude protein (% of DM) Net energy for lactation measured a 3 times maintenance (Mcal/lb) Non-effective NDF (% of DM) Rumen undegradable protein (% of CP) Digestibility of RUP (% of RUP) Total digestible nutrients at 1 time maintenance (% of DM) Total digestible nutrients at 3 times maintenance (% of DM) TDN from the crude protein fraction (% of DM) TDN from the ether extracts fraction (% of DM) TDN from the NDF fraction (% of DM) TDN from the NFC fraction (% of DM).

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Managing Heat Stress and its Impact on Cow Behavior

J.D. Allen1, S.D. Anderson2, R.J. Collier2, and J.F. Smith2 Northwest Missouri State University, ALLENJD@nwmissouri.edu 2 University of Arizona, Tucson, AZ, anderss@email.arizona.edu, rcollier@cals.arizona.edu, jfsmith@email.arizona.edu
1

SUMMARY Heat stress affects several aspects of the dairy industry including cattle behavior. Heat stress will increase an animals standing time as it tries to dissipate heat over its entire body surface. Increasing standing time or decreasing resting time reduces milk production. Prolonged standing further increases the risk of lameness. Understanding environmental and physiological parameters that affect standing behavior will improve industry efforts to minimize heat stress in dairy cattle. Core body temperature is correlated standing behavior, with cattle more likely to stand above a 102.07 F (39.2 C) core body temperature. Correlation between thermal heat index and cattle behavior has also been evaluated, although predictive capacity has yet to be established. However, cattle are more likely to stand above a THI of 68. INTRODUCTION The issue of environmental impacts on dairy production and cattle welfare has long been of interest to the industry. One environmental stressor which has commanded considerable research attention within the past several decades has been thermal stress. Production loss due to heat stress has been estimated at $900 million annually to U.S. dairy herd (St. Pierre et al., 2003). This interest in heat stress has coincided with the spreading demographic of the United States dairy industry from the Midwest to warmer and more arid climates, such as the desert Southwest as well as an increase to heat sensitivity due to a doubling of average production per cow. Improvements in warm weather dairy housing have provided more efficient technologies for cooling animals exposed to hot climates. However, heat stress remains an important environmental stressor on dairy cattle. Heat stress directly and indirectly affects feed intake, cow body temperature, maintenance requirements and metabolic processes, feed efficiency, milk yield, reproductive efficiency, cow behavior, and disease incidence (Thatcher, 1974; Cook et al., 2007; Tucker et al., 2007; Rhoads et al., 2009). These effects are well documented. It is only recently that researchers have attempted to understand the correlation of one of the most understood outcomes (increased body temperature) to one of the least understood outcomes (modified cow behavior) and its possible effect on bottom line production. HEAT STRESS AND DAIRY CATTLE Domestic animals have a core body temperature (CBT) range in which metabolism functions without modification, termed the thermoneutral zone. Typically, core body temperature is higher 68

than ambient temperature to ensure that heat generated by metabolism flows out to the environment (Collier et al., 2006). Deviation outside of this range which is relatively narrow leads to increases in resting metabolism, modifications to the biochemistry and cellular physiology as well as the behavior of the animal (Shearer and Beede, 1990). The thermoneutral zone lies between 41 and 77 F (5 and 25 C) for dairy cattle (Roenfeldt, 1998). Above 77 F (25 C), the body must modify physiology and behavior to keep CBT above the environment temperature. HEAT STRESS AND THERMAL HUMIDITY INDEX Temperature is not the only environmental factor that affects the intensity of heat stress. The temperature humidity index (THI) measures the combined effects of ambient temperature and relative humidity (RH) to ascertain heat load intensity (Berry et al., 1964). This index was later categorized into heat stress levels with an index above 72 THI [75 F (23.9 C) with 65% RH to 90 F (32.2 C) with 0% RH] established as the lower threshold of heat stress (Whittier, 1993; Armstrong, 1994). However, because of the increase of milk production per cow since the development of the THI, a 22 lbs/d (10 kg/d) increase will decrease the threshold for heat stress by 9 F (5 C; Berman, 2005). A recent re-evaluation of the THI has suggested that due to this improvement of milk production, the THI heat stress threshold should be lowered to 68 [72 F (22.2 C) with 45% RH to 80 F (26.7 C) with 0% RH; Zimbelman et al., 2009]. HEAT STRESS AND REPRODUCTION Elevated core body temperature (CBT) in dairy cows caused by heat stress can have detrimental effects on reproductive performance. An increase in rectal temperature of 1.8 F (1 C) occurring 12 h post-insemination decreased pregnancy rates by 16% (Ulberg and Burfening, 1967). Gwazdauskas et al. (1973) reported an increase in uterine temperature of 0.9 F (0.5 C) on the day of or the day after insemination resulted in decreased conception rates by 13% and 7%, respectively. Badinga et al. (1985) attributed decreased conception rates of lactating cows to their inability to maintain normal body temperature at high environmental temperatures [> 86 F (30 C)]. Ealy and colleagues (1993) further support this hypothesis by reporting that bovine embryos become more resistant to adverse effects of maternal heat stress as pregnancy progresses. Embryos are sensitive to deleterious effects on d 1 following artificial insemination but develop substantial resistance by d 3. Expression of estrous behavior is also depressed when cows become heat-stressed. Prolonged heat stress negatively affected reproduction by increasing estrous cycle length and decreasing duration of estrus (Abilay et al., 1975). A decrease in the frequency of pulsatile release of luteinizing hormone on d 5 of the estrous cycle was observed in heat-stressed cows compared to cooled cows (Wise et al., 1988). Follicular dynamics are altered and follicular dominance is depressed by heat stress (Wolfenson et al., 1995). Furthermore, fetal growth is negatively affected due to decreased uterine blood supply and the insufficiency of the placenta to provide maternal nutrients (Collier et al., 1982).

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HEAT STRESS AND MILK PRODUCTION When cows are subjected to heat stress, feed intake decreases. Simultaneously, maintenance requirements are increased due to activation of the thermoregulatory system. There is need to expend energy to maintain homeothermy that would otherwise be available for useful production (e.g. milk; Buffington et al., 1983). Mild to severe heat stress in dairy cattle has been estimated to cause an increase in maintenance requirements by 7 to 25% (NRC, 2001). By definition, heat-stressed cows are in a state of negative energy balance (NEBAL) since feed intake is not meeting energetic demands of maintenance and lactation. Decreased intake accounts for approximately 36% of the decrease in milk production due to shifts in postabsorptive metabolism and nutrient partitioning (Rhoads et al., 2009). Under thermoneutral conditions, cows experiencing NEBAL have increased rates of lipolysis. This is characterized by the presence of elevated plasma nonesterified fatty acid (NEFA) concentrations, while glucose is partitioned to the mammary gland for milk synthesis. However, heat-stressed cows have lower NEFA concentrations and a higher rate of peripheral glucose utilization, suggesting that glucose uptake by other tissues reduces the amount of glucose available for milk synthesis (Rhoads et al., 2009). A reduction in feed intake precedes a decrease in milk production when cows are subjected to heat stress (Rhoads et al., 2009). Spiers et al. (2004) showed that feed intake decreased within 1 d after initiation of heat stress, while milk yield decreased after d 2 of heat stress. Collier et al. (1981) demonstrated that maximum decrease in milk yield during heat stress occurs 48 hours after the initiation of the stress. Prolonged thermal stress negatively impacts somatotropin (growth hormone or GH) secretion from the anterior pituitary (Mitra et al., 1972). Depressed GH concentrations result in slower growth rates, reduced nitrogen retention, and contribute to decreased lactation performance in dairy cattle (Mitra et al., 1972). Johnson et al. (1963) reported that milk yield decreased by 4 lbs/d (1.8 kg/d) per cow for every 1 F (0.55 C) increase above a daily rectal temperature of 101.5 F (38.6 C). More recently, Igono et al. (1985) reported that a cow with a mean rectal temperature of 102.4 (39.1 C) produced 1.54 lbs/d (0.7 kg/d) less milk than a cow with a rectal temperature of 101.8 F (38.8 C). Zimbelman et al. (2009) also reported a negative relationship between rectal temperature and milk production. This is relationship is further complicated with higher internal heat production in high producing cows compared to low producing cows, regardless of environmental influence (Purwanto et al., 1990). HEAT STRESS AND PRODUCTION EFFICIENCY There are a number of behavioural, physiological and metabolic mechanisms which are employed by the cow to keep CBT above environmental temperature. Some of these are shown in Figure 1.

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Figure 1. Schematic of the general effects of heat stress in dairy cows. (Adapted from Atrian and Shahryar, 2012).

Energy production and expenditure through cellular maintenance produces excess metabolic heat. Thus, heat exchange from the animal to environment is necessary to maintain optimal CBT (Kadzere et al., 2002). A negative correlation between metabolic hormones (thyroid hormones, somatotropin, prolactin, etc.) has been reported (Mitra et al., 1972; Johnson et al., 1988; Lu 1989; Collier et al., 2006). These hormones are responsible for energy expenditure and heat production, including gut motility and blood flow to the digestive system (Hales et al., 1984; Johnson et al., 1988). A decrease in gut motility leads to slower passage rate, decreasing feed intake. West (2003) reported a 1.9 lbs (0.85 kg) decrease in dry matter intake with every 1.8 F (1 C) increase in ambient temperature above a cows thermal neutral zone. While digestion is improved, the lowered amount of feed within the digestive system is unable to meet requirements (Kadzere et al., 2002), decreasing feed efficiency. Physiological mechanisms which improve heat dissipation also lead to an increase in maintenance requirements because of an increase in nutrient needs. Examples include increased respiration rate, increased sweating, increased heart rate, and increased salivation (Atrian and Shahryar, 2012). These in turn lead to increased body fluid loss which further increases maintenance requirements to abate dehydration and blood homeostasis (Collier et al., 2006). While these actions may seem futile, 15% of total body heat loss can be realized through normal respiration (McDowell et al., 1976), and increased respiration rate can and does increase heat loss potential (Campos Maia, et al., 2005). Together, the cows adaptation to minimizing heat production and maximizing heat dissipation leads to economic issues. Milk production will decrease, but energy and nutrient usage by the cow will increase. With an increase in maintenance requirements compounded by a decrease in dietary nutrients, nutrients are diverted from systems not necessary for survival. Rhoads and others (2009) reported a significant repartitioning between dietary and body nutrients utilized 71

for milk production during heat stress, with decreased feed intake accounting for only 36% of milk production loss. Smith and others (2008) calculated that at a milk price of $18/cwt ($39.50/100 kg), a 2 to 12 lbs (0.9 to 5.5 kg) drop in milk production per cow per day due to heat stress could cost an operation between $32 [2 lbs/d (0.9kg/d) loss for 90 days] and $324 [12 lbs/d (5.5 kg/d) loss for 150 days]. HEAT STRESS AND HEALTH Other than the physiological issues that arise from heat stress adaptation, there is ample research linking heat stress to particular aspects of an animals health. Specifically, lameness incidence increases with an increase in ambient temperature (Cook et al., 2007). This coincides with the change of seasons as well; lameness prevalence is lower in cool months as compared to warm months (Sanders et al., 2009). These climatic and seasonal effects are also correlated to mastitis (Dohoo and Meek, 1982; Elvinger et al., 1991). Several trials have reported an increase of disease, particularly reproductive issues, during warmer months of the year due to the acceptable environment for pathogens and vectors (Collins and Weiner, 1968; Silanikove, 2000; Kadzere et al., 2002). Death losses also increase with an increase in THI (Vitali et al. 2009). Recent interest has also hinted at the effect of cow behavior on increased risk for locomotive diseases. HEAT STRESS AND COW BEHAVIOR Within the last decade, research efforts have turned to welfare of cattle experiencing heat stress. With an increase in ambient temperature or solar radiation, cattle are more likely to seek shades or other cooling structures (Tucker et al., 2008; Atrian and Shahryar, 2012). This change in behavior, aside from the physiological changes to decrease heat production mentioned above, suggests that dairy cows will also seek micro-environments that have a lower ambient temperature. Furthermore, cattle are more likely to seek optimum environments that have maximized cooling capacity. For instance, a recent study reported cattle were more likely to choose shade over a cooling system directed away from the shade, but were also likely to take advantage of shade that included a cooling system (Anderson et al., 2012). To maximize heat loss regardless of environment, dairy cattle in areas with elevated temperature often stand to increase available surface for heat dissipation (Igono et al., 1987; Anderson et al., 2012, Smith et al., 2012). Even a mild increase in ambient temperature can invoke an increase in standing time (Smith et al. 2012). Highest incidence of lameness (new cases) occurs when cattle stand longer than 45% of the day (Galindo and Broom, 2000), and locomotion scores increase during summer months relative to winter months (Cook et al., 2007). A negative correlation between time spent lying and incidence of lameness as well as time spent lying and temperature humidity index has also been reported (Leonard et al., 1996; Privolo and Riva, 2009). This suggests that cattle exposed to higher temperatures are more likely to stand to improve heat dissipation but are also more likely to experience periods of lameness during the same time frame. Reducing resting time has been reported to reduce milk production (Bach et al., 2008, Grant 2007). It was estimated that for each hour of increased resting time that milk production increased 3.7 lbs (1.7 kg).

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The effects of lameness on dairy production are compounded by and just as disconcerting as heat stress effects. Lameness affects resting and feeding behavior (Cook et al., 2007), decreases reproduction efficiency (Garbarino et al., 2004), and increases the likelihood for early removal from the herd (Collick et al., 1989). In hot climates, cattle are forced to risk lameness or risk overheating. CBT AND COW BEHAVIOR To further our understanding of behavioral conditions of heat stressed dairy cows, we combined three different data sets from heat stress trials conducted in Arizona (Anderson et al., 2012), California (S. Rungruang, unpublished), and Minnesota (Smith et al., 2012). In each trial, lactating dairy cows were fitted with 2 data loggers: one recorded CBT intra-vaginally, and one recorded angle of leg to determine lying status. All data were standardized to 5-minute intervals for CBT or 15-minute intervals for ambient conditions, and 2 hours per each milking period were removed to eliminate human interference and subsequent feeding. While mild temperatures in the Minnesota trial resulted in higher lying CBT, the 2 other climates revealed greater incidence of heat stress and higher standing CBT compared to lying CBT (Figure 2). Table 1 shows the narrow CBT range (0.11 F or 0.06 C) of cattle standing compared to cattle lying. Altogether, CBT during posture shift (lying to standing or standing to lying) was equal (Table 2), suggesting that dairy cattle may be cognizent of their fluctuating CBT and are reacting preemptively to battle a dramatic shift in CBT, regardless of time of day (Figure 3). Figure 2. Cumulative core body temperature relation to posture in lactating dairy cows. Treatments are designated as follows: 1 = Arizona with fixed fans and misters under drylot shade; 2 = Arizona with adjustable fans and misters under drylot shade; 3 = Minnesota within a cross-ventilated building; 4 = Minnesota within a cross-ventilated building with evaporative pads; 5 = California with feed-line soakers and fans; and 6 = California with hydrothermally cooled freestalls without feed-line soaking or fans. A treatment effect is observed (P < 0.0001). Columns within treatment with different letter designations differ (P < 0.01).

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Table 1. Core body temperature of lactating dairy cows in relation to posture 1 Posture Item: Standing Lying SEM P Value Core Body Temperature, F 103.834 101.912 0.0025 0.0001 (C) (39.908) (38.840) (0.0014) 1 Data is representative of cows in Arizona (n = 56), California (n = 37), and Minnesota (n = 64). Table 2. Core body temperature of lactating dairy cows in relation to posture 1 Posture Item: Continuance Continuance of SEM Initial of Stand Initial Lying Stand Lying Core Body Temperature, F 101.916b 101.847c 101.917b 0.0085 a 102.144 (C) (38.842) (38.804) (38.843) 0.0047 (38.969) a,b,c Letters in the same row with a different superscript differ ( P < 0.0001). 1 Initial lying is representative of the period in which the animal has transitioned from a standing to lying posture; continuance of lying is representative of the period after the animal has initially lied down. Data is representative of cows in Arizona (n = 56), California (n = 37), and Minnesota (n = 64). Figure 3. Effect of period of day on core body temperature of standing and lying bouts (Period effect: P < 0.01)

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An analysis of the entire data set (> 260,000 data points) provided a correlation (r 2 = 0.56, P < 0.0001) between CBT and cow posture, giving researchers and producers a more specific CBT in which to focus their attention when managing dairy cattle for heat stress and cow comfort. In this data set cattle were more likely to be standing at a CBT greater than 38.93 C (102.07 F; Figure 4). This does not suggest that cows with a CBT below this mark do not experience heat stress. However, with increased standing behavior as an indicator of heat stress, this CBT provides a point at which to improve our management efforts to alleviate the negative affects of heat stress, particularly in decreasing the amount of time a cow stands to dissipate body heat. Figure 4. Percent of animals standing in relation to core body temperature. Data is representative of cows in Arizona (n = 56), California (n = 37), and Minnesota (n = 64).

Data using THI as a predictor of stance were limiting in predictive power (r 2 < 0.20). This may be in part to the diminished number of THI data points (< 90,000) as compared to the CBT data points (> 260,000). However, the impact of THI on cattle behavior is measurable (Table 3) and appears to follow the same pattern as CBT. Although not specified, the 50 percent mark would occur just above a THI of 71, which is just above a THI of 68, the established threshold where heat stress begins in high producing dairy cows (Zimbelman et al., 2009).

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Table 3. Percent of cattle standing within THI categories1 THI Category2 80 to 89 90 to 98

Item: < 68 68 to 71 72 to 79 > 100 SEM Animals Standing, % 43.6 49.8 53.0 68.2 49.7 52.2 1.6 1 P < 0.0001. Data is representative of cows in Arizona (n = 56), California (n = 37), and Minnesota (n = 64). 2 Categories defined as thermal neutral (< 68), heat stress threshold (68 to 71), mild-moderate heat stress (72 to 79, moderate-severe heat stress (80 to 89), severe heat stress (90 to 98), and extremely severe heat stress (> 100). SUMMARY Heat stress is a major economic issue in the dairy industry. Its effects reach beyond milk production into reproduction, health, and welfare arenas through physiological and behavioral changes. Modifications to cow behavior are linked to overall production performance and should not be over-looked. Improving the cows comfort by reducing the amount of time it stands to dissipate heat can ultimately reduce the affect of heat stress on milk production. LITERATURE CITED Abilay, T. A., H. D. Johnson, and M. Madan. 1975. Influence of environmental heat on peripheral plasma progesterone and cortisol during the bovine estrous cycle. J. Dairy Sci. 58:1836-1840. Anderson, S. D., B. J. Bradford, J. P. Harner, C. B. Tucker, C. Y. Choi, J. D. Allen, L. W. Hall, S. Rungruang, E. Rajapaksha, R. J. Collier, and J. F. Smith. 2012. Effects of adjustable and stationary fans with misters on core body temperature and resting behavior of lactating dairy cows in a semi-arid climate. J. Dairy Sci. (Submitted) Armstrong, D. V. 1994. Heat stress interactions with shade and cooling. J. Dairy Sci. 77:20442050. Atrian, P., and H. A. Shahryar. 2012. Heat stress in dairy cows (a review). Res. in Zoology. 2:31-37. Bach, A., N. Valls, A. Solans, and T. Torrent. 2008. Associations between nondietary factors and dairy herd performance. J. Dairy Sci. 91:3259-3267. Badinga, L., R. J. Collier, W. W. Thatcher, and C. J. Wilcox. 1985. Effects of climatic and management factors on conception rate of dairy cattle in subtropical environment. J. Dairy Sci. 68:78-85. Berman, A. J. 2005. Estimates of heat stress relief needs for Holstein dairy cows. J. Anim. Sci. 83:1377-1384. Berry, I. L., M. D. Shanklin, and H. D. Johnson. 1964. Dairy shelter design based on milk production decline as affected by temperature and humidity. Trans. Am. Soc. Ag. Eng. 7:329-331. Buffington, D. E., R. J. Collier, and G. H. Canton. 1983. Shade management systems to reduce heat stress for dairy cows in hot, humid climates. Trans. Am. Soc. Agric. Eng. 26:1798:1803. 76

Campos Maia, A. S., R. Gomes DaSilva, and C. M. Battiston Loureiro. 2005. Respiratory heat loss of Holstein cows in a tropical environment. Int. J. Biometerol. 49:332-336. Collick, D. W., W. R. Ward, and H. Dobson, 1989. Associations between types of lameness and fertility. Veterin. Rec. 125:103-106. Collier, R. J., G. E. Dahl, and M. J. VanBaale. 2006. Major advances associated with environmental effects on dairy cattle. J. Dairy Sci. 89:1244-1253. Collier, R. J., D. K. Beede, W. W. Thatcher, L. A. Israel, and C. J. Wilcox. 1982. Influences of environment and its modification on dairy animal health and production. J. Dairy Sci. 65:2213-2227. Collier, R. J., R. M. Eley, A. K. Sharma, R. M. Pereira, and D. E. Buffington. 1981. Shade management in subtropical environment for milk yield and composition in Holstein and Jersey cows. J. Dairy Sci. 64:844-849. Collins, K. H., and H. S. Weiner. 1968. Endocrinological aspects of exposure to high environmental temperature. Physiol. Rev. 48:785-794. Cook, N. B., R. L. Mentink, T. B. Bennett, and K. Burgi. 2007. The effect of heat stress and lameness on time budgets of lactating dairy cows. J. Dairy Sci. 90:1674-1682. Dohoo, I. R., and A. H. Meek. 1982. Somatic cell counts in bovine milk. Can. Vet. J. 23:119125. Ealy, A. D., M. Drost, and P. J. Hansen. 1993. Developmental changes in embryonic resistance to adverse effects of maternal heat stress in cows. J. Dairy Sci. 76:2899-2905. Elvinger, F., P. J. Hansen, and R. P. Natzke. 1991. Modulation of function of bovine polymorphonuclear leukocytes and lymphocytes by high temperature in vitro and in vivo. Am. J. Vet. Res. 52:1692-1698. Galindo, F., and D. M. Broom. 2000. The relationships between social behavior of dairy cows and the occurrence of lameness in three herds. Res. Vet. Sci. 69:75-79. Garbarino, E. J., J. A. Hernandez, J. K. Shearer, C. A. Rixco, and W. W. Thatcher. 2004. Effect of lameness on ovarian activity in postpartum Holstein cows. J. Dairy Sci.87:41234131. Grant, R. 2007. Taking advantage of natural behavior improves dairy cow performance. Pages 225-236 in Proc. Western Dairy Management Conf., Reno, NV. Gwazdaukas, F. C., W. W. Thatcher, and C. J. Wilcox. 1973. Physiological, environmental, and hormonal factors at insemination which may affect conception. J. Dairy Sci. 56:873877. Hales, J. R. S., A. W. Bell, A. A. Fawcett, and R. B. King. 1984. Redistribution of cardiac output and skin AVA activity in sheep during exercise and heat stress. J. Therm. Biol. 9:113-116. Igono, M. O., H. D. Johnson, B. J. Steevens, G. F. Krause, and M. D. Shanklin. 1987. Physiological, productive, and economic benefits of shade, spray, and fan system versus shade for Holstein cows during summer heat. J. Dairy Sci. 88:2454-2461. Igono, M. O., B. J. Steevens, M. D. Shanklin, and H. D. Johnson. 1985. Spray cooling effects on milk production, milk and rectal temperatures of cows during a moderate summer season. J. Dairy Sci. 68:979-985. Johnson, H. D., P. S. Katti, L. Hahn, and M. D. Shanklin. 1988. Short-term heat acclimation effects on hormonal profile of lactating cows. In: Research Bulltetin No. 1061. University of Missouri, Columbia.

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Johnson, H. D., A. C. Ragsdale, I. L. Berry, and M. D. Shanklin. 1963. Temperature-humidity effects including influence of acclimation in feed and water consumption of Holstein cattle. Missouri Agr. Exp. St. Res. Bul. 846. Kadzere, C. T., M. R. Murphy, N. Silanikove, and E. Maltz. 2002. Heat stress in lactating dairy cows: a review. Livestock Prod. Sci. 77:59-91. Leonard, F. C., J. M. OConnell, and K. J. OFarrell. 1996. Effect of different housing conditions on behavior and foot lesions in Friesian heifers. Veterin. Rec. 134:490-494. Lu, C. D. 1989. Effect of heat stress on goat production. Small Rumin. Res. 2:151-162. McDowell, R. E., N. W. Hooven, and J. K. Camoens. 1976. Effects of climate on performance of Holsteins in first lactation. J. Dairy Sci. 59:965-973. Mitra, R. G., G. I. Christison, and H. D. Johnson. 1972. Effect of prolonged thermal exposure on growth hormone (GH) secretion in cattle. J. Anim. Sci. 34:776-779. NRC, National Research Council. 2001. Nutrient Requirements of Dairy Cattle. 7th rev. ed. Natl. Acad. Sci., Washington, DC. Privolo, G., and E. Riva. 2009. One year study of lying and standing behaviour of dairy cows in a freestall barn in Italy. J. Ag. Eng. 2:27-33. Purwanto, B. P., Y. Abo, R. Sakamoto, F. Furumoto, and S. Yuamamoto. 1990. Diurnal patterns of heat production and heart rate under thermoneutral conditions in Holstein Friesian cows differing in milk production. J. Agric. Sci. 114:139-142. Rhoads, M. L., R. P. Rhoads, J. J. VanBaale, R. J. Collier, S. R. Sanders, W. J. Weber, B. A. Crooker, and L. H. Baumgard. 2009. Effects of heat stress and plane of nutrition on lactating Holstein cows: I. Production, metabolism, and aspects of circulating somatropin. J. Dairy Sci. 92:1986-1997. Roenfeldt, S. 1998. You cant afford to ignore heat stress. Dairy Manage. 35:6 -12. Shearer, J. K., and D. K. Beede. 1990. Thermoregulation and physiological responses fo dairy cattle in hot weather. Agri-Practice 11:5-17. Sanders, A. H., J. K. Shearer, and A. De Vries. 2009. Seasonal incidence of lameness and risk factors associated with thin soles, white line disease, ulcers, and sole punctures in dairy cattle. J. Dairy Sci. 92:3165-3174. Silanikove, N. 2000. Effects of heat stress on the welfare of extensively managed domestic ruminants. Livestock Prod. Sci. 67:1-18. Smith, J. F., J. P. Harner, B. J. Bradford, K. C. Dhuyvetter, and M. Overton. 2008. Opportunities with low profile cross ventilated freestall facilities. Proceedings of the 2008 Housing of the Future, Sioux Falls, SD. Smith, J. F., B. J. Bradford, J. P. Harner, K. Ito, M. von Keyserlingk, C. R. Mullins, J. C. Potts, J. D. Allen, and M. W. Overton. 2012. Effect of cross ventilation with or without evaporative pads on core body temperature and resting time of lactating cows. J. Dairy Sci. (Submitted) Spiers, D. E., J. N. Spain, J. D. Sampson, and R. P. Rhoads. Use of physiological parameters to predict milk yield and feed intake in heat-stressed dairy cows. J. Therm. Biol. 29:759764. St-Pierre, N. R., B. Cobanov, and G. Schnitkey. 2003. Economic losses from heat stress by US livestock industries. J. Dairy Sci. 86:E52. Thatchet, W. W. 1974. Effects of season, climate, and temperature on reproduction and lactation. J. Dairy Sci. 57:360-368.

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Tucker, C. B., A. R. Rogers, and K. E. Shutz. 2007. Effect of solar radiation on dairy cattle behaviour, use of shade and body temperature in a pasture-based system. Appl. Anim. Behav. Sci. 109:141-154. Ulberg, L. C., and P. J. Burfening. 1967. Embryo death resulting from adverse environment on spermatozoa or ova. J. Anim. Sci. 26:571-577. Vitali, A., M. Segnalini, L. Bertocchi, U. Bernabucci, A. Nardone, and N. Lacetera. 2009. Seasonal pattern of mortality and relationships between mortality and temperaturehumidity index in dairy cows. J. Dairy Sci. 92:3781-3790. West, J. W. 2003. Effects of heat-stress on production in dairy cattle. J. Dairy Sci. 86:21312144. Wise, M. E., D. V. Armstrong, J. T. Huber, R. Hunter, and F. Wiersma. 1988. Hormonal alterations in the lactating dairy cow in response to thermal stress. J. Dairy Sci. 71:2480-2485. Whittier, J. C. 1993. Hot weather livestock stress. Univ. Missouri. Ext. Bull. G2099. Mt. Vernon. Wolfenson, D., W. W. Thatcher, L. Badinga, J. D. Savio, R. Meidan, B. J. Lew, R. Braw-Tal, and A. Berman. 1995. Effect of heat stress on follicular development during the estrous cycle in lactating dairy cattle. Biol. Repro.52:1106-1113. Zimbelman, R. B., R. P. Rhoads, M. L. Rhoads, G. C. Duff, L. H. Baumguard, and R. J. Collier. 2009. A re-evaluation of the impact of temperature humidity indx (THI) and black globe temperature humidity index (BGHI) on milk production in high producing dairy cows. Proceedings of the 24th Southwest Nutrition and Management conference, Tempe, AZ. pp. 158-168.

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Early Weaning Beef Calves to Improve Herd Productivity: Implications on Cow and Calf Nutrition
J. D. Arthington Range Cattle Research and Education Center, Ona University of Florida IFAS jarth@ufl.edu SUMMARY Early calf weaning is not new to the science of beef cattle production. The benefits of early weaning during instances of environmental strain, usually witnessed by a shortage of summer forage, has been understood and practiced for many years. However, the use of early weaning as a normal, annual management practice for first-calf cows is much less common. Our interest in the topic originated from the common production practice in the Southeast, which allowed heifers to develop for two years before being bred the first time. Calving heifers for the first time at three years of age is quite common across production regions that utilize a high percentage of Bos indicus lineage (i.e. Brahman). Compared to heifers of traditional English lineage (Bos taurus), Brahman-crossbred heifers have been shown to have lower calving rates when developed to calve at 24 months of age (DeRouen and Franke, 1989). The reason relates to the slow rate of maturity common to Bos indicus cattle, witnessed by both delayed onset of puberty (Rodrigues et al., 2002) and increased time required to achieve mature body size (Martin et al., 1992). Nevertheless, heifers that calve for the first time at three years of age have reduced lifetime economic efficiency than those managed to calve at two years of age. Although, calving at three years of age may increase repeatability of pregnancy, forcing all two year olds to remain non-productive an entire year decreases the overall economic efficiency of the cowherd (Nunez-Dominguez et al., 1991). INTRODUCTION In beef production systems, early weaning lacks an appropriate definition. In most regions of the US, beef calves are weaned from their dams at approximately six to eight months of age. Therefore, any calves weaned prior to six months of age may be considered, early weaned. For our purposes, early weaning refers to the permanent separation of the calf from its mother at approximately 90 days of age. This target age is used to ensure that the process of early weaning has an opportunity to impact the reproductive performance of the cow. Calves weaned at four and five months of age may be mistakenly referred to as early weaned, but this is truly a misnomer, as the cow will benefit little reproductively within a fixed breeding season. By this age, the breeding season has likely ended or almost over once the calf is weaned. To harvest the most efficiency from the effort, early weaning should always occur at the start, or very near the start, of the normal breeding season. It has been our experience, and the experience of others (Dr. Ron Lemenager, Purdue University; personal communication) that beef calves should not be weaned when they are less than 50 days of age. When weaning at ages less than 50 days, we have found that calves perform poorly and appear to be stunted, never recovering their normal body weight even many months later. This age threshold is supported by the common age at which dairy calves begin consuming significant amounts of dry feed. Dairy producers begin the 80

transition process from liquid milk to complete dry feeds when calves reach 45 to 50 days of age. Therefore, the target age for early weaning beef calves should be 50 to 90 days of age. THE COW Body Condition Assuming the beef herd is otherwise healthy, nothing impacts cowherd reproductive performance than prolonged post-partum anestrus and nothing impacts post-partum anestrus more than cow body condition. Low cow body condition is the primary reason for reduced conception rates and overall poor cowherd productivity. Cow body condition is a subjective estimate of the amount of fat on a cow and is the most reliable method for evaluating a nutritional program. Body condition typically declines after calving, when the nutritional demands of the cow are at a maximum. It is during this time that supplemental nutrition is most needed. Research from the University of Florida (Rae et al., 1993) has shown that cows with low body condition scores ( 4.0) have a 30% reduction in pregnancy rate compared to co ws in optimum body condition (5.0 to 6.0). The low body condition score cows that do conceive often do so late in the breeding season. This increase in post-partum interval results in later calves the following year. This is most pronounced in young cows, which possess higher nutritional demands to support both lactation as well as their own continued growth. When managing these young cows, producers are faced with a limited number of options, including, 1) provide adequate nutrient-dense supplementation, 2) early weaning, therefore removing the nutritional demands associated with lactation, or 3) breed heifers at 3 years of age when their own growth demands are lessened. We examined the influence of early weaning on first-calf cow body weight and body condition score change over two consecutive years (Arthington and Kalmbacher, 2003). In our study, early weaning resulted in a 2-point increase in cow body condition score compared to contemporary cows nursing their calves up to the time of normal weaning. (Table 1). This difference in body condition score allows the cows to calve in the following year with greater condition, which optimizes their chances to rebreed early resulting in older, heavier calves at weaning the next year.

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Table 1. Effect of early- versus normal-weaning on first-calf heifer BW and body condition (average of two consecutive years) Treatment Item EW NW Heifer BW, lb a January 922 c 955 d d April 988 946 c d August 1,083 997 c d Change 161 42 c b Heifer BCS January 4.28 4.28 April 5.42 d 4.46 c August 6.34 d 4.75 c a Individual heifer BW collected at the time of early weaning (EW; January), when calves came off ryegrass (April), and at the time of normal calf weaning (NW; August) (n = 50 and 58 for EW and NW, respectively). b Heifer body condition score (BCS) recorded as an average of two technicians at each collection date using a 1 to 9 scale (1 = emaciated and 9 = obese). c,d Treatment means within a row without a common superscript letter differ ( P < 0.05). Feed Intake Early weaning may be a practical and profitable management consideration for cow/calf operations. As early-weaned cows begin to stop lactating, their dry matter intake decreases by as much as 30%. Results from our research (Arthington and Minton, 2004) have shown that early-weaned, first-calf cows require approximately 50% less TDN to achieve and maintain a body condition score of 5.0 compared to lactating heifers of the same age and body condition (Figure 1). The intake values represented by these data show the amount of TDN consumed by a lactating first-calf cow, plus her calf, compared to an early-weaned first-calf cow without her calf. These data suggest greater than a 40% improvement in converting TDN into calf gain .

SEM 11.2 9.7 11.0 7.1 0.10 0.11 0.07

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Figure 1. Effect of early calf weaning on voluntary dry matter intake of TDN in first-calf cows. Early-weaned calves were removed on the first d of wk 1.

25

Early Weaned Normal Weaned

TDN intake, lb/d

20

15

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5
1 2 3 4 5 6 7 8 9 In itia l 10

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This voluntary decrease in dry matter intake has important practical implications for the cow/calf producer, whereas the cow can maintain or gain body weight with almost 30% less forage. In one study (Galindo-Gonzalez et al., 2007), the effect of cow parity (primi- versus multiparous) and early weaning on hay intake, cow body weight and condition change, and pregnancy rate was investigated. In that study, there was very little difference in the effect of parity when measuring cow response to early weaning. Our original hypothesis stated that young, primiparous cows would realize a greater production response to early weaning versus mature, multiparous cows when each were compared to normal-weaned contemporaries of a similar parity. This was not the case, as mature cows also experienced a considerable decrease in hay dry matter intake concurrent with an increase in body condition and pregnancy rate. In this study, cows with their calves consumed approximately 18% more hay than early-weaned cows. This value differs from the 30% decrease suggested earlier due to the presence of winter perennial pasture. The hay was a supplement to pasture and pasture forage intake was not measured. The response summary over two yeas (n = 96 cows) for both primi- and multiparous cows is provided in Table 2. Considering a 100 day winter hay supplementation period and hay valued at $100/ton, early weaning can save nearly $12 per cow in hay costs alone. This production efficiency estimate does not take into account the value of increased pregnancy rate and decreased post-partum interval, which are the primary benefits realized by early weaning.

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Table 2. Effect of early weaning on supplemental hay intake and performance of beef cows wintered on perennial bahiagrass pasture (average of two years; n = 96 cows). Item Early-weaned Normal-weaned SEM Hay intake, lb/cow/d a 14.2 16.7 0.5 Body condition change a,b + 0.5 - 0.5 0.08 Pregnancy rate, % 80.4 69.6 ----a Calves early weaned at an average age of 85 days. Hay intake and body condition change calculated for 75 days after early weaning, after which all cows were exposed to mature bulls as a single group for 45 days. b Body condition scored on a 1 to 9 scale (1 = emaciated and 9 = obese). Reproductive Performance The removal of a calf from a post-partum, anestrous cow results in an endocrine response initiating estrus. The common management system involving a 48-hour calf removal is targeted at initiating this response by removing the suckling stimuli. Early calf weaning creates the same scenario, but in this case the calf is not returned to the dam. In one study, we evaluated the postpartum interval of thin, first-calf heifers which were early-weaned or allowed to remain with their calf (Arthington and Minton, 2004). We fed each heifer individually to ensure similar amounts of body weight gain over 70 days. By the end of the feeding period, cows from both treatments gained a similar amount of body weight and body condition; however, more earlyweaned cows were cycling compared to normal-weaned contemporaries (Figure 2). These data suggest that the calf-removal response is an important factor affecting post-partum anestrus, independent of nutrition. Figure 2. Effect of early calf weaning on post-partum cyclicity of first-calf heifers. Earlyweaned calves were removed on the first d of wk 1. Date of return to estrus was determined as the first wk when progesterone concentrations were greater than 1 ng/mL for two consecutive weekly samples.
100 90 80 70 60 50 40 30 20 10 0
1 In itia l

P= < 0.01 < 0.01 0.23

** *

**

**

**

**

**

Cows cycling, %

Early Weaned Normal Weaned

Weeks following early weaning

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10

In some situations, producers may be unable or unwilling to permanently separate beef calves at these early ages. Recently, we compared the reproductive effects of 5 consecutive 48-h calf withdrawals (20 d apart) to permanent calf withdrawal (early weaning) and a traditional single 48-h calf withdrawal (Martins et al., 2012). In that study, first-calf cows that were early-weaned or exposed to multiple calf withdrawals attained post-partum cyclicity at the same rate and sooner than first-calf cows that were submitted to a single 48-h calf withdrawal (Figure 3). Although, the multiple calf withdrawals mimicked permanent separation in terms of hastened post-partum anestrus, cows with unweaned calves had lesser body weight gain and a greater body condition decline compared to early-weaned cows throughout the breeding season. Figure 3. Percentage of cows cycling during the 90-d breeding season. Early-weaned cows had their calves permanently removed at the start fo the breeding season (day 0); controlcows had their calves removed for 48 h once at the start of the breeding season; interval-weaned cows had their calves removed for 48 h five times, 20 d apart throughout the breeding season. a,b; P < 0.05.
100 90 80 70 Early-weaned Interval-weaned Control
a a a a

a
a

Percent cycling

60 50 40 30 20
ab
b

b b

a a
ab b b

10 0 0 10
b

20

30

40

50

60

70

80

90

Day

In any given year, the majority of cows in a producers non -pregnant category are heifers and young cows. The use of early weaning will allow these females to regain their lost body condition, and do so with less forage and supplemental feed. As well, the decrease in postpartum interval means these females will become pregnant earlier in the breeding season and produce calves that will be older and heavier at next years weaning. In a two -year study, investigating two-year old first calf heifers, we reported a greater pregnancy rate and a 21-day shorter calving interval in early-weaned versus normal-weaned cows of a similar age (89.5 versus 50.0 % pregnant for early- and normal-weaned cows, respectively; Arthington and Kalmbacher, 2003). The greatest economic advantage of early calf weaning is realized through increased pregnancy rate of otherwise anestrous cows. Although our data suggests that early weaning also improves the performance of mature cows, the major advantage to the system is allowing heifers to be bred as yearlings, calve at two-years of age without suffering losses in body weight and poor subsequent fertility as a lactating two-year old.

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THE EARLY-WEANED CALF Nutritional Management Depending on the region of production, producers may or may not have forage resources to graze early-weaned calves. Some of the research studying the effects of early weaning on calf productivity in the mid-west and high-plains regions of the US have focused on drylot feeding of the early-weaned calves. In warmer climates, producers may be able to graze calves on perennial or annual pastures throughout the year. In our experiences, opportunities to rear earlyweaned calves on high-quality pasture forage provide an important value toward the costs of maintaining an early-weaned calf. Early-weaned calves respond favorably to supplemental concentrate, even when they have are grazing highly nutritious pastures, such as annual winter ryegrass (Figure 4). In a study investigating the performance of early-weaned calves grazing winter ryegrass pastures (Vendramini et al., 2006), voluntary forage intake decreased and calf ADG increased as the rate of supplementation increased from 1.0, 1.5, and 2.0% of body weight (Table 3).

Table 3. Performance of early-weaned calves grazing winter rye-ryegrass pastures and supplemented with different levels of concentrate. a Item Concentrate, % BW SEM Response 1.0 1.5 2.0 Average daily gain, lb/d 1.63 1.79 1.96 0.07 Linear Forage OM intake, % BW b 1.8 1.3 1.1 0.01 Linear a Forage organic matter intake determined on grazing calves by the use of a sustained release bolus containing an indigestible. P= < 0.05 < 0.01

Recently, we evaluated the effects of 3 different early-weaned calf management systems on measures of calf performance and economic return. In this study (unpublished data), calves were early weaned at approximately 70 days of age and reared in 1 of 3 management systems for 84 days. The systems included; 1) grazing dormant winter perennial grass pasture (Bahiagrass) with 2% body weight concentrate supplementation, 2) Drylot with concentrate limit-fed at 3.5% body weight, or 3) Annual pasture grazing (Ryegrass) with 1% body weight supplementation. Our findings show that although greater body weight gain can be achieved with drylot rearing on concentrate diets, the efficiency of this gain is lacking when compared to calves grazing high-quality pasture with supplement (Table 4). Despite the method of calf management system adopted, each of these systems produced profitable performance results. Beef producers adopting early weaning systems with their young cows should consider harvesting the value of efficient feed conversions of these young calves by designing a 3 to 4 month rearing system that best fits their local resources.

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Table 4. Evaluation of management systems for the rearing of earlyweaned calves.1 Treatment1 Drylot

Item Bahiagrass Ryegrass SEM BW, lb d0 197 a 215 b 225 b 7.4 a b d 28 245 276 270 b 8.9 d 56 269 a 342 b 322 b 10.1 d 84 336 a 417 b 367 c 10.9 ADG, lb/d d 0 d 28 1.94 a 2.27 b 1.66 a 0.125 a c d 28 d 56 0.87 2.36 1.89 b 0.141 d 56 d 84 2.29 a 2.58 c 1.56 d 0.136 d 0 d 84 1.69 a 2.40 b 1.70 a 0.080 Feed Intake, lb/calf daily d 0 d 28 4.4 a 8.8 b 2.8 c 0.13 a b d 28 d 56 5.0 12.6 3.5 c 0.22 d 56 d 84 6.0 a 12.4 b 4.2 c 0.19 d 0 d 84 5.1 a 11.3 b 3.5 c 0.15 Economics Feed cost, $ BW gain 2 0.59 a 0.95 b 0.41 c 0.022 3 a b Calf value, $/calf 481 565 525 c 10.2 Return, $/calf 4 397 a 376 b 466 c 9.6 1 Calves ealy-weaned at approximately 70 days of age. 2% = calves grazing bahiagrass pastures and supplemented with concentrate at 2% of BW (n = 5 calves/pasture; 4 pastures); Feedlot = calves in drylot and limit-fed concentrate at 3.5% of BW (n = 5 calves/pen; 4 pens); Ryegrass = calves grazing ryegrass pastures and supplemented with concentrate at 1.0% of BW (n = 4 calves/pasture; 4 pastures). 2 Feed cost ($/calf) divided by total BW gain of each pen. 3 $1.40 per lb of calf BW. 4 Return = Calf value ($/calf) - feed cost ($/calf). In Florida, our fall-born, early-weaned calf management systems involve the establishment of winter annual ryegrass within Calf Nurseries for the rearing of calves. Over the past 10 years, we have grazed early-weaned calves at an average stocking rate of four to six calves/acre. Despite both dry and wet winters this stocking rate has proven acceptable. Optimal stocking rate should be defined as the rate which best utilizes the available forage for maximum animal body weight gain. On non-irrigated land, this target rate is highly dependent upon the amount of effective precipitation received during pasture establishment. Over six consecutive years, we have found a great deal of variability among ryegrass yield and calf performance (Table 5); however, a stocking rate of four to six calves per acre has proven to be acceptable to achieve rates of body weight gain similar to or greater than the gain achieved by non-weaned calves of a similar age. In each of the studies reported in Table 5, early-weaned calves were provided supplemental concentrate feed at a rate of 1% of body weight. Although we utilize annual ryegrass or rye-ryegrass blends in our Florida early-weaned calf nurseries, this system will not be practical for all regions of the country. For temperate regions of the United States, other grass 87

varieties should be considered. It is important to note that high-quality forage varieties that may not be tolerant to cow grazing may work well in an early-weaned calf grazing system. Young calves are much gentler on the pasture, consuming forage much like a deer or goat. As well, because the calves are smaller their dry matter intake is much less than a mature cow; therefore, moderate yielding, high-quality forages may be good candidates for use in an early-weaned calf nursery. In our system, a major shortcoming of the management of an early-weaned calf occurs once the winter annual ryegrass dies out in the spring. Once early-weaned calves are moved onto perennial, summer pastures their performance declines rapidly. Our annual ryegrass is grazed out by early to mid-May, leaving a 100 day deficit period until the time of normal weaning (early August). In our studies, performance of our early-weaned calves drops by an average of 25% in the summer versus winter periods. Although performance in the winter is similar among early-and normal-weaned calves, performance in the summer period is usually inferior for the early-weaned calf compared to those left with their dams. This decline in performance often results in a greater overall ADG for normal-weaned compared to early-weaned calves when calculated from January (time of early weaning) to August (time of normal weaning). We attribute this decline in summer performance to the lesser digestibility of our summer perennial pastures compared to the winter annual ryegrass (Figure 4). For Florida producers, these data would support the marketing of early weaned calves in late April or early May. Historically, calf markets are at their greatest at this time of the year. Consideration of regional variation in forage quality, quantity, and annual trends in market value should be considered when determining the optimal marketing time for early-weaned calves. Table 5. Performance of early weaned calves in both winter and summer grazing seasons over six consecutive years (average daily body weight gain stnd. dev.). a Stocking rate, calves/acre Winter Summer 2000 1.89 0.04 1.21 0.07 3.3 3.3 2001 2.08 0.06 ------3.3 ------2002 1.35 0.07 1.31 0.18 4.4 2.4 2003 1.60 0.06 1.34 0.06 4.0 1.2 2004 1.73 0.11 1.48 0.05 6.7 2.0 2005 2.15 0.10 ------5.3 ------Average 1.80 0.07 1.34 0.09 4.7 2.2 a Calves are approximately 60 to 90 days of age at the time of early weaning. All calves are provided supplemental feed at a target rate of 1.0% of body weight during both grazing seasons. A commercial feed (14 and 65 % CP and TDN, respectively) was utilized in 2000, 2001, 2002, and 2003 and a commodity blend of soybean hulls and cottonseed meal (85:15) was used in 2004 and 2005. b Winter and summer grazing periods each are approximately 100 days. Winter grazing always occurred on annual ryegrass. Ryegrass was typically fertilized twice using a complete fertilizer, once upon emergence and again approximately 50 days into grazing. Summer grazing consisted of established limpograss in 2000 (Arthington and Kalmbacher, 2001) and established stargrass in all other years. 88 Year Winter grazing b Summer grazing b

Figure 4. Average ryegrass an stargrass quality over two consecutive seasons (2003 and 2004). Average stocking rate = 1.6 calves/acre. Average SEM = 0.65 and 0.29, and 1.37 and 1.27 for ryegrass and stargrass crude protein and IVOMD, respectively.

Crude protein, % DM basis

25 20 15 10 5 0 90 80 70 60 50 40

IVOMD, % DM basis

Ja nu Fe ary br ua r M y ar ch A pr il M ay Ju ne Ju A ly ug us t

---------------

Ryegrass Stargrass

Feedlot Performance of Early-Weaned Calves Early weaning also has positive implications on the value of calves post-weaning. Researchers from the University of Illinois (Myers et al., 1999) investigated the effect of early weaning on carcass merit. In their studies, they reported that early weaning improved the percentage of calves grading USDA Choice or higher by over 30% compared to normal-weaned calves. In a comparison of weaning age (90, 150, or 210 days), they found that calves weaned at 90 days tended to produce higher quality carcasses. In many ranch settings, normal-weaned calves are shipped immediately after separation from the cow. When shipped as a complete group (not commingled) these calves typically perform well, nevertheless, buyers often discount fresh-weaned calves due to the potential for stressrelated disease. The use of early weaning, followed by growing period of 60 to 90 days, produces calves that have recovered from the stress of weaning and understand how to eat. Once received into the feed yard, these calves are likely to have fewer incidences of illness. In a study conducted in collaboration with our program and North Carolina State University, we examined the productivity of early- versus normal-weaned calves in the feedlot (Arthington et al., 2005). In that study, early-weaned calves were lighter at the time of normal weaning (492 versus 611 lb), but gained body weight at a faster rate during the feedlot receiving period (Figure 5). By d 28, body weight was similar (538 versus 617 lb for early- and normal-weaned calves, respectively). Overall, early-weaned calves gained over 1 lb/d more than normalweaned calves (Figure 5), despite no differences in daily feed dry matter intake (Table 6). The most striking response to early weaning in our feedlot study was the significant improvement in feed efficiency (Table 6). We have attributed this response to a lesser 89

Ja nu Fe ary br ua r M y ar ch A pr il M ay Ju ne Ju A ly ug us t

inflammatory reaction in early- versus normal-weaned calves in response to the stressors associated with weaning and transport. During normal stress events the early inflammatory reaction results in the production of acute phase proteins. In our study, early-weaned calves had a lesser acute phase protein response following transport and entry into the feedlot. Further, a relationship between plasma acute phase protein concentrations and daily body weight gain was observed in normal-weaned steers during the feedyard receiving period, whereas average ceruloplasmin concentrations were negatively associated with body weight gain in normalweaned (P < 0.01; R2 = 0.59), but not early-weaned (P > 0.05; R2 = 0.21) calves. Similarly, average haptoglobin concentrations were negatively associated with body weight gain in normal-weaned (P < 0.01; R2 = 0.40), but not early-weaned (P > 0.05; R2 = 0.10) calves. Other researchers have shown that feeder calf plasma haptoglobin concentrations, upon entry into the feedlot, are positively associated with the incidence of morbidity and subsequent number of medical treatments required (Berry et al., 2004; Carter et al., 2002). Table 6. Effects of early- versus normal weaning age on calf feedlot performance a Periodb Early-weaned Normal-weaned SEM c P= Receiving ADG, lb/d 1.92 0.88 0.22 0.03 DMI, lb/d 12.5 11.6 0.62 0.36 G:F 0.154 0.076 0.010 0.01 Growing ADG, lb/d 3.04 2.60 0.11 0.05 DMI, lb/d 19.4 19.6 0.77 0.84 G:F 0.157 0.133 0.006 0.06 Finishing ADG, lb/d 3.02 2.91 0.12 0.77 DMI, lb/d 19.2 20.2 0.64 0.33 G:F 0.157 0.144 0.007 0.35 Overall ADG, lb/d 2.71 2.76 0.24 0.82 Total BW gain, lb 650 589 20.5 0.10 Total DMI, lb 4,231 4,357 165.2 0.62 G:F 0.154 0.135 0.004 0.02 a Early-weaned calves were removed from their dams at 85 d of age. Normal-weaned calves remained with their dams until the day of normal weaning (average age = 300 d). b Receiving diet = d 0 to 28; Growing diet = d 28 to 112; and Finishing diet = d 112 to Table values are least square means. ADG = average daily body weight gain. c Largest SEM of least square means (n = four pens/treatment).

90

Figure 5. Percent change in body weight relative to weaning weight for early- and normalweaned calves. Calves were shipped during the first week in August. Early-weaned calves were weaned (early January) and retained on the ranch of origin until the time of normal weaning. Normal weaned calves were shipped the day of weaning. (Arthington et al., 2005).
Early weaned Normal weaned
Change in BW relative to BW at normal weaining, %

15 10 5 0 -5 -10 1 4 7 10 13 16 19 22 25 28

* *

70 60 50 40 30 20 * 10 0 28

* * *

40

52

64

76

88 100 112

Day in feedyard (Receiving period)

Day in feedyard (Growing period)

General Healthcare of the Early-Weaned Calf One common question related to weaning calves at this young age is health status. It is understandable that one would be concerned with the viability of calves of this age. In fact, ranch-derived calves at 70 to 90 days of age have a very high health status. This is related to the passive immunity that they obtained from their mothers through colostrum. This colostrum provides important immunity to calves of this age. In comparison, calves of normal weaning age (6 to 8 months) have little to no remaining passive immune protection. If normal-weaned calves are not properly vaccinated they will be more susceptible to succumbing to disease at the time of weaning compared to 70 to 90 day old early-weaned calves. We do not recommend vaccinating calves at the time of early weaning, as the vaccine will likely be neutralized by the calfs own passive immunity. Early-weaned calves should be vaccinated according to the same schedule used for the normal-weaned calves in the herd. One exception to this rule relates to producers that may gather early-weaned from multiple sources. In this situation, the producer often does not know the health status of the herds from which the calves are sourced. Further, the stress of transport and commingling may elicit the onset of disease. In these situations the producer should work with their veterinarian to develop a health-care plan that will take into consideration the balance between disease pressure and immune protection. One important difference that we have noticed in early-weaned calves is their susceptibility to internal parasites. We typically treat our early-weaned calves for internal parasites twice during the 200-day grazing period. By following this management schedule, we have noticed significant improvements in calf body weight gain following anthelmetic treatment.

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SUMMARY Early weaning must occur prior to the start of the breeding season to gain the full reproduction benefits associated with this management practice. a. Calves should not be less than 50 days of age at the time of early weaning. b. Breed yearling heifers 30 days before the mature cowherd so that the calves will be old enough to early wean at the start of the regular breeding season in the following year. Early-weaned cows will voluntarily consume approximately 30% less dry matter following early weaning. Their decrease in dry matter intake coupled with their concurrent decrease in nutrient demands translates into a 45% increase in nutrient efficiency of calf gain following early weaning. Early-weaned calves grow well on high quality annual pastures such as ryegrass, when provided supplemental grain at a rate of 1% of body weight. When high-quality pastures are not available, early-weaned calves will require access to greater rates of a highquality supplemental concentrate. At the time of early weaning (50 to 90 days of age), the crude protein requirement of the early-weaned calf diet may be as high as 20% on a dry matter basis. If planning to ship at the same time, vaccinate the early-weaned calves on the same schedule as the normal-weaned calves. Calves should not be vaccinated at the time of early weaning, as the vaccine will be neutralized by the calfs own passive immunity. Early-weaned calves are highly susceptible to internal parasites. Consider anthelmetic treatment every 50 to 60 days. In our experiences in Florida, we have been unable to maintain the high growth rate of the early-weaned calf into the summer. Depending on the region of the country, producers should carefully examine their pasture forage options and consider the efficiency of moving the calf to regions closer to feeding and finishing. When received into the feedlot at the time of normal weaning, early-weaned calves have greater feed efficiency compared to normal-weaned contemporaries. This is an important production response for producers to consider when evaluating retained calf ownership opportunities. Early-weaned calves have been shown to have carcasses of greater USDA quality score compared to normal-weaned contemporaries. This response is likely the result of being placed onto concentrate diets at an earlier age. Our early-weaned calves have similar USDA carcass quality scores as normal-weaned calves when grazed on pasture until the time of normal weaning. REFERENCES Arthington, J.D., and R.S. Kalmbacher. 2002. Use of ryegrass for grazing early-weaned calves in a subtropical environment. Soil Crop Sci. Soc. Florida Proc. 61:1-4. Arthington, J.D., and R.S. Kalmbacher. 2003. Effect of early weaning on beef cow and calf performance in the subtropics. J. Anim. Sci. 81:1136-1141. Arthington, J.D., and J.E. Minton. 2004. The effect of early calf weaning on feed intake, growth, and postpartum interval in thin, Brahman-crossbred primiparous cows. Prof. Anim. Sci. 20:34-38. 92

Arthington, J.D., and J.W. Spears. 2005. The effect of early weaning on feedlot performance and measures of stress in beef calves. J. Anim. Sci. 83:933-939. Berry, B. A., A. W. Confer, C. R. Krehbiel, D. R. Gill, R. A. Smith, and M. Montelongo. 2004. Effects of dietary energy and starch concentrations for newly received feedlot calves: II. Acute-phase protein response. J. Anim. Sci. 82:845-850. Carter, J. N., G. L. Meredity, M. Montelongo, D. R. Gill, C. R. Krehbiel, M. E. Payton, and A. W. Confer. 2002. Comparison of acute phase protein responses of cattle in naturally acquired respiratory disease: relationships to vitamin E supplementation and antimicrobial therapy. Am. J. Vet. Res. 63:1111-1117. Galindo-Gonzalez, S., J. D. Arthington, J. V. Yelich, G. R. Hansen, G. C. Lamb, and A. DeVries. 2007. Effects of cow parity on voluntary hay intake and performance responses to early weaning of beef calves. Livest. Sci. 110:148-153. DeRouen, S. M., and D. E. Franke. 1989. Effects of sire breed, breed type and age and weight at breeding on calving rate and date in beef heifers first exposed at three ages. J. Anim. Sci. 67:1128-1137. Martin, L. C., J. S. Brinks, R. M. Bourdon, and L. V. Cundiff. 1992. Genetic effects on beef heifer puberty and subsequent reproduction. J. Anim. Sci. 70:4006-4017. Martins, P. G. M. A., J. D. Arthington, R. F. Cooke, C. G. Lamb, D. B. Araujo, C. A. A. Torres, J. D. Guimaraes, and A. B. Mancio. 2012. Evaluation of beef cow and calf separation systems to improve reproductive performance of first-calf cows. Livest. Sci. 150:74-79. Myers, S. E., D. B. Faulkner, F. A. Ireland, and D. F. Parrett. 1999. Comparison of three weaning ages on cow-calf performance and steer carcass traits. J. Anim. Sci. 77:300310. Nunez-Dominguez, R., L. V. Cundiff, G. E. Dickerson, K. E. Gregory, and R. M. Koch. 1991. Lifetime production of beef heifers calving first at two versus three years of age. J. Anim. Sci. 69-3467-3479. Rae, D. O., W. E. Kunkle, P. J. Chenoweth, R. S. Sand, and T. Tran. 1993. Relationship of parity and body condition score to pregnancy rates in Florida beef cattle. Theriogenology 39:1143-1152. Rodrigues, H. D., J. E. Kinder, and L. A. Fitzpatrick. 2002. Estradiol regulation of luteinizing hormone secretion in heifers of two breed types that reach puberty at different ages. Biol. Reprod. 66:603-609. Vendramini, J. M. B., L. E. Sollenberger, J. C. B. Dubeux, Jr., S. M. Interrante, R. L. Stewart, Jr., and J. D. Arthington. 2006. Concentrate supplementation effects on forage characteristics and performance of early weaned calves grazing rye-ryegrass pastures. Crop Sci. 46:1595-1600.

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BH in the Rumen: The Gateway to Seeing Benefits From Fat Supplements

T. C. Jenkins Department of Animal & Veterinary Sciences Clemson University Clemson, SC 29634 Corresponding author: tjnkns@clemson.edu SUMMARY BH is a microbial pathway in ruminal contents designed to reduce unsaturation of lipids found within plant matter, and is likely an evolutionary adaptation to protect the microbial population from antimicrobial effects of unsaturated fatty acids. The major intermediates include an array of trans-C18:1 and conjugated linoleic acid isomers, but most published pathways have traditionally ignored the majority of minor intermediates and present only a superficial view of BH. Dietary lipid drives BH and the accumulation of bioactive intermediates in the rumen. Fatty acids originate from intake of forages and grains, oilseeds, byproducts, and fat supplements. The impacts of BH on animal performance include protection of ruminal fermentation from antimicrobial effects of unsaturated fatty acids, loss of dietary omega fatty acids needed for optimal reproduction and immune function, and accumulation of conjugated intermediates with physiological activity, such as the trans-10,cis-12 isomer that inhibits mammary lipogenesis and causes milk fat depression. INTRODUCTION The major accomplishment of BH (BH) in ruminal contents is to convert unsaturated fatty acids in plant matter to more saturated endproducts that flow to the intestines. The reason the microbial population developed the capacity for BH has been debated. Disposal of H has been considered, but seems unlikely given that lipid BH as a H sink in the rumen is small (< 5%) in comparison to methane and propionate production. Considering the sensitivity of many microbial species to cytotoxic effects of unsaturated fatty acids, it seems more likely that BH was an evolutionary adaptation to protect the microbial population from the antimicrobial effects of double bonds. The direct addition of hydrogen to a double bond is energetically costly and proceeds only slowly without the presence of a catalyst, as in the case of commercial hydrogenation of oils to raise melting point. The BH pathway in the rumen overcame this energetically unfavorable step by first forming trans double bonds that lower the energy of activation for subsequent functioning of the reductases. Thus, a variety of trans-monoene and conjugated dienes containing one or more trans double bonds emerged as intermediates. Over the decades, most published accounts of BH ignored early reports of complex intermediates and presented oversimplified pathways that included only predominant trans intermediates. Recent work is now being directed at accounting for all possible trans-18:1 and CLA intermediates, including identifying their physiological biopotentcy. The impact of BH on human health and animal performance is immense. The accumulation of the saturated fatty acids in animal tissue has had tremendous consequences on per capita consumption of meat and milk, following continual scrutiny of saturated fatty acids for their role 94

in etiology of atherosclerosis and other human health risks. In dairy cows, fat supplements can increase energy supply to enhance lactation performance of the cow and improve profitability. Biohydrogenation plays a role in assisting the benefits of these fat supplements by regulating the accumulation of rumen fatty acids that are linked to carbohydrate digestion, feed intake, and milk fat production. Likewise, BH greatly depletes the supply of essential fatty acids to ruminant tissues, which recent studies suggests compromises reproductive performance and immune function of the host animal in some circumstances. Notwithstanding are the potent physiological and metabolic functions of certain BH intermediates, especially the various conjugated linoleic acid (CLA) isomers produced. Notable effects of CLA include their anticarcinogenic properties and their ability to inhibit fat synthesis. This paper will begin with a brief overview of BH and its intermediates. This will be followed by a description of the fat sources fed to dairy cows that drive the process of BH, and how beneficial effects of fat supplements are modulated by the pathways of BH. The paper concludes with a discussion on regulation of BH that might enhance utilization of fat supplements by dairy cows. OVERVIEW OF BH Food consumed by ruminants first passes through the largest of the four stomach compartments or rumen, which acts like a fermentation vat. Countless numbers of bacteria, protozoa, and fungi in the rumen ferment the feed releasing end products that are utilized by the host animal for maintenance and growth of body tissues. The microbial population in the rumen also is responsible for extensive transformation of dietary lipid. Lipid transformations include lipolysis to release free fatty acids from complex plant lipids, followed by BH to convert unsaturated fatty acids in plant matter to more saturated lipid end products. The BH of linoleic acid in the rumen (Figure 1) begins with its conversion to CLA. In this initial step, the number of double bonds remains the same but one of the double bonds is shifted to a new position by microbial enzymes. Normally, the double bonds in linoleic acid are separated by two single bonds, but in CLA, the double bonds are only separated by one single bond. Many types of CLA are produced in the rumen of dairy cows (Bauman and Lock, 2006), but a common CLA produced from BH of linoleic acid is cis-9, trans-11 C18:2.

95

Figure 1. Major steps in the BH of linoleic and oleic acids by ruminal microbes.

Linoleic Acid

CLA

Trans C18:1

Oleic Acid

Stearic Acid

As BH progresses, double bonds in the CLA intermediates are then hydrogenated further to trans fatty acids having only one double bond. A final hydrogenation step by the ruminal microbes eliminates the last double bond yielding stearic acid as the final end product. Trans double bonds only differ from cis double bonds in the placement of the hydrogens (Figure 2). The hydrogens are located on opposite sides of the double bond for trans fatty acids, but on the same side of the double bond for cis fatty acids. Although the difference in structure between trans and cis fatty acids appears small, it causes significant differences in their physical and metabolic properties. Figure 2. Structural differences between cis and trans fatty acids.

In cows on a typical forage diet, the major trans C18:1 present in ruminal contents is trans-11 C18:1. Most of the remaining isomers have double bonds distributed equally among carbons 12 96

through 16 (Bickerstaffe et al., 1972). The exact pathways for the production of these positional isomers are not known. Linoleic and linolenic acids are converted to several trans C18:1 and C18:2 intermediates during BH. Mosley et al. (2002) showed that the BH of oleic acid by mixed ruminal microorganisms involves the formation of several positional isomers of trans C18:1 rather than direct BH to form stearic acid as previously thought. The BH process of linolenic acid in the rumen has not been researched as extensively as oleic or linoleic acids. In most publications, linolenic acid initially is converted to cis-9 trans-11 cis-15 C18:3 by ruminal BH (Figure 3). Then, trans-11 cis-15 C18:2 was produced through the reduction of the cis-9 double bond. This was further hydrogenated to trans-11 C18:1 to produce stearic acid. Trans-11 cis-15 C18:2 can also hydrogenate to trans-15 or cis-15 C18:1. Figure 3. Steps commonly reported in the BH of linolenic acid. From Kellens et al. (1986).

In a study conducted by Loor et al. (2004), linolenic acid was isomerized into three C18:3 intermediates: cis-9 trans-12 cis-15 C18:3, cis-9 trans-12 trans-15 C18:3, and trans-9 trans-12 trans-15 C18:3. Low concentrate diets (65:35 forage to concentrate) and the addition of linseed oil in the diet increased the duodenal flow of these three C18:3 isomers. In addition, Destaillats et al. (2005) reported that 0.3% of milk fat was the cis-9 trans-11 cis-15 C18:3 and the cis-9 trans-13 cis-15 C18:3 isomers, suggesting that these two isomers were the initial intermediates of linolenic acid BH. Subsequently, cis-9 trans-11 cis-15 C18:3 isomer was reduced to cis-9 trans-11 C18:2 and trans-11 cis-15 C18:2, and cis-9 trans-13 cis-15 C18:3 isomer to cis-9 97

trans-13 C18:2 and trans-13 cis-15 C18:2. Both cis-9 trans-11 C18:2 and trans-11 cis-15 C18:2 were able to hydrogenate to trans-11 C18:1, reducing to stearic acid. The cis-9 trans-13 C18:2 and trans-13 cis-15 C18:2 hydrogenated to trans-13 C18:1, which was then subsequently reduced to stearic acid. They suggested that two CLA isomers ( cis-9 trans-11 CLA; trans-13 cis-15 CLA) were produced from linolenic acid. However, no evidence supporting this route was given in this study. Wsowska et al. (2006) reported that cis-9 trans-11 cis-15 C18:3 and trans-9 trans-11 cis-15 C18:3 were accumulated by linolenic acid BH in strained rumen fluid. They also found that the trans-11 cis-15 C18:2 originated from linolenic acid, but cis-9 trans-11 CLA was not observed in their study. In a recent study by Lee et al. (2011), a stable isotopic tracer study was done to investigate the BH intermediates, including CLA, of 13C-linolenic acid incubated in cultures of mixed ruminal microorganisms. Results from the study showed a total of eight CLA isomers had carbons that originated from linolenic acid. The authors pointed out that these results do not prove that linolenic acid will yield all these CLA isomers in the live animal under all circumstances, but only proves the capability of the anaerobic population to convert linolenic acid to CLA. The production of CLA isomers in ruminal contents varies with the predominating microbial species, such that changes in diet offered the animal could dictate the presence or absence of a particular CLA isomer. DIETARY FAT SOURCES THAT DRIVE BH Grain and Forage Lipids The fatty acid content of most cereal seeds and forages typically ranges from 10 to 30 g/kg DM, with the majority of the fatty acids classified as unsaturated (predominately oleic, linoleic, and linolenic acids). Among the unsaturated fatty acids, linolenic acid is the predominant fatty acid in most forage species followed by linoleic acid (Hatfield et al., 2007). In the cereal seeds, fatty acids are comprised mainly of linoleic acid followed by oleic acid. Fatty acid concentrations in some pasture can exceed 50 g/kg DM, depending on plant species, stage of maturity, environment, etc. Fatty acid content of annual ryegrass pasture that was clipped in the field, immediately immersed in liquid nitrogen, and then freeze dried contained as much as 68 g/kg DM total fatty acids (Freeman-Pounders et al., 2009). Cattle grazing some species of immature pasture, in effect, may be consuming a high fat diet. Much lower concentrations are usually seen in hay and silage prepared from the same plant species. This is partially due to loss of plant leaves where chloroplast lipid is concentrated, but also due to plant metabolism of stored energy sources. Plant enzymes can continue to function in dried forage containing as little as 5 to 10% moisture. Plant maturity has a definite impact on both fatty acid content and fatty acid composition. Fatty acid content (g/kg DM) generally is highest in the spring and fall seasons and lowest in the summer months. For example, fresh perennial ryegrass contained 32 g/kg DM total fatty acids during primary growth in May, but only 12 g/kg DM at the beginning of second regrowth (Bauchart et al., 1984). Linolenic acid follows a similar seasonal pattern (Bauchart et al., 1984). As linolenic acid declines over the summer months, percentages of palmitic and linoleic acid increases. It is not only the amount of fatty acids in grains and forages that determine BH effects in the rumen, but also the form of the fatty acids. Information is just starting to become known about 98

accumulation of free fatty acids in forages. Fatty acids in forages are usually bound to glycerol making them less susceptible to disrupt fermentation in the rumen. Recent data are showing increased free fatty acids in forages in some circumstances. For example, free fatty acids in ryegrass were reported in one study to increase from 2% to as much as 73% of total fatty acids. Also, work done at The University of Georgia at Tifton (Cooke et al., 2007) reported seeing free fatty acids in whole cottonseed as high as 30% compared with a normal free fatty acid content of <7%. Replacing the normal cottonseed with the high free fatty acid cottonseed reduced milk fat from 4.2 to 3.6%. Distillers Grains Fermentation of corn mash produces ethanol, which is distilled to remove the ethanol and centrifuged to remove as much excess liquid as possible. The liquid fraction can be dehydrated to produce condensed solubles and the solid fraction may be sold directl y as wet distillers grains or dehydrated to produce dried distillers grain. The condensed soluble can be blended with the distillers grains to produce wet distillers grains + soluble (WDGS) or dried distillers grains + soluble (DDGS). Table 1. Nutrient composition of corn co-products. Nutrient DM, % CP, % Fat, % NDF, % TDN, %
a

WDG 25-35 30-35 8-12 30-50 70-110

MDGS 50 30-35 8-12 30-50 70-110

DDG 88-90 25-35 8-10 40-45 77-88

DDGS 88-90 25-32 8-10 39-45 85-90

Wet distillers grain (WDG); Modified distillers grains + soluble (MDGS); Distillers dried grains (DDG); Distillers dried grains + soluble (DDGS) Adapted from Tjardes and Wright. (2002). Large quantities of DDGS are now available throughout the United States as a dairy feed ingredient due to the rapid growth of ethanol plants primarily in the Midwest. Maximum feeding levels of DDGS in dairy diets can approach 20% or more of the feed dry matter (Schingoethe et al., 2002). With DDGS containing 25-35% crude protein and 8-10% fat (Table 1), its inclusion in the diet can substantially replace other protein supplements and elevate total ration fat content. Modified distillers grains + solubles (MDGS) contain even higher protein (30 -35%) and fat (8-12%) concentrations. Fat concentrations in MDGS can reach 15% or higher. Variability in the fat content of DDGS both within and across production plants is an important consideration that should be taken into account when formulating diets.

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Oilseeds Oilseeds contain relatively high amounts of fatty acids, but by nature of their hard outer seed coat, might invoke less microbial shift and CLA accumulation than expected. Evidence of some oilseed protection from ruminal BH can be seen by their ability to alter milk fatty acid composition. Feeding whole oilseeds (i.e. whole soybeans, whole cottonseeds, whole sunflower seeds, etc) to cows increases tissue and milk unsaturation according to some reports. When diets containing 0, 10, 15, or 20% whole cottonseed were fed to cows, 18:1 in milk steadily increased from 23.5 to 32.0% of total fatty acids (DePeters et al., 1985). However, there were no changes in milk 18:2 or 18:3 as cottonseed increased in the ration. Processing of the seed can affect the degree of protection from ruminal BH and the extent that milk fatty acids are altered. Disruption of the seed coat exposes the oil to the microbial population and the potential for fermentation problems and BH shifts. The seed coat can be sufficiently broken by chewing and rumination, or through a variety of processing techniques such as extrusion or grinding. Roasting of cottonseed was reported to reduce BH (Pires et al., 1997). In a meta-analysis of published data examining how ruminal fatty acid losses compare among fat sources (Jenkins and Bridges, 2007), oilseeds on average increased duodenal flow but reduced apparent ruminal losses only for oleic acid. Oleic acid ruminal losses were lower for oilseeds than for unprotected fats in 12 of the 15 oilseed observations. Protection by oilseeds was not as effective for polyunsaturated fatty acids as it was for oleic acid. Only 5 of the 20 oilseed observations were below the prediction interval for linoleic acid loss as defined by the unprotected fats. The oilseed data falling in the protected region were whole soybeans, with lower protection reported when the seeds were processed. The remaining oilseed data points, consisting mainly of cotton and canola seeds, fell within the prediction intervals indicating that their responses were more similar to unprotected fats. In the case of linolenic acid, only 2 of the 15 oilseed data points had ruminal losses below the prediction interval, which came exclusively from whole soybeans. Fat Supplements A useful way to classify fat supplements for dairy rations is based on their expected rumen response. Terminology varies widely for classifying fat sources according to nutritional effects, but most groupings consider the extent that a fat source depresses digestibility of the basal feed ingredients and the extent that the fat source resists BH. On this basis, fats can be classified as rumen-active, rumen-inert, or protected. The term rumen-inert has been assigned to fats that were specifically designed to have little, if any, negative effect on feed digestibility when fed to dairy cattle. Rumen-inert fats often have the added advantage of being dry fats that are easily transported and can be mixed into the diet without the need for specialized equipment. Rumeninert fats are often high in calcium salts of fatty acids, saturated fatty acids, or hydrogenated fats. Fats in this category have also been referred to as bypass fats.

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The rumen-active fats have the potential to interfere with microbial fermentation in the rumen and reduce feed digestibility to varying degrees. Digestibility of the fibrous carbohydrate fraction is especially susceptible to antimicrobial effects of rumen-active fats. Generally, unsaturated fatty acids depress fiber digestibility more than saturated fatty acids. Rumen-active fats include fats of animal origin (tallow, grease, etc), plant oils (soybean oil, canola oil, etc), oilseeds (cottonseeds, soybeans, etc), and high fat byproducts such as residues from food processing plants. Rumen-active fats undergo BH by ruminal microbes and generally have little impact on modifying milk fatty acid profile. The term protected fat is most applicable to fat sources specifically designed to resist BH by ruminal microbes and modify fatty acid profile of body tissues and milk. Many of the protected fats are based on surrounding unsaturated fatty acids by a protective capsule that acts to shield the internal fatty acids from BH. Another strategy for protection is chemical modification of unsaturated fatty acids to forms that resist BH, such as the conversion of fatty acids to fatty amides. A single fat source may overlap two, or even all three fat groups to some extent. For example, at normal levels of supplementation, some rumen-active fats, such as tallow, are fed to dairy cows without evidence of consistent problems with fiber digestion. Even whole oilseeds help to lessen the severity of digestion problems by encapsulation of antimicrobial fatty acids within their hard outer seed coat. However, classification according to ruminal digestion is better defined at high levels of supplementation, where the frequency of digestibility problems for tallow and oilseeds is much greater than for the rumen-inert fats. The oilseeds may also overlap as protected fats in instances where their hard outer seed coat provides protection from BH. However, disruption of the outer seed coat by chewing and rumination often leads to oilseeds having little ability to enhance unsaturated fatty acids in milk. BH REGULATION OF FAT BENEFITS Lactation Performance Elevating fatty acid concentration in ruminal contents may cause a number of changes in ruminal characteristics that determine the success of the fat supplement in achieving its desired outcome. For example, fatty acids are antimicrobial agents that can disrupt normal function of the rumen microbial ecosystem. Disruption of fermentation is most severe for fat sources high in unsaturated fatty acids, which inhibit growth and function of ruminal microbes more than saturated fatty acids (Jenkins et al., 2008). Unsaturated fatty acids exert cytological damage by adhering to the plasma membrane of microbial cells, followed by penetration into the lipid bilayer. The fatty acids can then cause disorganization of the membrane lipid bilayer that leads to inactivation of membrane-bound enzymes (Jenkins, 2002). An alternative explanation is the coating of feed particles by lipid, which blocks the action of cellulolytic enzymes. However, many recent studies suggest that lipid binding to feed particles protects against antimicrobial effects of fatty acids by reducing their accumulation onto the plasma membrane of microbial cells (Jenkins, 2002). In addition to disruption of ruminal fermentation, fat added to dairy rations also can reduce feed intake, which can greatly reduce or even eliminate a positive milk response. Any boost in 101

energy density of the ration from added fat does little to increase energy for milk if it is accompanied by reduced consumption of total feed. Several causes for the depression in feed intake by added fat are under consideration. These include reduced gut motility, reduced acceptability of diets with added fat, release of gut hormones, and oxidation of fat in the liver (Allen, 2000). Refer to Allen (2000) for a description of each factor and a comparison of fat sources. Gut hormones continue to receive considerable attention as regulators of food intake. Depressed feed intake in cows fed fat supplements has been attributed to changes in cholecystokinen (Choi and Palmquist, 1996) and glucagon-like peptide 1 (Benson and Reynolds, 2001). Other peptides of gut origin, such as peptide YY, pancreatic glucagons, glicentin, and oxyntomodulin, have been linked to reduced feed intake patterns in animals fed fat (Holst, 2000). Biohydrogenation has a possible link to regulation of feed intake because previous work has shown that abomasal infusion of unsaturated fatty acids causes greater feed intake depression than infusion of saturated fatty acids (Drackley et al., 1992; Bremmer et al., 1998). Dietary factors that enhance rates of BH and lessen the flow of unsaturated fatty acids to the intestine in turn might also maintain higher feed intakes. A study by Litherland et al. (2005) showed that the intake depression was greater following abomasal infusion of unsaturated free fatty acids than it was following infusion of unsaturated triglycerides. Also, as intake declined in the study by Litherland et al., (2005), the concentration of plasma glucagon-like peptide 1 increased but plasma concentration of cholecystokinen did not change. Another connection of BH with lactation performance is diet-induced milk fat depression (MFD), which continues to have major economic impact in the dairy industry and a priority for finding solutions. Current thinking links MFD with CLA produced from BH by the mixed microbial population. It was discovered that a CLA, namely the trans-10, cis-12 isomer, was closely associated with milk fat depression. This led to the BH theory of MFD that suggested feeding management was linked to an abnormal ruminal fermentation causing accumulation of the trans-10, cis-12 isomer. According to a study conducted by Baumgard et al. (2000), trans-10 cis-12 CLA decreased the lipogenic rate and milk fat synthesis of dairy cows, showing a 42% decrease in milk fat content and a 48% reduction in milk fat yield. These researchers also found that lipogenic activity decreased 82% using a radio-labeled acetate, and the activity of acetate oxidation to carbon dioxide was reduced to 61% in dairy cows inoculated with trans-10 cis-12 CLA. Additionally, the mRNA expression of all measured enzymes decreased from 54 to 39% after dosing with trans-10 cis-12 CLA. The results suggested that the trans-10 cis-12 CLA inhibited milk fat synthesis by decreasing enzyme activity through the inhibition of gene expression affecting de novo fatty acid synthesis, uptake, and transport. Trans-9 cis-11 CLA and cis-10 trans-12 CLA have also been reported as potential inhibitors of milk fat synthesis (Sb et al., 2005; Perfield II et al., 2007) with the former being associated with a 15% reduction in milk fat yield.

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Figure 4. The shift in intermediates produced from BH of linoleic acid in ruminal contents as a result of a diet-induced microbial shift.

Meeting Essential Fatty Acid Demands The -6 and -3 parent compounds (linoleic and linolenic acids, rspectively) cannot be synthesized by body tissues and, therefore, must be supplied in the diet. Thus, linoleic and linolenic acids are regarded as essential because they are required for normal tissue function but cannot be synthesized by body tissues. A typical total mixed ration of grains and forages generally contains adequate essential fatty acids to meet the needs of the animal. However, the majority of the dietary essential fatty acids are destroyed by BH. Interest continues on finding ways to bypass unsaturated fatty acids from BH and increase their delivery to body tissues. Part of the interest in protecting omega fatty acids from BH is to enhance their concentration in milk for value-added opportunities, and part is to enhance their concentration in body tissues of the cow to enhance production and health. Nutritional control of milk fatty acid profile continues to receive attention whether the goal is to improve manufacturing properties of milk or to enhance the concentration of fatty acids having beneficial health effects in humans. The key objective is usually to increase one or more unsaturated fatty acids in milk. For instance, increasing oleic acid content in milk enhances the plasticity and softness of milk fat, which has interested processors attempting to improve the spreadability of butter. Also, market pressures are aimed at enhancing the concentration of the unsaturated fatty acids in milk known to enhance human health. This has led to considerable interest in finding ways to shield dietary unsaturated fatty acids from BH in order to enhance their absorption and delivery to the mammary gland. Oleic acid concentration in milk fat normally varies from 18 to 24% of total fatty acids. Various rumen-protected fats, some commercialized and some not, pushed oleic acid in milk to as much as 48%. The effects of protected fats on milk linoleic acid concentration have been less dramatic. Linoleic acid 103

concentration in milk normally ranges from 1.5 to as much as 4% when cows are fed control diets with no added fat. Feeding rumen-protected fats increased the upper range of linoleic acid concentration to about 6.5%. Another significant finding bringing a great deal of attention to BH intermediates in milk fat was the discovery that CLA had beneficial effects on human health, most notably cancer-fighting properties. It was the cis-9, trans-11 CLA isomer in particular that received the most attention for its anticarcinogenic properties. The recent interest in enhancing BH intermediates in milk propagated research to determine the origin and possible enhancement of beneficial fatty acid isomers produced in the rumen. Reducing BH and increasing tissue delivery of unsaturated fatty acids also has improved reproductive performance of dairy cows in some studies. Reported improvements of reproductive performance from added fat include higher conception rates (Schneider et al., 1988; Sklan et al., 1989; Ferguson et al., 1990), increased pregnancy rates (Schneider et al., 1988; Sklan et al., 1991), and reduced open days (Sklan et al., 1991). Staples (2006) summarized results from 16 published studies that reported a significant (P < 0.10) improvement in conception rate across a wide range of fat sources. Conception rate across the 16 studies improved from 50 to 71% when fat was fed. The mechanism of how fat supplements alter reproductive performance is not clear, but fats as a source of essential fatty acids remains a possibility. Supplemental omega-3 fatty acids have been proposed to enhance reproduction by providing parent omega fatty acids for the synthesis of adequate prostaglandins involved in reproduction. Despite strong evidence of a link between reproduction and the omega fatty acids, questions remain regarding the proper amounts, types, and timing of feeding additional fatty acids for maximum reproductive response. According to Staples (2006), cows showing a positive reproductive response to added fat were being fed fat supplements at a minimum of 15 g/kg diet DM. Based on this observation, Staples (2006) suggested that fat supplementation at 15 g/kg was adequate to obtain a reproductive response. However, it is not clear as to which fatty acids (linoleic, linolenic, DHA, EPA) or combination of fatty acids provides the maximum response, or if levels of supplementation below 15 g/kg diet DM were sufficient. Intermediates of BH also might influence immune responses and disease resistance in animals. For example, CLA decreased the growth rate in chicks and rats after they were injected with endotoxin (lipopolysaccharide; LPS). This probably was caused by release of cytokines and the prevention of the catabolic effects (Cook et al., 1993). Miller et al. (1994) examined endotoxininduced growth suppression in mice fed with 0.5 % fish oil and CLA. The fish oil fed-group lost twice as much body weight after the inoculation with endotoxin than the CLA-fed groups. These researchers found that the CLA in the endotoxin injection inhibited anorexia (a decreased sensation of appetite) and increased splenocyte blastogenesis, concluding that it might inhibit arachidonic acid synthesis preventing the catabolism of tissue by removing eicosanoid precursors. In addition, Bontempo et al. (2004) examined the effects of CLA on the immunological variables of lactating sows and piglets fed with a 0.5 % CLA diet. They found that CLA-fed sows exhibited increased colostrum IgG and serum leptin, and IgG and lysozyme. Nursing piglets of CLA-fed sows also exhibited higher levels of IgG and lysozyme. As these results show, dietary CLA enhanced the effect of immunological variables in lactating sows and piglets.

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INFLUENTIAL FACTORS OF BH Changes in diets offered to ruminants affect the extent of BH in the rumen. The rumen environmental factors such as pH, soluble carbohydrates, microbial growth factors, solid dilution rate, and fatty acids have differential effects on BH in addition to diet supply (Hungate, 1966; Church, 1969; Martin and Jenkins, 2002). Martin and Jenkins (2002) examined continuous culture incubations at liquid dilution rates of 0.05 and 0.10/h with pH values of 5.5 and 6.5, and 0.5 and 1.0 g/L of mixed soluble carbohydrate. They found that the major environmental factor that influenced formation of CLA and trans-C18:1 from linoleic acid BH was culture pH. At pH of 5.5, the concentration of transC18:1 and CLA were significantly reduced. Similar effects were observed by TroegelerMeynadier et al. (2003). They found that the amounts of BH products were always lower at pH 6.0 than at pH 7.0 at 24 h from in vitro incubations in ruminal fluid. Low amounts of CLA at pH 6.0 could be due to low isomerase activity or to high reductase activity. Moreover, they found that low pH (pH 6.0) resulted in lower amount of trans-11 C18:1 at all incubation times compared with higher pH (pH 7.0), but concentration of trans-10 C18:1 was higher at 16 to 24 h of incubation. Low pH inhibited initial isomerization and the second reduction ( trans-11 C18:1 to stearic acid), leading to an accumulation of trans-11 C18:1 in ruminal cultures (TroegelerMeynadier et al., 2006). Choi et al. (2005) reported that cis-9 trans-11 CLA are produced at pH higher than 6.2 by rumen bacteria, but trans-10 cis-12 CLA are produced more than cis-9 trans11 CLA at lower pH. They concluded that trans-10 cis-12 CLA producing bacteria may be more aero and acid-tolerant than cis-9 trans-11 CLA producing bacteria. Oleic acid BH was also affected by ruminal pH and dilution rate. AbuGhazaleh et al. (2005) reported that low pH and dilution rate are restricted to formation of trans-C18:1 and increased the concentration of stearic acid from oleic acid. They conducted the experiment using 13Clabeled oleic acid to determine BH intermediates in mixed ruminal microorganisms grown in continuous cultures at different pH and liquid dilution rate. At pH 6.5 and 0.10/h dilution rate, 13 C enrichment was detected in trans-6 through 16 C18:1 isomers. However, at pH 5.5 and 0.05/h dilution rate, 13C enrichment was not detected in any trans isomers with a double bond position over C10. Qiu et al. (2004) reported that reduced ruminal pH can affect microbial populations, especially cellulolytic bacteria. Total cellulolytic bacteria numbers are reduced, accompanied by reduced acetate-to-propionate ratio and BH when pH was low. The rumen pH also influenced fungal growth and metabolism. Culturing rumen fungi at pH 6.0 and pH 7.0 slowed BH compared pH 6.5. CLA production was increased by pH 7.0 compared to pH 6.0 and pH 6.5. Therefore, optimum pH was 6.5 and 7.0 for BH and CLA production, respectively, by ruminal fungi (Nam and Garnsworthy, 2007a). Vitamins effects on BH remains a possibility but many questions remain. Pottier et al. (2006) determined that vitamin E supplementation to cows increased milk fat content, decreased trans10 C18:1 concentration in milk, and increased the concentration of cis-9 trans-11 CLA and trans-11 C18:1 in milk. Not much is known, however, what vitamin E mechanism is responsible for the BH process. Researchers have suggested four possibilities: 1) Vitamin E might act as an electron donor for the reduction of the cis bond of the CLA. 2) Vitamin E might be metabolized 105

to become electron donors by ruminal microorganisms 3) Vitamin E might act as an electron donor to restore -tochopherolquinol and deoxy--tocopherolquinol. 4) Vitamin E could inhibit the growth and function of trans-10 C18:1 producing bacteria. In a later study, Nam and Garnsworthy (2007b) reported that the addition of VFA and vitamin E did not affect the pattern or extent of BH by mixed ruminal fungi in in vitro cultures. Other antioxidants could also affect BH. Finally, the concentration of fatty acid substrates affects the BH process. Polan et al. (1964) found that high amounts of stearic acid were produced from BH in cultures inoculated with low concentrations of linoleic acid. As the linoleic acid concentration increased, the conversion to stearic acid decreased. Similarly, Harfoot et al. (1973) found that linoleic acid inoculated at less than 1.0 mg/ml of ruminal contents resulted in stearic acid being the primary end product. When the concentration of linoleic acid increased to more than 1.0 mg/ml of ruminal contents, trans11 C18:1 was the primary end product. Over the years, results have shown that high amounts of long chain fatty acids irreversibly inhibit the final BH step of C18:1 to stearic acid. TroegelerMeynadier et al. (200, 2006) confirmed that a high concentration of linoleic acid inhibited both the reduction of CLA to trans-C18:1 and the reduction of trans-C18:1 to stearic acid, thus increasing CLA concentration in the rumen. High amounts of linolenic acid, however, inhibited linoleic acid isomerization and led to lower CLA and higher trans-11 C18:1 concentrations. Fish oil fatty acids also influence the pattern of BH. AbuGhazaleh et al. (2002,2003) reported that lactating dairy cows fed fish oil produced milk with higher concentrations of cis-9 trans-11 CLA and trans-11 C18:1 These changes may have been due to the inhibited reductase activity of ruminal microorganisms, causing the accumulation of CLA and trans-C18:1. Wsowska et al. (2006) found that the major fatty acids in fish oil, EPA or DHA, only partially inhibited ruminal BH. REFERENCES AbuGhazaleh, A. A., M. B. Riley, E. E. Thies, and T. C. Jenkins. 2005. Dilution rate and pH effects on the conversion of oleic acid to trans C18:1 positional isomers in continuous culture. J. Dairy Sci. 88:4334-4341. AbuGhazaleh, A. A., D. J. Schingoethe, A. R. Hippen, and L. A. Whitlock. 2002. Feeding fish meal and extruded soybeans enhances the conjugated linoleic acid (CLA) content of milk. J. Dairy Sci. 85:624-631. AbuGhazaleh, A. A., D. J. Schingoethe, A. R. Hippen, and K. F. Kalscheur. 2003. Milk conjugated linoleic acid response to fish oil supplementation of diets differing in fatty acid profiles. J. Dairy Sci. 86:944-953. Allen, M. S. 2000. Effects of diet on short-term regulation of feed intake by lactating dairy cattle. J. Dairy Sci. 83:1598-1624. Bauchart, D., R. Verite, and B. Remond. 1984. Long-chain fatty acid digestion in lactating cows fed fresh grass from spring to autumn. Can. J. Anim. Sci. 64:330-331. Baumgard, L. H., B. A. Corl, D. A. Dwyer, A. Saebo, and D. E. Bauman. 2000. Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis. Am. J. Physiol. Regul. Integr. Comp. Physiol. 278:R179-R184. Bauman, D. E. and A. L. Lock. 2006. Concepts in lipid digestion and metabolism in dairy cows. Proc. Tri-State Dairy Nutr. Conf. pp. 1-14. Available at: http://tristatedairy.osu.edu/.

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Benson, J. A., and C. K. Reynolds. 2001. Effects of abomasal infusion of long-chain fatty acids on splanchnic metabolism of pancreatic and gut hormones in lactating dairy cows. J. Dairy Sci. 84:1488-1500. Bickerstaffe, D., D. E. Noakes, and E. F. Annison. 1972. Quantitative aspects of fatty acid BH, absorption and transfer into milk fat in the lactating goat, with special reference to the cisand trans- isomers of octadecenoate and linoleate. Biochem. J. 130:607-617. Bontempo, V., D. Sciannimanico, G. Pastorelli, R. Rossi, F. Rosi, and C. Corino. 2004. Dietary conjugated linoleic acid positively affects immunologic variables in lactating sows and piglets. J. Nutr. 134:817-824. Bremmer, D. F., L. D. Ruppert, J. H. Clark, and J. K. Drackley. 1998. Effects of chain length and unsaturation of fatty acid mixtures infused into the abomasum of lactating dairy cows. J. Dairy Sci. 81:176-188. Choi, B. R., and D. L. Palmquist. 1996. High fat diets increase plasma cholecystokinin and pancreatic polypeptide, and decrease plasma insulin and feed intake in lactating dairy cows. J. Nutr. 126:2913-2919. Choi, N. J., J. Y. Imm, S. J. Oh, B. C. Kim, H. J. Hwang, and Y. J. Kim. 2005. Effect of pH and oxygen on conjugated linoleic acid (CLA) production by mixed rumen bacteria from cows fed high concentrate and high forage diets. Anim. Feed Sci. Tech. 123-124:643-653. Church, D. C. 1969. Digestive physiology and nutrition of ruminants. Vol. 1. O. S. U. Book Sotres Inc, Corvallis, OR. USA. Cook, M. E., C. C. Miller, Y. Park, and M. Pariza. 1993. Immune modulation by altered nutrient metabolism: nutritional control of immune-induced growth depression. Poult. Sci. 72:13011305. Cooke, K. M., J. K. Bernard, C. D. Wildman, J. W. West, and A. H. Parks. 2007. Performance and ruminal fermentation of dairy cows fed whole cottonseed with elevated concentrations of free fatty acids in the oil. J. Dairy Sci. 90:2329-2334. DePeters, E. J., S. J. Taylor, A. A. Franke, and A. Aguirre. 1985. Effects of feeding whole cottonseed on composition of milk. J. Dairy Sci. 68: 897-902. Destaillats, F., J. P. Trottier, J. M. G. Galvez, and P. Angers. 2005. Analysis o f -linolenic acid BH intermediates in milk fat with emphasis on conjugated linolenic acids. J. Dairy Sci. 88:3231-3239. Drackley, J. K., T. H. Klusmeyer, A. M. Trusk, and J. H. Clark. 1992. Infusion of long-chain fatty acids varying in saturation and chain length into the abomasums of lactating dairy cows. J. Dairy Sci. 75:1517-1526. Ferguson, J. D., D. Sklan, W. V. Chalupa and D. S. Kronfeld. 1990. Effects of hard fats on in vitro and in vivo rumen fermentation, milk production, and reproduction in dairy cows. J. Dairy Sci. 73:2864. Freeman-Pounders, S. J., Hancock, D. W., Bertrand, J. A., Jenkins, T. C., and B. W. Pinkerton. 2009. The fatty acid profile of rye and annual ryegrass pasture changes during their growth cycle. Forage and Grazinglands, 30 January 2009. (www.plantmanagementnetwork.org/fg). Harfoot, C. G., R. C. Noble, and J. H. Moore. 1973. Factors influencing the extent of BH of linoleic acid by rumen micro-organisms in vitro. J. Sci. Fd. Agric. 24:961-970. Hatfied, R., H. G. Jung, G. Broderick, and T. C.Jenkins. 2007. Nutritional chemistry of forages. In Forages, Volume 2. The Science of Grassland Agriculture, Sixth Edition. R. F. Barnes (Ed.), Blackwell Publishing, Iowa State Press, Ames Holst, J. J. 2000. Gut hormones as pharmaceuticals. From enteroglucagon to GLP-1 and GLP-2. Reg. Peptides 93:45-51. 107

Hungate, R. E., 1966. The rumen and its microbes. Academic Press, New York, USA. Jenkins, T. C. 2002. Lipid transformations by the rumen microbial ecosystem and their impact on fermentative capacity. pp 103-117 in Gastrointestinal Microbiology in Animals, S. A. Martin (Ed.), Research Signpost, Kerala, India. Jenkins, T. C., and W. C. Bridges, Jr. 2007. Protection of fatty acids against ruminal BH in cattle. Eur. J. Lipid Sci. Technol. 109:778-789. Jenkins, T. C., R. J. Wallace, P. J. Moate, and E. E. Mosley. 2008. Board-invited review: Recent advances in BH of unsaturated fatty acids within the rumen microbial ecosystem. J. Anim. Sci. 86:397-412. Kellens, M. J., H. L. Goderis, and P. P. Tobback. 1986. BH of unsaturated fatty acids by a mixed culture of rumen microorganisms. Biotechnol. Bioeng. 28:1268-1276. Lee, Y. J. and T. C. Jenkins. 2011. Biohydrogenation of linolenic acid to stearic acid by the rumen microbial population yields multiple intermediate conjugated diene isomers. J. Nutr. 141:1445-1450. Litherland, N. B., S. Thire, A. D. Beaulieu, C. K. Reynolds, J. A. Benson, and J. K. Drackley. 2005. Dry matter intake is decreased more by abomasal infusion of unsaturated free fatty acids than by unsaturated triglycerides. J. Dairy Sci. 88:632-643. Loor, J. J., K. Ueda, A. Ferlay, Y. Cgilliard, and M. Doreau. 2004. BH, duodenal flow, and intestinal digestibility of trans fatty acid and conjugated linoleic acids in response to dietary forage:concentrate ratio and linseed oil in dairy cows. J. Dairy Sci. 87:2472-2485 Martin, S. A., and T. C. Jenkins. 2002. Factors affecting conjugated linoleic acid and trans-C18:1 fatty acid production by mixed ruminal bacteria. J. Anim. Sci. 80:3347-3352. Miller, C. C., Y. Park, M. W. Pariza, and M. E. Cook. 1994. Feeding conjugated linoleic acid to animals partially overcomes catabolic responses due to endotoxin injection. Biochem. Ciophys. Res. Commun. 198:1107-1112. Mosley, E. E., G. L. Powell, M. B. Riley, and T. C. Jenkins. 2002. Microbial BH of oleic acid to trans isomers in vitro. J. Lipid Res. 43:290-296. Nam, I. S., and P. C. Garnsworthy. 2007a. BH of linoleic acid by rumen fungi compared with rumen bacteria. J. Appl. Microbiol. 103:551-556. Nam, I. S., and P. C. Garnsworthy. 2007b. Factors influencing BH and conjugated linoleic acid production by mixed rumen fungi. J. Microbiol. 45(3):199-204. Perfield II, J. W., A. L. Lock, A. Sb, J. M. Griinari, D. A. Dwyer, and D. E. Bauman. 2007. Trans-9, cis-11 conjugated linoleic acid (CLA) reduces milk fat synthesis in lactating dairy cows. J. Dairy Sci. 90:2211-2218. Pires, A. V., M. L. Eastridge, J. L. Firkins, and Y. C. Lin. 1997. Effects of heat treatment and physical processing of cottonseed on nutrient digestibility and production performance by lactating cows. J. Dairy Sci. 80:1685-1694. Polan, C. E, J. J. McNeill, and S. B. Tove. 1964. BH of unsaturated fatty acids by rumen bacteria. J. Bacteriol. 88:1056-1064. Pottier, J., F. Focant, C. Debier, G. De Buysser, C. Goffe, E. Mignolet, E. Froidmont, and Y. Larondelle. 2006. Effect of dietary vitamin E on rumen BH pathways and milk fat depression in dairy cows fed high-fat diets. J. Dairy Sci. 89:685-692. Qiu, X., M. L. Eastridge, K. E. Griswold, and J. L. Firkins. 2004. Effects of substrate, passage rate, and pH in continuous culture on flows of conjugated linoleic acid and trans-C18:1. J. Dairy Sci. 87:3473-3479.

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Saebo, A., P. C. Saebo, J. M. Griinari, and K. J. Shingfield. 2005. Effect of abomasal infusions of geometric isomers of 10,12 conjugated linoleic acid on milk fat synthesis in dairy cows. Lipids. 40:823-832. Schingoethe, D. J. 2002. Using distiller's grains in the dairy ration. Proc. National corn Growers Association Ethanol Co-Products workshop, Lincoln, NE. Schneider, P., D. Sklan, W. Chalupa, and D. S. Kronfeld. 1988. Feeding calcium salts of fatty acids to lactating cows. J. Dairy Sci. 71: 2143-2150. Sklan, D., E. Bogin, Y. Avidar and S. Gur-Arie. 1989. Feeding calcium soaps of fatty acids to lactating cows: effect on production, body condition and blood lipids. J. Dairy Res. 56:675682. Sklan, D., U. Moallem and Y. Folman. 1991. Effect of feeding calcium soaps of fatty acids on production and reproduction responses in high producing lactating cows. J. Dairy Sci. 74:510-517. Staples, C. R. 2006. Milk fat depression in dairy cows - Influence of supplemental fats. Florida Ruminant Nutrition Symposium, Gainesville, Florida. Tjardes, K., and C. Wright. 2002. Feeding corn distiller's co-products to beef cattle. Extension Extra, South Dakota State University Cooperative Extension Service. Troegeler-Meynadier, A., M. C. Nicot, C. Bayourthe, R. Moncoulon, and F. Enjalbert. 2003. Effects of pH and concentrations of linoleic and linolenic acids on extent and intermediates of ruminal BH in vitro. J. Dairy Sci. 86:4054-4063. Troegeler-Meynadier, A., L. Bret-Bennis, and F. Enjalbert. 2006. Rates and efficiencies of reactions of ruminal BH of linoleic acid according to pH and polyunsaturated fatty acids concentrations. Reprod. Nutr. Dev. 46:713-724. Wsowska, I., M. R. G. Maia, K. M. Niedwiedzka, M. Czauderna, J. M. C. Ramalho Ribeiro, E. Devillard, K. J. Shingfield and R. J. Wallace. 2006. Influence of fish oil on ruminal BH of C18 unsaturated fatty acids. Brit. J. Nutr. 95:1199-1211.

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Why Cows Die on Dairies

SEPTEMBER 2012 1 Dairy Sessions Moderators: Matt Dodd, Kirk Smith, Jim Bennett Franklyn Garry,1 DVM, MS, Dipl ACVIM; Craig McConnel,2 DVM, PhD 1 Department of Clinical Sciences Colorado State University 300 West Drake Road Fort Collins, CO 80521 2 Charles Sturt University SAVS, Boorooma Street Wagga Wagga, NSW 2678, Australia ABSTRACT On-farm death of adult dairy cows is a significant problem for both economic and animal welfare reasons. Adult cow mortality losses on dairies have increased in recent years. These losses and their causes are not carefully monitored or evaluated on most dairies, leaving producers and veterinarians without the information needed to manage them. The reasons cows die are multiple and complex, necessitating an improved approach to diagnosis, information management and analysis. RSUM La mort la ferme de vaches laitires adultes constitue un problme important pour des raisons la fois conomiques et de bien-tre des animaux. Les pertes dues la mortalit des vaches adultes dans les laiteries ont augment au cours des dernires annes. Ces pertes et leurs causes ne font pas lobjet dun suivi rigoureux et dune valuation dans la plupart des fermes laitires, les producteurs et les vtrinaires ne disposent donc pas de linformation dont ils ont besoin pour assurer la gestion de ces pertes. Les raisons pour lesquelles les vaches meurent sont multiples et complexes, ce qui ncessite une meilleure approche en matire de diagnostic, de gestion de linformation et danalyse. INTRODUCTION Death losses have not been studied very intensively in the dairy industry. Yet, mortality rates in the dairy industry are much higher than those in the cow-calf or feedlot industries. Estimates of these death losses are variable. Unless they focus on monitoring cow deaths, dairy producers may underestimate the amount of adult cow death loss on their operations. The SDA:APHIS:VS National Animal Health Monitoring System (NAHMS) Dairy 2007 survey reported that 5.7% of dairy cows die on-farm across the country each year, an increase from 4.8% of the January 2002 inventory, and 3.8% of the January 1996 inventory.14,15 Information from computerized dairy record systems suggests that mortality rates have continually increased over the last 10 years. In some states, adult cow mortality exceeds 10% 110

per year.2,4 Few formal studies have focused on this issue, yet dairy cattle death losses are an extremely important problem. Not only are these losses an economic disaster, they also represent very substantial problems with animal well-being. Adult cow death loss is an issue that should be very important to producers and veterinarians. But rising rates of occurrence across the industry suggests that veterinarians and producers do not have the information required to manage the problem appropriately. The purpose of this presentation is to critique the information we have, consider what information we need, and suggest changes in information gathering for dairy herds that would help diminish losses. WHY DO DAIRY COWS DIE? Most studies of dairy cow mortality have come from outside the United States. Studies from the US on this issue have been primarily focused on culling and herd turnover rates, rather than death losses per se. The 2007 national survey of dairies in the US14 showed that approximately 23.6% of dairy cows left herds permanently during 2007, and that approximately 5.5% of these cows were sold to other dairies, while 94% were culled (i.e. sold and not returned to milk production, sent for slaughter). The reasons cows were culled included reproductive failure (26.3% of culled cows), mastitis and udder problems (23%), lameness or injury (16%), other disease (3.7%), and poor milk production not related to these other problems (16%), while other miscellaneous reasons accounted for about 8% of culling. Therefore, on average, the overwhelming majority of dairy cows leaving farms are not fit for sale as dairy production animals, and approximately 50% of these cows leave because of disease or injury problems, rather than being selectively removed because of low fertility or milk productivity. Adult cow death losses appear to be attributable to reasons similar to those for culling cows. A recent literature review identified 19 studies between the years 1965 and 2006 that focused on dairy cow mortality in countries with relatively intensive dairy production.13 While 10 of the 19 studies provided information about causes of death, none of the diagnoses were founded on necropsy evaluation. Only a single study discriminated between cows that were euthanized or died unassisted. The categories used to describe causes of death were relatively uniform across studies and were presented as accidents, calving disorders, digestive disorders, locomotor disorders, metabolic disorders, udder/teat disorders, other known reasons, and unknown reasons. The NAHMS Dairy 2007 survey recorded causes of death similarly to those established through the literature review, documenting the percentage of cow deaths due to euthanasia due to lameness or injury (20.0%), mastitis (16.5%), calving problems (15.2%), respiratory problems (11.3%), scours, diarrhea, or other digestive problems (10.4%), lack of coordination or severe depression (1.0%), poison (0.4%), other known reasons (10.2%), and unknown reasons (15.0%).14 Lets consider what the preceding information means. First, it suggests that historically the careful tracking of causes of mortality on dairies has not been seen as a high priority. Such an attitude would make sense if deaths occur very infrequently and appear to have little to do with the health of the remaining herd. It makes a lot less sense when 5 to 10% of standing herd inventory is lost to death each year. This information also speaks to the diverse health challenges seen on dairies. Dairy cows are complex animals that go through multiple life stages

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in the course of their residence on a farm. This is very different than a beef feedlot where most of the animals are young and growing, somewhat equivalent to dairy heifers. In these populations, infectious respiratory disease is far and away the number one health challenge that predisposes to euthanasia and death. For adult dairy cows there is no single predominant lifethreatening disease. It is also worth noticing that the categorization systems used on dairies and reported in the literature are not very helpful when it comes to instituting corrective actions. For example, if you consider the category of lameness as a cause of death, there are so many potential causes of lameness that it would be difficult to institute a specific corrective action that would decrease the numbers in this category. Similarly, consider the wide range of disease problems that could be categorized as digestive death. HOW GOOD IS OUR INFORMATION ABOUT CAUSE OF DEATH? Cause of death entered in dairy record systems is usually based on producer assessment and diagnosis. It appears that most dairy veterinarians are minimally involved in the diagnosis of cause of death, and relatively few US dairy operations perform necropsies in an effort to determine the cause of cow death. The NAHMS Dairy 2007 study reported that necropsies were performed on only 13% of operations, and only 4.4% of cow deaths received a postmortem examination.14 Therefore, historically almost all studies of dairy cow mortality are based on producer assessment rather than veterinary diagnosis, and the causes of death are described using broad categories that do not provide much information about specific cause. Dairy record systems appear to be an unreliable source of information concerning cause of death in individual animals. We have been studying the phenomenon of dairy cow mortality over the last several years. Our findings suggest that dairy producer assessment of the proximate cause of death is inaccurate approximately 50% of the time. Our results also validate that there are multiple causes of dairy cow death.9 It seems reasonable to suggest that numerous health problems in dairy cows are not recognized early enough or treated appropriately to promote an optimal outcome, but this type of information cannot be retrieved from record systems. Furthermore, without good descriptors and records of the reasons that cows die, preventive measures that should decrease disease and death are not modified or improved to address the problem. No specific reason has been identified for the increase in dairy cow death rates. In conversation with producers and veterinarians, some have questioned whether the federal regulations regarding down dairy cows and neurologic disease may have artificially increased recorded death rates. While this will contribute to recorded mortalities, death rates were increasing prior to the implementation of this rule.11 Furthermore, if euthanized down cows represent more than a small fraction of dairy mortalities, we need to ask why there are so many down cows that need to be euthanized. Others have suggested that specific disease problems, such as hemorrhagic bowel disease, may be increasing death rates. This could certainly be true on an individual dairy, but the increased mortality rates across the industry exceed the incidence of any specific disease problem.

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Any conjectures on the cause of increased mortality are difficult to validate without specific diagnoses. Determining the cause of death would provide invaluable information for preventing future deaths and improving herd health.7 The fact that very few dairy cow deaths are evaluated by necropsy leaves a serious information gap in any analysis of cow mortality. EPIDEMIOLOGICAL ASSOCIATIONS WITH DAIRY COW MORTALITY Although record systems as they are currently designed and used are not particularly helpful in managing adult cow death losses, they do demonstrate associations between high death rates and herd health problems. Analyses of large data sets demonstrate that herds with high rates of disease and culling also have higher death rates.1,3,10 More specifically, high mortality in dairy herds is related to high rates of lameness and large proportions of cows that are removed due to lameness or injury. Mortalities tend to occur much more frequently in the early part of lactation, coincident with increases in other health problems.2 Death losses are related to the occurrence of respiratory disease, diarrhea, and mastitis.10 These findings should not be surprising, as they suggest that herds that have poor ability to control lameness, injury, and infectious disease also have increased likelihood of cow death. It is important to recognize that these epidemiologic associations do not inform us of specific causes, and rather show that herds with certain types of problems also have higher rates of death. The problem for the producer and dairy consultant lies in how to determine specific actions that decrease disease prevalence and risk of death. WHAT CAN BE DONE TO DECREASE DAIRY COW DEATHS? Most decisions in a low-cost production dairy model are made with input cost as the primary driving force, and potential negative impacts on the animals in the production system are seen as problems that must be managed as a consequence. For example, it is common that large-scale expansion of a dairy will capture production cost efficiencies, but often with the caveat that expansions are accompanied by substantial problems with animal health. During the time that large numbers of animals are being imported to the herd, it is routine that disease introduction is occurring. Numerous animal health problems are prevalent, and even increase with time.5,16 Because there are compelling reasons for dairies to expand, there is a real need for the dairy industry and dairy veterinarians to re-evaluate dairy management systems with a focus on optimum animal health. An overview of the health challenges faced by dairy cows needs to recognize that some changes in the modern dairy industry may result in systematic problems with animal care. The labor force on most dairies is primarily composed of low-wage workers without extensive, preexisting dairy cow management skills. The ability of dairy personnel to adequately identify disease in individual animals and respond with prompt individual animal attention is limited by the extent of their experience and training. The overwhelming majority of sick cows on dairies are identified, diagnosed, and treated by farm workers rather than veterinarians. Poor outcomes may be an issue of poor clinical disease management in addition to any pre-existing problems with cow physiology. Farm necropsy examinations should be an invaluable tool to help assess cause of adult cow death.7 Necropsy of dead animals to assess and monitor cause of death is rarely performed on 113

dairies.14 This is in sharp contrast to other intensive livestock management systems, including poultry, swine, and feedlot enterprises where necropsy monitoring is routine. Most dairy veterinarians focus considerable effort on dairy reproduction, or udder health and milk quality, but little time on mortality evaluation. This presents a very significant liability to the dairy industry, because efforts to effectively decrease mortality losses are hampered by a lack of monitoring and information necessary to accurately assess the problem. We believe that dairy workers could be trained to more effectively monitor death losses, and to perform on-farm necropsy examinations in consultation with veterinarians when the veterinarian cannot be present to perform the examination on a freshly dead carcass. We have presented this recommendation to producer groups and produced an on-line training program for that purpose on our website.12 Very few producers or veterinarians have pursued this approach, attesting to the notion that monitoring actual cause of death has not been seen as a valuable pursuit. Necropsy examinations provide good information, but we also need to develop new recording systems that allow the necropsy results to be recorded as usable information. On their own, necropsy diagnoses provide great detail about the specific cause of death, but do not necessarily provide information about why that specific cause occurred. Therefore, necropsy information needs to be combined with other historical information about the affected animals to help direct management changes.8 Our studies suggest that more than 50% of cow death losses are attributable to causes that could be mitigated with proper management.8 Because of the complex nature of dairy management systems a variety of causes are responsible for high disease and mortality rates, with different rates of occurrence on different operations. The wide range of lactational incidence risk for common diseases (milk fever: 0.03%-22.3%, retained placenta (RP): 1.3 39.2%, metritis: 2.2-37.3%, ketosis: 1.3-18.3%, left-displaced abomasum (LDA): 0.3-6.3, lameness: 1.8-30%) attests to the complexity of dairy systems.6 To adequately address such complexity requires more accurate information about current losses, followed by management alterations that address the underlying problems. This will require changing the nature of information used in dairy management systems. An example of mastitis prevalence can illustrate this point: the specific infectious organism that causes a clinical mastitis episode can have a dramatic impact on outcome, and appropriate preventative or therapeutic measures need to be tailored to the specific cause, e.g. gram-negative vs grampositive, environmental vs contagious, Escherichia coli vs Staphylococcus aureus. Assessments and record systems that track mastitis without identifying other specific details provide less information than needed to establish effective interventions. Similarly, monitoring death losses with generic terms such as lameness or mastitis and performing this monitoring on the basis of presumption will not allow correction of management problems that may underlie the death. SPECIFIC RECOMMENDATIONS TO DECREASE DEATH LOSSES We have proposed an approach to monitoring death losses that should help producers identify management changes to improve cow health and survival.8 The first step is to identify the magnitude of the problem on a dairy and commit to improving outcomes. Like any other substantial management change on a dairy, if the owner or manager is not committed to change it will not actually happen. Therefore, simple analysis of the incidence of on-farm death and an assessment of its importance to the dairy and the well-being of the cows is critical. 114

Second, we recommend performing necropsy examinations to identify specific causes of death. This information needs to be considered along with other cow information such as preceding health problems, treatments, and individual cow circumstances as part of a complete postmortem evaluation. It is unrealistic to assume that 100% of all dead cows will be examined by necropsy. Our experience suggests that routine necropsy examination is important, but that targeting cases is useful. For animals euthanized due to obvious trauma, or where the cause of death is obvious based on priority veterinary assessment, necropsy examination usually will not provide much more information. Alternatively, for unexpected deaths or animals without simple specific antemortem diagnoses, necropsy can help not only define the cause of death but also inform farm workers about the types of problems that occur on the farm. We have developed a conceptual model to help assign cause of death to categories that have more meaning than those simple categories that assign cause of death to an organ system that the owner perceives was affected by disease. Necropsy is a key tool for assigning cause of death, if the information obtained is also matched with other animal information. Dairy workers who are involved in animal care should be included in the discussion of the necropsy and cause of death. The monitoring and focus on cause of death as an important component of dairy animal monitoring increases owner and worker focus on the actions needed to prevent future death losses. We recommend maintaining hard copy records of each case of death. When a particular category of death is seen to be problematic, the details of the individuals in that category can be reviewed. As with all records, they need to be used to inform management if they are to be any use at all. Therefore, we recommend periodic meetings between farm managers and veterinarians to consider death losses and what can be done to improve outcomes. More focus needs to be placed on evaluating subclinical disease problems. One of the problems with current record systems is that health events are only entered when they are obvious and prompt a treatment. Subclinical disease does not fit this category, and therefore information about subclinical cow problems cannot be retrieved to be compared with assessment of death losses. Consider for example the assessment of lameness on dairies. As noted above, high rates of lameness are strongly associated with high rates of death losses. However, most record systems monitor lameness only when cows receive specific treatment. It is unusual for dairies to do routine locomotion scoring that detects cows with more modest degrees of lameness. It is likely that management changes targeted to improving overall cow locomotion will also improve other aspects of cow health, and ultimately lead to decreased death losses. CONCLUSIONS There will not be a single simple answer to the problem of high mortality on dairies. Steps toward managing this challenge will require recognizing and defining the problem, improving information systems to provide details necessary to take action, and monitoring appropriate metrics that promote ongoing attention to management corrections.

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REFERENCES 1. Bascom SS, Young AJ. A summary of the reasons why farmers cull cows. J Dairy Sci 1998;81:2299-2305. 2. Dechow CD, Goodling RC. Mortality, culling by sixty days in milk, and production profiles in high- and low-survival Pennsylvania herds. J Dairy Sci 2008;91:4630-4639. 3. Dechow CD, Smith EA, Goodling RC. The effect of management system on mortality and other welfare indicators in Pennsylvania dairy herds. Animal Welfare 2011;20:145-158. 4. DHI Computing Services, Inc. P.O. Box 51427, Provo Utah 84605-1427, 800-453-9400. 2010. 5. Faust MA, Kinsel ML, Kirkpatrick MA. Characterizing biosecurity, health, and culling during dairy herd expansions. J Dairy Sci 2001;84:955-965. 6. Kelton DF, Lissemore KD, Martin RE. Recommendations for recording and calculating the incidence of selected clinical diseases of dairy cattle. J Dairy Sci 1998;81: 2502-2509. 7. Mason GL, Madden DJ. Performing the field necropsy examination. Vet Clin North Am Food Anim Pract 2007;23:503-526. 8. McConnel CS, Garry FB, Hill AE, Lombard JE, Gould DH. Conceptual modeling of postmortem evaluation findings to describe dairy cow deaths. J Dairy Sci 2010;93:373386. 9. McConnel CS, Garry FB, Lombard JE, Kidd JA, Hill AE, Gould DH. A necropsy-based descriptive study of dairy cow deaths on a Colorado dairy. J Dairy Sci 2009;92:19541962. 10. McConnel CS, Lombard JE, Wagner BA, Garry FB. Evaluation of factors associated with increased dairy cow mortality on United States dairy operations. J Dairy Sci 2008;91:1423-32. 11. Miller RH, Kuhn MT, Norman HD, Wright JR. Death losses for lactating dairy cows in herds enrolled in dairy herd improvement test plans. J Dairy Sci 2008;91:3710-3715. 12. Severidt JA, Madden DJ, Mason GL, Garry FB, Gould DH. 2002; Available from: http://www.cvmbs.colostate.edu/ilm/proinfo/necropsy/notes/index.html. Integrated Livestock Management, Colorado State University. 13. Thomsen PT, Houe H. Dairy cow mortality. A review Vet Q. 2006;28:122-129. 14. USDA. 2007. Dairy 2007, Part 1: Reference of dairy cattle health and management practices in the United States, 2007. USDA-APHISVS, CEAH, Fort Collins, CO. 15. USDA. 2007. Dairy 2007, Part II: Changes in the US dairy cattle industry, 1991-2007. USDA-APHIS-VS, CEAH, Fort Collins, CO. 16. Weigel KA, Palmer RW, Caraviello DZ. Investigation of factors affecting voluntary and involuntary culling in expanding dairy herds in Wisconsin using survival analysis. J Dairy Sci 2003;86:1482-1486.

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An Update on Hypocalcemia on Dairy Farms

G. R. Oetzel Department of Medical Sciences School of Veterinary Medicine University of Wisconsin-Madison Corresponding author: groetzel@wisc.edu SUMMARY Hypocalcemia (low blood calcium) is an important determinant of fresh cow health and milk production. Most second and greater lactation cows experience a transient hypocalcemia around the time of calving. Subclinical hypocalcemia (low blood calcium without obvious clinical signs) results in decreased early lactation milk yield, increased risk for displaced abomasum, and reduced fertility at first service. Subclinical milk fever is of greater economic importance than clinical cases of milk fever because it affects a higher proportion of cows. Measures to control hypocalcemia include nutritional approaches and strategic treatment with oral calcium around calving. INTRODUCTION Hypocalcemia is particularly amenable to strategies tailored to individual cows or targeted groups of cows. First, a substantial proportion of cows are affected by hypocalcemia. Average blood calcium concentrations noticeably decline in second or greater lactation cows around calving, with the lowest concentrations occurring about 12 to 24 hours after calving (Figure 1, Kimura et al., 2006; Goff, 2008). A cow does not necessarily have to become recumbent (down) to be negatively affected by hypocalcemia. With or without obvious clinical signs, hypocalcemia has been linked to a variety of secondary problems in post-fresh cows (Goff, 2008; Oetzel, 2011). This happens because blood calcium is essential for muscle and nerve function - particularly functions that support skeletal muscle strength and gastro-intestinal motility. Problems in either of these areas can trigger a cascade of negative events that ultimately reduce dry matter intake, increase metabolic diseases, and decrease milk yield (Goff, 2008). Subclinical hypocalcemia can be defined as low blood calcium concentrations without clinical signs of milk fever. Subclinical hypocalcemia affects about 50% of second and greater lactation dairy cattle fed typical pre-fresh diets. If anions are supplemented to reduce the risk for milk fever, the percentage of hypocalcemic cows is reduced to about 15 to 25% (Oetzel, 2004).

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Figure 1.Plasma concentrations of total calcium before and after calving in mature Jersey cows with or without clinical milk fever (Kimura et al., 2006).

Subclinical hypocalcemia is more costly than clinical milk fever because it affects a much higher percentage of cows in the herd (Oetzel, 2011). For example, if a 2000-cow herd has a 2% annual incidence of clinical milk fever and each case of clinical milk fever costs $300 (Guard, 1996), the loss to the dairy from clinical cases is about $12,000 per year. If the same herd has a 30% annual incidence of subclinical hypocalcemia in second and greater lactation cows (assume 65% of cows in the herd) and each case costs $125 (an estimate that accounts for milk yield reduction and direct costs due to increased ketosis and displaced abomasum), then the total herd loss from subclinical hypocalcemia is about $48,750 per year. This is about 4 times greater than the cost of the clinical cases. A recently published, large multi-site study shows that hypocalcemia around calving is most strongly associated with reduced milk yield (Chapinal et al., 2012) and increased risk for displaced abomasum (Chapinal et al., 2011). These studies also demonstrated that the cutpoint for serum total calcium is higher (about 8.5 mg/dl) than was previously assumed (see Figures 2 and 3).

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Figure 2.Effect of serum total calcium on milk yield for the first 4 DHI tests after calving. Different cutpoints were derived for serum samples collected on weeks -1, 1, 2, and 3 after calving. Data are from 2,365 cows in 55 Holstein herds in Canada and the US (Chapinal et al., 2012).

Figure 3.Effect of serum total calcium on the odds for displaced abomasum after calving. Different cutpoints were derived for serum samples collected on weeks -1, 1, 2, and 3 after calving. Data are from 2,365 cows in 55 Holstein herds in Canada and the US (Chapinal et al., 2011).

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CLINICAL SIGNS AND TREATMENTS FOR HYPOCALCEMIA Clinical Signs of Hypocalcemia Clinical signs of milk fever in dairy cattle around calving may, for convenience, be divided into three stages. Stage I milk fever is early signs without recumbency. It may go unnoticed because its signs are subtle and transient. Affected cattle may appear excitable, nervous, or weak. Some may shift their weight frequently and shuffle their hind feet (Oetzel, 2011). Some cows become hypocalcemic at times other than calving and exhibit clinical signs similar to those described above for Stage I milk fever. Such non-parturient hypocalcemias are often triggered by periods of unusual stress or decreased dry matter intake. This condition is most commonly seen in cows in the first 2 to 10 days of lactation, cows that are in heat, cows with severe digestive upsets, or cows suffering from severe (toxic) mastitis (Oetzel, 2011). Treatment Options for Hypocalcemia Oral calcium supplementation is the best approach for hypocalcemic cows that are still standing, such as cows in Stage 1 hypocalcemia or who have undetected subclinical hypocalcemia (Oetzel, 2011). A cow absorbs an effective amount of calcium into her bloodstream within about 30 minutes of supplementation (Goff and Horst, 1993). Blood calcium concentrations are supported for only about four to six hours afterwards (Goff and Horst, 1993; 1994) for most forms of oral calcium supplementation. Intravenous (IV) calcium is not recommended for treating cows that are still standing (Oetzel, 2011). Treatment with IV calcium rapidly increases blood calcium concentrations to extremely high and potentially dangerous levels (Goff, 1999). Extremely high blood calcium concentrations may cause fatal cardiac complications and (perhaps most importantly) shut down the cow's own ability to mobilize the calcium she needs at this critical time (Oetzel, 2011). Cows treated with IV calcium often suffer a hypocalcemic relapse 12 to 18 hours later (Curtis et al., 1978; Thilsing-Hansen et al., 2002). The problems with IV calcium treatment are illustrated in Figure 4. Cows in Stage II milk fever are down but not flat out on their side. They exhibit moderate to severe depression, partial paralysis, and typically lie with their head turned into their flank. Stage III hypocalcemic cows are flat out on their side, completely paralyzed, typically bloated, and are severely depressed (to the point of coma). They will die within a few hours without treatment (Oetzel, 2011). Stage II and Stage III cases of milk fever should be treated immediately with slow IV administration of 500 ml of a 23% calcium gluconate solution. This provides 10.8 grams of elemental calcium, which is more than sufficient to correct the cows entire deficit of calcium (about 4 to 6 grams). Giving larger doses of calcium in the IV treatment has no benefit (Doze et al., 2008). Treatment with IV calcium should be given as soon as possible, as recumbency can quickly cause severe musculoskeletal damage.

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Figure 4.Effect of IV calcium treatment with 10.5 g of elemental calcium on serum total calcium concentrations in a mature Jersey cow with clinical milk fever (Goff, 1999).

To reduce the risk for relapse, recumbent cows that respond favorably to IV treatment need additional oral calcium supplementation once they are alert and able to swallow, followed by a second oral supplement about 12 hours later (Thilsing-Hansen et al., 2002; Oetzel, 2011). Transient hypocalcemia can occur in cows whenever they go off feed or have periods of decreased intestinal motility (DeGaris and Lean, 2008). It can be difficult to tell which comes first - the hypocalcemia or the gastrointestinal stasis. Whatever the case, the two problems can positively reinforce each other. During the experimental induction of hypocalcemia (Huber et al., 1981), ruminal contractions ceased well before the onset of clinical signs of milk fever. Offfeed cows, particularly in early lactation, are very likely to benefit from prompt oral calcium supplementation. Oral Calcium Supplementation Strategies Even herds with successful anionic salts programs and minimal clinical cases of milk fever will benefit from strategic use of oral calcium supplements (Oetzel and Miller, 2012). Start by supplementing all standing cows who have clinical signs of hypocalcemia and all down cows following successful IV treatment. For herds with a high incidence of hypocalcemia, it may also be economically beneficial to strategically supplement all fresh cows with oral calcium. Finally, cows with high milk yield in the previous lactation (>105% of herd average ME milk production) and lame cows have the best response to oral calcium supplementation (Oetzel and Miller, 2012). These cows gave 6.8 lbs more milk at first DHI test compared to unsupplemented cows. 121

The source of calcium in an oral supplement and its physical form greatly influence calcium absorption and blood calcium responses. A series of experiments has shown that calcium chloride has the greatest ability to support blood calcium concentrations (Goff and Horst, 1993; 1994). This can be explained by its high calcium bioavailability and its ability to invoke an acidic response in the cow, which causes her to mobilize more of her own calcium stores. Providing a typical amount of elemental calcium chloride (e.g., 50 grams of elemental calcium) in a small oral dose (e.g., 250 ml water) provided the best absorption (Figure 5). Administering 100 grams of elemental calcium from calcium chloride in water resulted in an excessive increase in blood calcium concentrations - perhaps enough to shut down the cows own calcium homeostatic mechanisms and to invoke a calcitonin response to protect her from hypercalcemia. Figure 5.Effect of two different doses of oral calcium chloride on plasma total calcium concentrations, expressed as percent of baseline values (Goff and Horst, 1993).

The risk of aspiration is great when thin liquids are given orally, and calcium chloride is very caustic to upper respiratory tissues. Calcium propionate is more slowly absorbed (presumably because it is not acidogenic) and must be given at higher doses of elemental calcium (usually 75 to 125 grams - see Figure 6). Calcium propionate has the property of being glucogenic as well as providing supplemental calcium. Calcium carbonate in water did not increase blood calcium concentrations at all (see Figure 7, Goff and Horst, 1993). This may be explained by its poorer bioavailability and by the alkalogenic response it can invoke.

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Figure 6.Effect of oral calcium chloride and oral calcium propionate on plasma total calcium concentrations, expressed as percent of baseline values (Goff and Horst, 1993; 1994) (Goff and Horst, 1993, 1994).

Figure 7. Effect of oral calcium chloride and oral calcium carbonate on plasma total calcium concentrations, expressed as percent of baseline values (Goff and Horst, 1993).

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A combination of calcium chloride and calcium sulfate delivered in a fat-coated bolus (Bovikalc, Boehringer Ingelheim Vetmedica, St. Joseph, MO) resulted in more sustained improvements in blood calcium concentrations (see Figure 8) than were observed in previous studies with oral calcium chloride or calcium propionate in water (Sampson et al., 2009). This encapsulated version of calcium salts had the advantages of not having an unpleasant taste to the cow, having little to no waste of the oral formulation, no risk for aspiration pneumonia, and a more prolonged release of the oral calcium (Pehrson and Jonsson, 1991). These workers reported a 4-fold reduction in the odds for developing clinical milk fever in cows that were supplemented with 4 boluses around calving (Pehrson and Jonsson, 1991). Figure 8.Effect of administration of two Bovikalc boluses on blood ionized calcium concentrations (expressed as percent of baseline) at calving and 12 hours later. Experimental animals were Holstein cows (n=20) with hypocalcemia at calving (Sampson et al., 2009).

Subcutaneous Calcium Treatment Subcutaneous calcium can be used to support blood calcium concentrations around calving, but has substantial limitations (Goff, 1999). Absorption of calcium from subcutaneous administration requires adequate peripheral perfusion. It may be ineffective in cows that are severely hypocalcemic or dehydrated. Subcutaneous calcium injections are irritating and can cause tissue necrosis; administration should be limited to no more than 75 ml of a 23% calcium gluconate solution (about 1.5 g elemental calcium) per site. Calcium solutions that also contain glucose should not be given subcutaneously. Glucose is very poorly absorbed when given by this route. Abscessation and tissue sloughing may result when glucose is given subcutaneously. The kinetics of subcutaneously administered calcium indicate that it is well-absorbed initially, but that blood concentrations fall back to baseline values in about 6 hours (see Figure 9, Goff, 124

1999). Thus, repeat doses would be necessary to equal the sustained blood calcium support that is possible with oral calcium boluses. Figure 9. Effect of subcutaneous administration of 500 ml of 23% calcium gluconate on plasma total calcium, expressed as percent of baseline. The 500 ml solution was divided into 10 different sites (Goff, 1999).

Timing of Oral Calcium Supplementation Relative to Calving Strategies for giving oral calcium supplements around calving should include at least two doses; one at calving calving and a second dose the next day. The expected nadir in blood calcium concentrations occurs between 12 and 24 hours after calving (Goff, 1999; Sampson et al., 2009). Giving only one oral calcium supplement around calving time leaves the cow without support when her blood calcium concentrations are naturally the lowest. It is interesting to note that the original protocols for oral calcium supplementation called for 4 doses - one about 12 hours before calving, one at calving, one 12 hours post-calving, and one 24 hours post-calving. It was very difficult to predict when at cow was in fact about 12 hours from expected calving, and many cows calved without receiving this dose (Oetzel, 1996). The dose at calving is not practically challenging to administer, and providing a dose sometime the day after calving will provide critical support around the time of nadir and can still be practical in large dairies where the post-fresh pen is locked up just once daily. CONCLUSIONS New research information has refined and updated our understanding of hypocalcemia on dairy farms. We now have a better understanding of the prevalence and impact of hypocalcemia (particularly subclinical hypocalcemia) on fresh cow performance. Strategic oral 125

supplementation can mitigate the impacts of hypocalcemia, provided the oral supplements are in the correct form and are administered at the proper time. REFERENCES Chapinal, N., M. Carson, T. F. Duffield, M. Capel, S. Godden, M. Overton, J. E. Santos, and S. J. LeBlanc. 2011. The association of serum metabolites with clinical disease during the transition period. J. Dairy Sci. 94:4897-4903. Chapinal, N., M. E. Carson, S. J. LeBlanc, K. E. Leslie, S. Godden, M. Capel, J. E. Santos, M. W. Overton, and T. F. Duffield. 2012. The association of serum metabolites in the transition period with milk production and early-lactation reproductive performance. J. Dairy Sci. 95:1301-1309. Curtis, R. A., J. F. Cote, M. C. McLennan, J. F. Smart, and R. C. Rowe. 1978. Relationship of methods of treatment to relapse rate and serum levels of calcium and phosphorous in parturient hypocalcaemia. Can. Vet. J. 19:155-158. DeGaris, P. J., and I. J. Lean. 2008. Milk fever in dairy cows: A review of pathophysiology and control principles. Vet. J. 176:58-69. Doze, J. G., R. Donders, and J. H. van der Kolk. 2008. Effects of intravenous administration of two volumes of calcium solution on plasma ionized calcium concentration and recovery from naturally occurring hypocalcemia in lactating dairy cows. Am. J. Vet. Res. 69:13461350. Goff, J. P. 1999. Treatment of calcium, phosphorus, and magnesium balance disorders. Vet. Clin. North Am. Food Anim. Pract. 15:619-639. Goff, J. P. 2008. The monitoring, prevention, and treatment of milk fever and subclinical hypocalcemia in dairy cows. Vet. J. 176:50-57. Goff, J. P., and R. L. Horst. 1993. Oral administration of calcium salts for treatment of hypocalcemia in cattle. J. Dairy Sci. 76:101-108. Goff, J. P., and R. L. Horst. 1994. Calcium salts for treating hypocalcemia: Carrier effects, acidbase balance, and oral versus rectal administration. J. Dairy Sci. 77:1451-1456. Guard, C. L. 1996. Fresh cow problems are costly; culling hurts the most. Hoard's Dairyman 141:8. Huber, T. L., R. C. Wilson, A. J. Stattleman, and D. D. Goetsch. 1981. Effect of hypocalcemia on motility of the ruminant stomach. Am. J. Vet. Res. 42:1488-1490. Kimura, K., T. A. Reinhardt, and J. P. Goff. 2006. Parturition and hypocalcemia blunts calcium signals in immune cells of dairy cattle. J. Dairy Sci. 89:2588-2595. Oetzel, G. R. 1996. Effect of calcium chloride gel treatment in dairy cows on incidence of periparturient diseases. J. Am. Vet. Med. Assoc. 209:958-961. Oetzel, G. R. 2004. Monitoring and testing dairy herds for metabolic disease. Vet. Clin. North Am. Food Anim. Pract. 20:651-674. Oetzel, G. R. 2011. Non-infectious diseases: Milk fever. Pages 239-245 in Encyclopedia of Dairy Sciences. Vol. 2. J. W. Fuquay and P. L. H. McSweeney, ed. Academic Press, San Diego. Oetzel, G. R., and B. E. Miller. 2012. Effect of oral calcium bolus supplementation on early lactation health and milk yield in commercial dairy herds. J. Dairy Sci. 95:7051-7065. Pehrson, B., and M. Jonsson. 1991. Prevention of milk fever by oral administration of encapsulated Ca-salts. Bov. Pract. 26:36-37.

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Sampson, J. D., J. N. Spain, C. Jones, and L. Carstensen. 2009. Effects of calcium chloride and calcium sulfate in an oral bolus given as a supplement to postpartum dairy cows. Vet. Ther. 10:131-139. Thilsing-Hansen, T., R. J. Jrgensen, and S. stergaard. 2002. Milk fever control principles: A review. Acta Vet. Scand. 43:1-19.

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Performance of Calves in Different Calf Feeding Systems

Mark Hill, Gale Bateman, II, Jim Quigley, Jim Aldrich, and Rick Schlotterbeck Nurture Research Center, Provimi North America, Brookville, OH 45309 Corresponding author: mhill@provimi-na.com SUMMARY A large percentage of US dairy calves are fed approximately 1 lb of milk or milk replacer (MR) solids (i.e. 1 gallon) daily. Calf growth in the first month of life can be increased by feeding calves approximately twice this liquid rate but delays intake of starter and may reduce growth in the weeks around weaning unless the liquid is gradually reduced over approximately 3 weeks to allow rumen development. Intermediate amounts (i.e. 1.5 gallons) of liquid diets are easy to manage and less apt to interfere with starter intake or weaning growth. Nutrient densities of the liquid diet for optimal growth differ depending upon amount of liquid fed. For example, a MR fed at 1.5 to 2 lb (1.5 to 2 gallons) should have a greater CP concentration than a MR fed at 1 lb (1 gallon). Balancing diets correctly to not over-feed or underfeed nutrients typically reduced the cost of body weight gain and with some nutrients and situations the reduced costs are substantial. INTRODUCTION A large percentage of US dairy calves are fed approximately 1 lb of milk or MR solids (i.e. 1 gallon) daily. The number of operations feeding more than this amount of milk or MR solids has increased over the last 10 plus years (USDA, 2012) much because of research from Dr. Mike Van Amburghs laboratory at Cornell University (Diaz et al., 2001). This has not been without controversy as to what was nutritionally correct, economical, or humanely appropriate. Some in the industry would suggest that marketing and opinion has driven the increase in milk or MR fed. Nonetheless, it has been a great challenge for the industry to consider how calves are fed. This paper and presentation will review the published literature from the last 10 to 15 years on dairy calf feeding programs for calves less than approximately 4 months of age. Strengths, weaknesses, and nutrients provided with different feeding programs will be mentioned and based on published research trials. CONVENTIONAL PROGRAMS Two quarts of milk or reconstituted MR per feeding has been popular for over 40 years in the US. Conventional MR are typically 22 to 24% CP (DM basis; 20 to 22% CP as-fed basis) and 22% fat (DM basis). Milk is typically 25 to 28% CP and even higher in fat (DM basis). Feeding 1 lb of a 22% CP and 22% fat MR provides sufficient ME and CP for maintenance and limited body weight gain (approximately 0.4 lb/day) under thermoneutral conditions. This low feeding rate promotes an early appetite for starter feed and allows weaning as early as 28 days of age. Indeed, early weaning programs developed in the 1950s and 1960s wer e predicated on 128

limited MR, early and aggressive intake of starter and early weaning. One important feature of early weaning programs was lower cost, since saleable milk or MR is expensive compared to dry starter. Additionally, labor costs associated with feeding and managing calves in individual housing fed a liquid diet are high compared to older weaned calves in group housing fed a dry starter (Tozer and Heinrichs, 2001). Most calves are raised in individual pens or hutches. Numerous studies have shown that reducing or eliminating calf-to-calf contact prior to weaning reduces predisposition to disease, improves health and growth of calves. Individual plastic hutches that are positioned to eliminate calf contact are very effective in reducing disease transmission as long as basic biosecurity procedures are followed. See a review of individual housing research by Quigley (2001) for this and more information. Calf pens in barns may use solid or wire panels to separate calves. Solid petitions, while eliminating calf-to-calf contact may compromise ventilation within the barn. See the recent paper of Hill et al. (2011a) for a partial review of these data. Recent unreviewed data suggest that single or small group housing does not affect markers of immunity (Hulbert et al., 2012abc). INTENSIVE FEEDING PROGRAMS As more milk or MR is fed pre-weaning, ADG increases (Jasper and Weary, 2002; Cowles et al., 2006; Hill et al., 2006ab, 2007d). The amount of increased ADG is a function of the nutrients provided and will be addressed in a subsequent section. However, when the amount of milk or MR fed is more than approximately 1.5 lb DM, post-weaning ADG will be compromised compared to calves that are hand-fed greater amounts of milk (Bar-Peled et al., 1997; Jasper and Weary, 2002), hand-fed non-acidified MR (Cowles et al., 2006; Hill et al., 2006ab, 2007d), or allowed free-choice access to acidified MR (Nocek and Braund, 1986; Hepola et al., 2008). Two laboratories have measured total tract apparent DM digestion postweaning in calves fed low or high amounts of MR and both reported 6 to 9% lower digestion in calves fed the high level of MR (Terre et al., 2006, 2007; Hill et al., 2010). The low digestion in calves fed high levels of MR was associated with less development of the rumen (Terre et al., 2006, 2007; Suarez-Mena et al, 2011). So the conundrum is how to feed more milk or MR and maintain the increased body weight after the calf transitions to starter. One option is to not feed more than about 1.5 lb DM per day via the liquid diet. Another approach is to gradually wean calves over 14 to 25 days (Khan et al., 2007; Hill et al., 2007d, 2012b; Sweeney et al., 2010). Gradual reduction of the liquid diet forces the calf to find energy from other sources, including the starter. The calf requires about 14 days for the rumen to adapt to increasing starter intake, so gradual weaning from high volumes of liquid require 2 weeks or more. We assembled data from 10 peer-reviewed studies at 6 different laboratories where a conventional (control) MR was fed along with one or more intensified MR treatments (Table 1). A simple covariate analysis was conducted. Note the moderate and intensive MR treatments were 26.5% and 29% CP, respectively, compared to 21.4% for calves fed the control treatments. Calves fed the moderate rate of MR gained 29% better and calves fed the intensive treatments gained 41% better than calves fed the control milk replacer treatments during the pre-weaning period. Starter intake of calves fed the intensive treatment were 67% of the calves fed the control. Feed efficiency (gain to feed) of calves fed the intensive treatment were 29% better than calves fed the control. Starter intake and feed efficiency of calves fed the moderate 129

treatment were 4% less and 9% better than the calves fed the control. However, during postweaning periods calves fed the intensive treatments gained less weight than calves fed the control and calves the moderate treatment gained more weight than calves fed the control. Overall (115 total days) calves fed the moderate and intensive treatments gained 12% and 6% better, respectively, the rate of calves fed the control (Table 2). These studies in this database, individually, and when combined make a compelling case that feeding more than approximately 1 lb of solids from milk or MR has merit for improving early life growth in calves. However, they also clearly point to issues with feeding over 1.5 lb of solids from milk or MR. Table 1. Average milk replacer composition, intake, and pre-weaning performance from 10 peer-reviewed studies where a conventional milk replacer was fed along with a moderate and/or intensive milk replacer. Item Days MR intake, lb/day MR CP, % MR fat, % Starter intake, lb/day ADG, lb/day Gain/Feed Control 44.1 1.06 21.4% 20.4% 1.34 1.08 0.450 Moderate 41.1 1.54 26.5% 16.9% 1.28 1.39 0.493 Intensive 45.9 1.98 29.0% 17.8% 0.90 1.52 0.528

References: Cowles et al., 2006; Hill et al., 2006b; Raeth-Knight et al., 2009; Hill et al., 2010; Davis Rinker et al., 2010; Stamey et al., 2012; Osorio et al., 2013. Table 2. Intake, body weight gain, and feed efficiency from 10 peer-reviewed studies where a convention milk replacer was fed along with a moderate and/or intensive milk replacer (total days = 115.6). Control 47 546 212 0.357 Moderate 63 617 239 0.351 Intensive 91 535 226 0.361

Item MR, lb DM Dry feed DM, lb Body weight gain, lb Gain/Feed

References: Cowles et al., 2006; Hill et al., 2006b; Raeth-Knight et al., 2009; Hill et al., 2010; Davis Rinker et al., 2010; Stamey et al., 2012; Osorio et al., 2013. IMPACT OF SPECIFIC NUTRIENTS The optimum protein to energy ratio for ADG in calves fed only MR has been estimated to be approximately 48 g CP/Mcal ME in 2 trials (Bartlett et al., 2005; Blome et al., 2003). A similar estimate was made by Donnelly and Hutton (1976) in calves fed only MR. Calves in the MR only trials were over 2 weeks of age when the trials began and were managed in conditions close to thermoneutrality, which may have influenced the results. Hill et al. (2009b) estimated optimum CP:ME ratio in calves fed starter and MR at 2 rates to achieve different ME intakes. 130

Optimum protein to energy ratios were 52 (low ME intake) and 55 (high ME intake) g CP/Mcal ME. Calves in these trials were 2 to 3 days of age when the trials began and were housed in a nursery mostly below thermoneutrality on straw bedding. Also, calves were vaccinated and dehorned during the trial. These trials demonstrate the need for more than 22% CP (DM basis) in MR fed at rates over the conventional rate fed in the U.S. (approximately 1 lb DM from MR/calf day). This has also been shown in MR program trials where calves were fed both MR and starter (Hill et al., 2006ab). Increased fat and ME intake from MR has frequently resulted in either no change or reduced ADG, protein accretion, and measures of structural growth (Bascom et al., 2007; Van den Borne, 2007; Hill et al., 2009cd). Hill et al. (2009c) reported reduced digestion of most nutrients as fat increased from 14 to 23% in 27% CP MR in a trial conducted in winter months with ambient temperatures well below thermoneutrality. While milk is typically high in fat, MR does not have to be formulated that way. Milk replacers that fall in the range of optimum protein to energy ratios, as discussed above, are approximately 25 to 27% CP and 16 to 17% fat (each DM basis; Tikofsky et al., 2001; Bascom et al., 2007; Hill et al., 2009b). Yet most MR contain 20% fat whether conventional or intensive (22 to 29% CP) and therefore have a lower than optimum CP to energy ratio. Milk replacers for calves in the US are typically formulated with animal fat and are relatively low in butyric acid, medium chain fatty acids, and linolenic acid. Additionally, calf starters and growers are relatively low in these fatty acids. In research where these fatty acids were supplemented to calves, ADG, hip width growth, and feed efficiency were increased approximately 7+% and days with abnormally loose feces were reduced (Hill et al., 2007bce, 2009a, 2011ab; Fokkink et al., 2009). Additionally, calves supplemented with butyric, medium chain, and linolenic have improved markers of both short and long term immunity (Hill et al. 2011bc). Recent estimates of amino acid requirements have been made in 11 trials with calves fed both starter and MR (Hill et al. 2007c, 2008a, 2011d). The benefit from balancing MR for amino acids vs. CP was approximately 9% more ADG and feed efficiency in calves fed 22% CP (DM basis) MR at 1 lb DM/day and over 15% for calves fed high protein MR (> 25% CP) at 1.5 lb DM/day. IMPORTANCE OF STARTER INTAKE Equations from the Dairy NRC (2001) suggest that energy limits ADG in typical dry diets fed to weaned calves. As forage is increased in the weaned calfs diet, energy becomes more limiting compared to diets containing no or low forage. Recent publications from different labs each using over 900 calves report that ADG of the dairy calf between birth and 2 mo of age is positively related to starter intake (Heinrichs and Heinrichs, 2011; Bateman et al., 2012). The analysis of Bateman et al. (2012) was very dynamic across many feeding rates, compositions of MR programs, and seasons of the year. Starter intake is affected by many factors, including particle size of the diet (Lassiter et al., 1955; Gardner, 1967; Kertz et al., 1979; Bateman et al., 2009), roughage concentration and amount fed (Warner et al., 1956; Stobo et al., 1966; Jahn and Chandler, 1976; Porter et al., 131

2007; Hill et al., 2008b, 2009e), amount of milk (Bar-Peled et al., 1997; Jasper and Weary, 2002) or MR fed (Kertz et al., 1976; Terre et al., 2006, 2007; Suarez-Mena at al., 2011; Stamey et al., 2012), and weaning program utilized (Hill et al.,2007d, 2012b; Kahn et al., 2007; Sweeney et al., 2010). Because so many factors impact starter intake, and because starter intake is positively related to growth of dairy calves (Heinrichs and Heinrichs, 2011; Bateman et al., 2012), it is important to design feeding programs and diets to maximize starter intake in the transition calf. Few new papers have addressed CP requirements of the calf during the transition to dry feed. Optimum concentrations of CP for starters fed to calves on conventional milk and MR programs from birth to shortly after weaning were19 to 20% CP (DM basis; Lucini et al., 1991; Akayezu et al., 1994) which is similar to the Dairy NRC (2001). Calves fed conventional milk or MR at approximately 1 lb DM/day or MR (> 25% CP) at > 1.5 lb DM/day had optimum ADG with starters containing 20.5% CP (63 g CP/Mcal ME; Hill et al., 2007a, 2008c; Stamey et al., 2012). The optimum CP in starters for weaned calves from 2 to 4 mo of age was 17% (to maximize ADG) to 18% (to maximize feed efficiency; 52 to 56 g CP/Mcal ME: Hill et al., 2008c). GROUP HOUSING PROGRAMS Group housing of calves is an area of interest in North America. Most of the interest has been in eastern Canada, New York, and New England states. Several researchers have evaluated the effect of ad libitum MR feeding on intake and growth of calves (Thickett et al., 1983; Nocek and Braund, 1986; Fallon and Harte, 1988; Richard et al., 1988; Hepola et al., 2008; Hill et al., 2013). Where post-weaning measurements were made (Nocek and Braund, 1986; Hill et al., 2013), calves fed the ad libitum plane of nutrition had greater ADG pre-weaning, but less ADG post-weaning compared to calves fed conventionally (approximately 1 lb DM/day) in individual pens. Patterns in MR intake, starter intake, and growth in ad libitum group-housed systems were similar to calves hand-fed intensive MR in individual housing. Hill et al. (2013) reported when calves were fed MR ad libitum, they consumed over 95% of the MR between 6 to 8 AM and 4 to 6 PM and standing behavior was not greatly altered compared to calves fed hand-fed 1.5 lb DM of MR/day. INTRINSIC BENEFITS OF ENHANCED NUTRITION There are 7 peer-reviewed trials comparing 2 or more levels of milk or MR nutrition on the impact of future milk production (Bar-Peled et al., 1997; Shamay et al., 2005; Morrison et al., 2009; Raeth-Knight et al., 2009; Terre et al., 2009; Moallem et al., 2010; Davis Rincker et al., 2011). Only 2 of these trials report significant increases in milk production because of enhanced nutrition (Shamay et al., 2005; Moallem et al., 2010). Those trials both compared an inferior MR program to milk. Four of the 5 other trials reported trends for improved milk yield. More details for these trials were summarized by Heinrichs and Jones (2011) along with some non peer-reviewed trials. We gathered the treatment means from the 7 peer-reviewed trials and used them in regression analysis. There were no statistically significant or biologically meaningful relationships between amount of fat or protein fed to the calves and future milk fat or milk protein yield. However, there was a significant relationship between pre-weaning ADG and future milk yield. 132

Van Amburgh (2012) performed a meta-analysis on a similar set of peer-reviewed trials but also included several non-reviewed trials. He reported a positive relationship between feeding calves more nutrients from milk or MR and future milk production, as well as, a positive relationship with increased pre-weaning ADG and future milk. There are 3 additional peer-reviewed studies correlating calf ADG on future milk production. Each took very different approaches to collecting data and the statistical analysis and because of these differences, creates concerns when attempting to group their results with those of the other 7 studies used in our statistical analysis. Bach and Ahedo (2008) correlated calf ADG to future milk from a calf ranch in Spain with 900 heifers and found no statistical relationship. In this analysis, calf ADG varied from 0.8 to 2.5 lb/day. Heinrichs and Heinrichs (2011) collected farm data from a county in PA, USA over several years. The data set comprised over 900 animals. Starter intake during the first 2 months of life was positively correlated with calf ADG during the first 2 months of life and future milk production. Amount of milk or MR fed did not correlate to future milk yield. This data set was predominately calves fed approximately 1 lb of DM from milk or a conventional MR. Soberon et al. (2012) collected data (approximately 1,800 animals) from the Cornell University herd and a nearby commercial herd. The university calves were fed either a 28% CP, 15% fat MR powder or a 28% CP, 20% fat MR powder at approximately 2 to 2.2 lb DM per calf daily. The commercial farm calves were fed a 28% CP, 15% fat MR powder at approximately 1.9 lb DM per calf daily. The amount of MR fed at each site was fixed and not varied to change calf ADG. Even though the amount of MR was fixed, calf ADG varied from 0.2 to 3.5 lb/day. Calf ADG was positively correlated with future milk production. This paper did not describe several key factors that could relate calf ADG differences to future milk such as starter intake and colostrum consumption (or immunoglobulin absorption) of the calves. However, the final relationship was for 1,541 lb more milk in the first lactation per increase in pre-weaning ADG by 1 lb (a linear relationship). Several laboratories have compared feeding calves conventional MR at approximately 1 lb DM daily to high 29% CP MR at approximately 2 lb DM daily and measured markers of innate immunity. Few if any differences in innate immunity between the 2 programs were observed and the few differences were post-weaning (Nonnecke et al., 2003; Foote et al., 2005, 2007; Hengest et al., 2012; Ballou, 2012). We are not aware of trials that have reported reductions in digestive or respiratory sickness using intensive or moderately intensive milk or MR programs compared to conventional programs, however some report more sickness with intensive programs (Cowles et al., 2006; Hill et al., 2006a; Quigley et al., 2006; Hengest et al., 2012). During periods of cold or heat stress the maintenance requirement of calves increase (NRC, 2001). Housing situations where the bedding does not insulate the calf (i.e. concrete, metal, wood, rock, sand, shavings, or any type of wet bedding) increase the maintenance requirements of the calves in cool and cold weather. Housing and bedding management ameliorates cold stress to a significant degree (Hill et al., 2007d, 2011a, 2012a). Bateman et al. (2011) conducted a meta-analysis of 20 trials across many feeding rates, compositions of MR programs, and seasons of the year with ambient temperatures in the unheated nursery ranging from near 0 to 100 F (average trial temperatures ranged from 22 to 78 F). In this meta analysis, calf ADG increased as ambient temperature decreased. Heat stress appears harder to ameliorate than cold stress with cooling using fans reported to reduce panting and increase ADG during summer heat stress (Hill et al., 2011a, 2012a). Moderate increases of (25 to 50%) in the amount of milk or 133

MR fed when using conventional programs feeding 1 lb of DM to 1.25 to 1.5 lb of DM are justified in extreme cold or heat stress (Hill et al., 2007d, 2011a, 2012a). However, management of environment with insulating, dry bedding and draft (wind) protection are critical to the calf before more calories are fed (Hill et al., 2007d). ECONOMIC CONSIDERATIONS Costs can be applied to the performance summary of the 10 trial summary in Table 2 to partially access the economics of MR feeding rate programs. In this example, costs of $1.00/lb for control MR, $1.18/lb for moderate MR, $1.24/lb for intensive MR, and $0.23/lb for dry feed were used. Total feed costs and cost per lb of body weight gain were lowest for the control ($171/calf, $0.81/lb), intermediate from the moderate program ($215/calf, $0.90/lb), and highest ($234/calf, $1.04/lb) for the intensive program. Other economic considerations could be based on the value of body weight gain and calculate the value of body weight gain over the total feed cost. Medical and health associated costs should be similar among feeding programs, possibly higher with intensive vs. moderately intensive or control programs (Cowles et al., 2006; Hill et al., 2006a; Quigley et al., 2006; Hengest et al., 2012). The economics of waste milk would be different since the cost of waste milk is presumed less than MR. The intrinsic values less certain to estimate are future milk, less days to first calving, or the ability of larger calves to compete as heifers. Prorating the 1,541 lb of increased milk in the first lactation per 1 lb of ADG estimated by Soberon et al. (2012) to the 10 trial summary data in Table 1 yields an estimate of 478 and 678 more lb of milk in the first lactation for moderate and intensive programs, respectively, compared to the control group. This translates to $91 and $121 for more income from milk in the first lactation for moderate and intensive programs, respectively, compared to the control group. This estimated increase in income more than offsets the greater costs of the moderate and intensive programs compared to the control group and suggests value to moderate and intensive programs. Labor costs of group housed, free-choice feeding programs might be lower than individually housed, hand-fed programs. Karszes (2012) estimated costs in the first 13 weeks of age on 22 farms in NY, USA. Labor costs in group-housed programs were estimated to be $0.83/calf daily compared to $1.36/calf daily with individually housed calves. Additionally, other non-feed and labor costs were $1.25/calf daily in group-housed programs compared to $1.05/calf daily in individually housed programs. Practically on farms, there are some opportunities to improve the fat and fatty acid profile of calf diets. Minimize or avoid addition of fat and oil to calf starters and growers will typically lower the cost of a feed. Minimize or avoid using high fat and oil ingredients like soybeans and distillers grains. These options will help to minimize the linoleic acid content of the diet that is typically high. Supplement commercial sources of butyric acid, medium chain fatty acids, and linolenic acid to milk or MR, starters, and grower feeds. The cost is less than $1 per bag to milk formulas and less than $15/ton to starters and growers. This calculates to less than $1 per calf when supplemented to the milk formula or the starter feed pre-weaning and approximately $3 per calf when supplemented post-weaning. Correcting the fatty acid concentrations of the calf 134

diets in these ways reduce the costs associated with health issues, lowers the cost of body weight gain approximately 4 to 5% when improving calf ADG approximately 7 to 10% over the first 4 months of life. The amino acid profile of a MR is also practical to improve with little increase in feed costs. A 9% improvement in ADG pre-weaning in conventional MR programs with no change in feed cost is an 8% reduction in feed cost per unit body weight gain. A 15% improvement in ADG pre-weaning in moderate intensive MR programs with no change in feed cost is a 13% reduction in feed cost per unit body weight gain. Formulating a MR for amino acid requirements versus CP allows for a MR that is 2 to 4 percentage points lower in CP, another way to lower costs while maintaining calf performance. FINAL COMMENTS Feed costs to raise calves can be substantial. Milk replacer is only a portion of the total costs to rear a dairy calf to a milking cow but it has a high cost per lb. Understanding of how to select the right milk replacer nutrient profile and feeding rate has been addressed with an abundance of research over the last 10 to 15 years. Additionally, correcting for underfed nutrients (i.e. protein, amino acids, and fatty acids) can substantially reduce feed costs per unit body weight gain. During cold and heat stress, conventional programs based on approximately 1 gallon of liquid milk or MR (or 1 lb of DM) provides inadequate nutrients in many rearing and management programs. Moderate and intensive programs have been controversial but do increase calf ADG and possibly future milk production. When considered, they need to provide the proper protein (amino acid) to energy ratio to achieve protein tissue and frame growth, and this type of growth is what is possibly linked with future enhancement in milk production. Housing cannot be overlooked. Housing that allows drafts should be evaluated in the cool and cold weather since maintenance requirements are increased considerably in these situations. Housing situations where the bedding does not insulate the calf (i.e. concrete, metal, wood, rock, sand, shavings, or any type of wet bedding) increase the maintenance requirements of the calves in cool and cold weather. During periods of hot weather, heat stress reduces calf ADG and heat abetment systems (i.e. fans or ways to gain more airflow such as elevating the back side of poly hutches) should be implemented. Labor (including management) is a high cost along with many liquid diets (saleable milk or MR) of the milk fed calf. Finding a balance between labor and feed costs is important and can be challenging with various calf programs. Group housing, weaning age, and liquid feeding program each impact cost and growth of calves.

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REFERENCES Akayezu, J. M., J. G. Linn, D. E. Otterby, W. P. Hansen, and D. G. Johnson. 1994. Evaluation of calf starters containing different amounts of crude protein for growth of Holstein calves. J. Dairy Sci. 77:1882-1889. Bach, A., and J. Ahedo. 2008. Record keeping and economics for dairy heifers. Veterinary Clinics of North America - Food Animal Practice 24:117-138. Ballou, M. A. 2012. Immune responses of Holstein and Jersey calves during the preweaning and immediate postweaned periods when fed varying planes of milk replacer. J. Dairy Sci. 95:7319-7330. Bar-Peled, U., B. Robinzon, E. Maltz, H. Tagari, Y. Folman, I. Bruckental, H. Voet, H. Gacitua, and A. R. Lehrer. 1997. Increased weight gain and effects on production parameters of Holstein heifer calves that were allowed to suckle from birth to six weeks of age. J. Dairy Sci. 80:25232528. Bartlett, K. S., F. K. McKeith, M. J. VandeHaar, G. E. Dahl, and J. K. Drackley. 2006. Growth and body composition of dairy calves fed milk replacers containing different amounts of protein at two feeding rates. J. Anim. Sci. 84:1454-1467. Bascom, S. A., R. E. James, M. L. McGilliard, and M. Van Amburgh. 2007. Influence of dietary fat and protein on body composition of Jersey bull calves. J. Dairy Sci. 90:5600-5609. Bateman, II, H. G., T. M. Hill, J. M. Aldrich, and R. L. Schlotterbeck. 2009. Effects of corn processing particle size and diet form on performance of calves in bedded pens. J. Dairy Sci. 92:782-789. Bateman, II, H. G., T. M. Hill , J. M. Aldrich, R. L. Schlotterbeck, and J. L. Firkins. 2012. Meta analysis of the impact of initial serum protein concentration and empirical prediction model for growth of neonatal Holstein calves through eight weeks of age. J. Dairy Sci. 95:363-369. Blome, R. M., J. K. Drackley, F. K. McKeith, M. F. Hutjens, and G. C. McCoy. 2003. Growth, nutrient utilization, and body composition of dairy calves fed milk replacers containing different amounts of protein. J. Anim. Sci. 81:1641-1655. Cowles, K. E., R. A. White, N. L. Whitehouse, and P. S. Erickson. 2006. Growth characteristics of calves fed an intensified milk replacer regime with additional lactoferrin. J. Dairy Sci. 89:4835-4845. Davis Rincker, L. E., M. J. VandeHaar, C. A. Wolf, J. S. Liesman, L. T. Chapin, and M. S. Weber Nielsen. 2011. Effect of intensified feeding of heifer calves on growth, pubertal age, calving age, milk yield, and economics. J. Dairy Sci. 94:3554 3567. Diaz, M. C., M. E. Van Amburg, J. M. Smith, J. M. Kelsey, and E. L. Hutten. 2001. Composition of growth of Holstein calves fed milk replacer from birth to 105-kilogram body weight. J. Dairy Sci. 84:830-842. Donnelly, P. E., and J. B. Hutton. 1976. Effects of dietary protein and energy on the growth of Friesian bull calves. I. Food intake, growth, and protein requirements. N. Z. J. Agric. Res. 19:289-297. Fallon, R. J., and F. J. Harte. 1988. Effect of normal or acidified milk replacer offered ad libitum on calf performance. Irish J. Agric. Res. 27:123-130. Fokkink, W. B., T. M. Hill, H. G. Bateman, II, J. M. Aldrich, and R. L. Schlotterbeck. 2009. Selenium yeast for dairy calf feeds. Anim. Feed. Sci. Tech. 153:228-235.

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Shamay, A., D. Werner, U. Moallem, H. Barash, and I. Bruckental. 2005. Effect of nursing management and skeletal size at weaning on puberty, skeletal growth rate, and milk production during first lactation of dairy heifers. J. Dairy Sci. 88:1460 1469. Soberon, F., E. Raffrenato, R. W. Everett, and M. E. Van Amburgh. 2012. Preweaning milk replacer intake and effects on long-term productivity of dairy calves. J. Dairy Sci. 95:783793. Stamey, J. A., N. A. Janovick, A. F. Kertz , and J. K. Drackley. 2012. Influence of starter protein content on growth of dairy calves in an enhanced early nutrition program. J. Dairy Sci. 95:3327-3336. Stobo, I. J. F., J. H. B. Roy, and H. J. Gaston. 1966. Rumen development in the calf. 1. The effect of diets containing different proportions of concentrates to hay on rumen development. Br. J. Nutr. 20:171-191. Suarez-Mena, F. X., T. M. Hill, A. J. Heinrichs, H. G. Bateman, II, J. M. Aldrich, and R. L. Schlotterbeck. 2011. Effects of including corn distillers dried grains with solubles in dairy calf feeds. J. Dairy Sci. 94:3037-3044. Sweeney, B. C., J. Rushen , D. M. Weary , and A. M. de Passill. 2010. Duration of weaning, starter intake, and weight gain of dairy calves fed large amounts of milk. J. Dairy Sci. 93:148-152. Terre, M., M. Devant, and A. Bach. 2006. Performance and nitrogen metabolism of calves fed conventionally or following an enhanced-growth feeding program during the preweaning period. Livest. Sci. 105:109-119. Terre, M., M. Devant, and A. Bach. 2007. Effect of level of milk replacer fed to Holstein calves on performance during the preweaning period and starter digestibility at weaning. Livest. Sci. 110:82-88. Terre, M., C. Tejero, and A. Bach. 2009. Long-term effects on heifer performance of an enhanced-growth feeding programme applied during the preweaning period. J. Dairy Res. 76:331339. Thickett, W. W., N. H. Cuthbert, T.D.A. Brigstocke, M. A. Lindeman, and P. N. Wilson. 1983. A note on the performance and management of calves reared on cold acidified milk replacer fed ad libitum. Anim. Prod. 36:147-150. Tikofsky, J. N., M. E. Van Amburgh, and D. A. Ross. 2001. Effect of varying carbohydrate and fat content of milk replacer on body composition of Holstein bull calves. J. Anim. Sci. 79:2260-2267. Tozer, P. R. and A. J. Heinrichs. 2001. What affects the costs of raising replacement dairy heifers: a multiple-component analysis. J. Dairy Sci. 84:1836-1844. USDA. 2012. Dairy Heifer Raiser, 2011, an overview of operations that specialize in raising dairy heifers. Accessed on November 28, 2012. http://www.aphis.usda.gov/animal_health/nahms/dairy/downloads/dairyheifer11/Heifer Raiser.pdf Van Amburgh, M. E. 2012. Management and environment factors that affect productivity of calves. Pages 52-58 in proceeding of Group-Housed Dairy Calf Systems Symposium. December 12-13, 2012. Pro-Dairy Program, Cornell University. Syracuse, NY. Van den Borne, J. J. G. C., G. E. Lobley, M. W. A. Verstegen, J. Muijlaert, S. J. J. Alferink, and W. J. J. Gerrits. 2007. Body fat deposition does not originate from carbohydrates in milk-fed calves. J. Nutr. 137: 2234-2241. Warner, R. G., W. P. Flatt, and J. K.Loosli. 1956. Dietary factors influencing the development of the animals stomach. J. Agric. Food Chem. 4:788 -792. 140

Update on Milk Fat and Human Health

A. L. Lock* and D. E. Bauman * Department of Animal Science, Michigan State University Department of Animal Science, Cornell University Corresponding author: allock@msu.edu

SUMMARY For over a half-century the concept of healthy eating has become synonymous with avoiding fat, especially saturated fat and this remains a centerpiece in nutritional advice of medical societies and government agencies worldwide. Investigations have shown the science behind this advice, however was based on incomplete and in some cases flawed investigations. Nutritional science has advanced rapidly and evidence now demonstrates that the proportion of total energy from fat or saturated fat is largely unrelated to the risk of cardiovascular diseases or other chronic diseases. Indeed there appears to be an enormous disconnect between the evidence from longterm prospective studies and public perceptions of harm from the consumption of milk fat and dairy products. This clearly represents a new paradigm requiring a major shift in recommendations by national advisory groups. Furthermore, milk and dairy products are key components in dietary patterns chosen for optimum health maintenance and the prevention of chronic diseases. INTRODUCTION The importance of animal products in meeting global needs for food security is well established, and public health organizations around the world include milk and other dairy products in recommendations for a healthy, well-balanced diet. Dairy products are an important source for many vital nutrients including high quality protein, energy, and many essential minerals and vitamins (Bauman and Capper, 2011). Dairy products, however are also a major food source of saturated fat, accounting for 20-30% of the saturated fat intake for the US population (USDA/USDHHS, 2010). For over a half-century, saturated fat has been demonized as the major cause of cardiovascular vascular disease (CVD) and public health recommendations are to reduce dietary intake of saturated fat and food products containing saturated fatty acids (FA). As a consequence, the perception of the public, and much of the scientific community, is that milk fat is a negative component of dairy products and typical dietary advice is to consume only reduced-fat or no-fat dairy products. The net effect of this recommendation over the last 50 years has been a progressive reduction in fluid milk consumption and a shift toward fluid milk products containing less fat (Table 1). Recent estimates indicate that approximately 30% of our dietary intake of saturated fat comes from dairy products with cheese being the major source (Ervin et al., 2004). The term CVD includes coronary heart disease, cerebrovascular disease, and other related disorders of the heart and blood vessels. CVD is the leading cause of death in the US and globally, accounting for about one-third of all deaths (Roger et al., 2011). Strategies to reduce CVD involve reducing leading risk factors that include smoking, hypertension, and high 141

cholesterol; doing so reduces the burden of CVD. The U.S. Center for Disease Control estimates that about 50% of the US population has at least one of these three risk factors with CVDrelated health care costs in the US at over $400 billion annually (CDC, 2011). New research and re-evaluation of previous research is challenging long-held dogma on the relationship between saturated fat, milk fat, and CVD. While national organizations, health professionals, and dietitians will be cautious in revising decade-old recommendations, there is no doubt that the last few years have provided game-changing scientific evidence that will revise the paradigm for the connection between saturated fat and CVD. Furthermore, these new studies and re-evaluations have convincingly demonstrated the important role that milk and dairy products play in health maintenance and the prevention of chronic diseases. In the following sections we will deal briefly with some background and historical aspects related to saturated fat and CVD, and then highlight recent findings related to the role of milk and milk fat in human health. Table 1. U.S. per capita availability of fluid milk. Adapted from Huth and Park (2012). Total Consumption Liters 133.6 125.6 115.8 99.7 94.7 81.4 73.4 Whole Reduced fat Low fat Nonfat milk (2%) (1%) (<0.05%) ----------------- Percent of total fluid milk ---------------99.0 0 0 0.9 95.6 0.7 0 3.1 83.8 11.0 0.7 4.5 62.7 24.9 7.0 5.3 41.4 37.9 9.6 11.0 36.5 34.2 12.5 16.2 30.2 39.1 14.0 16.6

Year 1950 1960 1970 1980 1990 2000 2009

BACKGROUND AND HISTORICAL ASPECTS Ancil Keys (University of Minnesota) played a central role in labeling dietary fat, specifically saturated fat, as a major public health concern for CVD. Using population data obtained from WHO reports, Keys (1953) reported a curvilinear relationship between the intake of fat and deaths from coronary heart disease for six countries (open circles in Figure 1). Keys was featured on the cover of TIME magazine and this was the beginning of the diet -heart hypothesis proposing a sequence of etiologic relations between dietary saturated fat, circulating cholesterol, and the development of CVD. Yerushalmy and Hilleboe (1957), however examined the publication by Keys (1953) and concluded that many aspects were flawed including the fact that the 6 countries had been cherry-picked from a data set for 22 countries (Figure 1). This represented an extraordinary critique, and based on a rigorous analysis of Keys publication, they concluded The association between the percent of fat calories available for consumption in national diets and degenerative heart disease (reported by Keys) is not valid; the association is specific neither for dietary fat nor for heart disease mortality (Yerushalmy and Hilleboe, 1957). Despite this, Keys (1980) continued promoting the diet-heart hypothesis and followed-up with a seven country comparison showing that dietary intake of saturated fat as a percentage of calories was strongly correlated with coronary death rates (r = 0.84). Again, other scientists pointed out major flaws in his study, e.g. countries chosen by Keys to represent low saturated fat 142

intake and low incidence of CVD were in fact less industrialized and differed in many ways including smoking habits, physical activity, and obesity (Willett, 2012). Nevertheless, the dietheart hypothesis was well received by many health practitioners and policy makers. Historically, serum cholesterol has served as a surrogate marker for the risk of CVD. Lowdensity lipoprotein cholesterol (LDL-C) is correlated with risk of CVD; thus, the discovery that saturated fat resulted in an increase in LDL-C provided support for the diet-heart hypothesis, and some concluded that saturated fat must be the major cause of CVD (Ordovas, 2005). This particularly impacted public perception of dairy products because dairy fat contains 60 to 70% saturated FA (Table 2). Additionally, it was discovered that individual FA differ in their effects on serum LDL-C; whereas most saturated FA were neutral, lauric acid (C12:0), myristic acid (C14:0), and palmitic acid (C16:0) caused pronounced increases in LDL-C (Hegsted et al., 1965) and these three FA represent about 40% of total milk fat (Table 2). This led to the development of an atherogenic index which ranked foods based on their content of these three FA (Ulbricht and Southgate, 1991); needless to say, the atherogenetic index ranked dairy products as problematic with respect to CVD risk. Figure 1. Relationship between percent of calories from fat and mortality from atherosclerotic and degenerative heart disease. Six countries (open circles) selected by Keys (1953) from a WHO data set for 22 countries (unselected countries shown by solid circles). Adapted from Yerushalmy and Hilleboe (1957) and Maijala (2000). Relationship for the six selected countries represented the foundation for Keys diet-heart hypothesis to explain cause of cardiovascular disease.

143

Public health recommendations in the 1970s and 1980s were to dramatically reduce the intake of saturated fat. This included recommendations to reduce intake of dairy products and/or a shift to low-fat or no-fat dairy products. One specific recommendation was to replace butter with margarine, and this was particularly unfortunate as it markedly increased the risk of CVD for millions of people; during this era margarine had high levels of trans FA and subsequent research has established that intake of industrial sources of trans FA are a major risk for CVD (Gebauer et al., 2011; Lock and Bauman, 2011). Much of the case against dairy foods is linked to an imperfect understanding of cholesterol as a surrogate marker of CVD risk. In addition to effects on LDL-C, saturated FA, in particular lauric acid, myristic acid, and palmitic acid cause an increase in serum high-density lipoprotein-cholesterol (HDL-C) and this cholesterol fraction reduces the risk of CVD. Thus, when changes in both LDL-C and HDL-C are considered, saturated FA have no adverse effect on serum cholesterol as a risk factor for the incidence of CVD. A meta-analysis by Mensink et al. (2003) provides convincing evidence for this; using data from 60 clinical studies, they evaluated CVD risk on the basis of ratio of serum total cholesterol:HDL-C. As illustrated in Figure 2, ratios for lauric acid, myristic acid, and palmitic acid provide little or no evidence for an atherogenic effect when compared to an isoenergetic carbohydrate substitution; in fact the ratio for lauric acid was significantly decreased. When compared by fat type, the meta-analysis revealed no effect of saturated FA when compared to carbohydrate substitution on an isoenergetic basis (Figure 2). Nonetheless, changes in the ratio of serum total cholesterol:HDL-C were indicative of the well-established beneficial effects of monosaturated and polyunsaturated FA and the increased atherogenic risk of trans FA. Thus, this classic meta-analysis utilizing cholesterol-related surrogate markers provides no support for saturated fat in general or the major individual saturated FA in milk fat as risk factors in CVD. Table 2. Fatty acid composition of retail milk samples in the United States. Adapted from ODonnell-Megaro et al. (2011). Milk samples were obtained from 56 milk processing plants representing all US regions and seasons over a 12-month period.
30 25

g/100 g Fatty Acids

20 15 10 5 0
1 18 8:0 :1 tr an 18 s :1 c 18 is :2 n18 6 :3 n3 C LA O th er s 4: 0 6: 0 8: 0 10 :0 12 :0 14 :0 16 :0

Fatty Acid

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For the last 50 years medical societies and government agencies have embraced the concept that nutrition, specifically saturated fat, is a major player in the epidemic of CVD. As discussed previously, the diet-heart hypothesis and the use of LDL-C as a surrogate marker were perceived to offer support for this concept. Acceptance by the scientific community, however, was far from unanimous (e.g. Ravnskov, 2002; Weinberg, 2004; Ordovas, 2005) and a growing body of scientific studies offered no support (see discussion in Lock and Bauman, 2011). The classic study by Mensink et al. (2003) discussed above was of special importance in challenging the dogma on the relationship between dietary saturated fat, serum cholesterol, and incidence of CVD. An important recent investigation was that by Siri-Tarino et al. (2010). This meta-analysis of 21 prospective epidemiologic studies covered a 5 to 23 year follow-up of 347,747 subjects and again results indicated there is no significant evidence that dietary saturated fat is associated with an increased risk of coronary heart disease or CVD (Siri-Tarino et al., 2010). Clearly, the relationship of fats including saturated fats, cholesterol, and CVD is more complex than initially thought and the risk of CVD is multifaceted. Figure 2. Meta-analysis (n = 60 trials) to examine changes in serum ratio of total cholesterol to HDL-cholesterol when carbohydrates constituting 1% of energy are replaced isoenergetically with fatty acids (Mensink et al., 2003). Panel A represents comparison of saturated, cismonounsaturated, cis-polyunsaturated, and trans-monounsaturated fatty acids (*=P<0.05; =P<0.001). Panel B represents comparison of lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), and stearic acid (18:0) (*=P<0.001). Mensink et al., 2003).

A
0.06

B
0.06

Total:HDL Cholesterol

0.04

Total:HDL Cholesterol

0.04

0.02

0.02

0.00

0.00

-0.02

-0.02

-0.04

-0.04

*
Lauric acid Myristic acid Palmitic acid Stearic acid

Saturated fatty acids cis Monounsaturated fatty acids cis Polyunsaturated fatty acids trans Monounsaturated fatty acids

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RECENT DEVELOPMENTS IN SATURATED FAT AND MILK FAT Dietary recommendations by nationally recognized bodies have a substantial impact on the nutrition and health community. The 2010 Dietary Guidelines for Americans recommended restricting the consumption of saturated fat to less than 10% of total dietary calories (USDA/USDHHS, 2010). In a peer-reviewed publication Hite et al. (2010) challenged this report, including its specific conclusions regarding saturated fat, and pointed out that recommendations were based on science that was inaccurately represented and summarized (as we have discussed in the previous section), and even more serious that the report failed to include the full body of relevant research. Hoenselaar (2012) took this a step further in a peerreviewed publication by examining recommendations on saturated fat from the report by USDA/USDHHS (2010) as well as reports from two additional important US and European advisory committees the Institute of Medicine (IOM, 2005), and the European Food Safety Authority (EFSA, 2010). He summarized the recommendations from these advisory committees as follows: 1. 2. 3. Consume less than 10% of calories from saturated FA, replacing them with monounsaturated and polyunsaturated FA (USDA/USDHHS, 2010). Keep the intake of saturated FA as low as possible while consuming a nutritionally adequate diet (IOM, 2005). Saturated fat intake should be as low as possible (EFSA, 2010).

These advisory committees cited studies to support their recommendations, but Hoenselaar (2012) pointed out their conclusions were not based on a valid representation of the scientific literature. For example, the effect of saturated fat on LDL-cholesterol and its connection to an increased CVD risk was cited in all three reports, but concurrent beneficial effects of saturated fat to increase HDL-cholesterol thereby reducing CVD risk was systematically ignored. It is unfortunate that due to a focus on the small rise in blood cholesterol with milk consumption, the debate on milk fat has never achieved a reasonable balance in the evaluation of risks and benefits. The overall conclusions by Hoenselaar (2012) were Results and conclusions about saturated fat in relation to CVD, from leading advisory committees, do not reflect the available scientific literature. Two additional public health advisory committees are the World Health Organization (WHO) and American Heart Association (AHA). Although recommendations by these two bodies were not included in the critiques by Hite et al. (2010) and Hoenselaar (2012), their assessment would also apply to them. The WHO recommends saturated fat intake be reduced to less than 10% of dietary calories and the 2009 Guidelines by AHA recommends saturated fat be reduced to an even lower <7% of total calories (Huth and Park, 2012). Support for a paradigm shift in conclusions for the relationship between saturated fat, milk fat, and cardiovascular health also comes from recent reviews of the published literature. The comprehensive reviews of Parodi (2009), Givens and Minihane (2011), Kratz et al. (2012), and Huth and Park (2012) reached a similar conclusion that the majority of observational studies have failed to support an adverse association between the intake of dairy products and CVD, regardless of milk fat levels, and in many cases a long-term beneficial effect was observed. Several excellent investigations have been reported in the last three years and key studies are summarized in Table 3. de Oliveira Otto et al. (2012) conducted a multi-ethic study examining 146

the relationship between consumption of saturated dairy fat and CVD; they concluded that a higher intake of saturated fat was associated with lower CVD risk . Importantly, the authors found that associations between saturated fat and incident CVD depended on the food source, with the consumption of dairy saturated fat being inversely associated with risk (risk was 0.62 when 5% of energy was from dairy saturated fat; de Oliveira Otto et al., 2012). These findings raise the possibility that associations of foods that contain saturated fat with health may depend on specific FA present in these foods or the complex mixture of other food constituents, in addition to saturated fat (de Oliveira Otto et al., 2012). Elwood et al. (2010) conducted a meta-analysis of prospective cohort studies to examine associations between the intake of milk and dairy products and the incidence of ischemic heart disease and stroke. Results indicated a reduction in risk in subjects with the highest dairy consumption relative to those with the lowest intake; relative risk values were 0.92 for ischemic heart disease and 0.79 for stroke (Figure 3; Elwood et al., 2010). Goldbohm et al. (2011) reported results from a large cohort study designed to examine the association between the intake of dairy products and mortality; data covered a 10 year period for 120,852 men and women, and results indicated no association between dairy product consumption and stroke mortality for men or women. Likewise, there was no association between total milk intake and ischemic heart disease mortality in men, whereas a small positive association was observed for women (relative risk = 1.07; Goldbohm et al., 2011). Soedamah-Muthu et al. (2011) conducted a similar meta-analysis comparing intake of dairy products and the risk of CVD (including coronary heart disease, stroke, and mortality); their meta-analysis gained greater analytical power by including different dairy food categories and different ranges of intake. Results demonstrated that milk intake was not associated with total mortality, but may be inversely associated with overall CVD risk; relative risk for the later was 0.94 (Soedamah-Muthu et al., 2011).

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Table 3. Summary of recent prospective cohort studies. Reference de Oliveira Otto et al. (2012) Description/Objective Multi-ethnic study of saturated dairy fat intake and incidence of CVD; 5209 individuals for 10 yr period. Meta-analysis of cohort studies of dairy intake in relation to cardiac health. Multivariate survival analysis of case cohorts to examine association between dairy product intake and risk of cardiac-related death. Data collected over 10 yr (n=120,853 patients). Dose-response meta-analysis of dairy consumption and incidence of CVD and all-cause mortality. Conclusion A higher intake of saturated fat from dairy products was associated with lower CVD risk. Greater intake of milk and dairy products reduced incidence of ischemic heart disease and stroke. Dairy product intake had neutral effects on mortality in men, but in women dairy fat intake was associated with slightly increased mortality from ischemic heart disease. Milk intake was associated with reduction in overall CVD risk, but no relationship to total mortality.

Elwood et al. (2010)

Goldbohm et al. (2011)

Soedamah-Muthu et al. (2011)

These findings are in broad agreement with the recently reported outcome of a remarkable 61year follow up of the Boyd-Orr cohort. This study involved the recruitment of 4,999 children in England and Scotland in 1937-39 with causes of death recorded from 1948 (van der Pols et al., 2009). Results demonstrated that a family diet in childhood, which was high in dairy products, did not give rise to a greater risk of CVD or stroke mortality. Indeed all-cause mortality was lowest in those with the highest dairy product and milk intake (basic hazard ratio for both, 0.69; 95% CI 0.57 to 0.84; P for trend <0.002). These findings are therefore suggestive that despite milk fat being rich in saturated FA, milk has properties that are beneficial in reducing the risk of CVD.

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Figure 3. The numbers of deaths in England and Wales in 2008 from various causes and the risks for these causes in subjects with the highest milk/dairy consumption, relative to subjects with the lowest milk/dairy consumption. Adapted from Elwood et al. (2010).

There are a limited number of studies that report disease rates in subjects who consume natural dairy foods, and in those who consume reduced fat dairy foods. Results from such studies are often confounded however due to the adoption of other health-related behaviors. The appropriate question to ask is: Do fat-reduced milks and dairy foods provide any additional advantage, or does the reduction in fat reduce the benefits of whole milk? Interestingly, a recent study from Australia reported that full fat (but not low-fat) dairy consumption was inversely associated with cardiovascular mortality and this effect was significant (Bonthuis et al., 2010). Compared with participants in the lowest intake group, participants in the highest full-fat dairy intake group had a multivariable hazard ratio of 0.33 (95% CI: 0.130.81; P for trend = 0.05). Conversely, a meta-analysis of prospective cohort studies by Soedamah-Muthu et al. (2012) suggests that low-fat dairy and milk could contribute to the prevention of hypertension, whereas total dairy intake was not significantly associated with hypertension incidence. Therefore, a statement by German and Dillard (2004) is appropriate: Hypotheses [about fat-reduced milks] are the basis of sound scientific debate; however they are not the basis of sound public health policy. DAIRY PRODUCTS AND HUMAN HEALTH Consumers are increasing aware of the connection between diet and health, and scientists are being asked to clarify the role of specific foods in health maintenance and the prevention of chronic diseases. Multidisciplinary studies in developing countries demonstrate that when diets of schoolchildren had little or no animal source foods, the intake of essential micronutrients was inadequate resulting in negative health outcomes including severe problems such as poor growth, impaired cognitive performance, neuromuscular deficits, psychiatric disorders and even death (Nuemann et al., 2002; Randolph et al., 2007). Continued recommendations to reduce milk fat intake may result in inadequate intakes of key nutrients in certain population groups.

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Long-term effects of milk and dairy products on health and the prevention of chronic diseases of the general population are also of interest, and these would best be determined in randomized controlled trials. There have been no such trials and realistically none are likely because of the required number of subjects and the long latency period associated with chronic diseases. The best evidence, therefore, comes from prospective cohort studies with disease events or death as the outcome. There have a number of prospective cohort studies that have evaluated the association between intake of milk and dairy products and the incident of chronic diseases. Results of meta-analysis of such studies provide convincing evidence that milk and dairy products are associated with beneficial effects for long-term health maintenance and the prevention of chronic diseases. The beneficial effects in reducing the risk of CVD was discussed earlier, and additional examples of chronic diseases for which consumption of dairy products reduces risk include: diabetes, obesity, metabolic syndrome and many types of cancer (Elwood et al., 2008; 2010; Tremblay and Gilbert, 2009; Kliem and Givens, 2011; Grantham et al., 2012; Korhonen, 2012; Kratz et al., 2012). Overall, the science clearly demonstrates the importance of milk and dairy products in childhood development, health maintenance, and the prevention of chronic diseases. Indeed, linking the benefits of milk consumption with deaths from key chronic diseases led Elwood et al. (2008) to conclude that high milk consumers have an overall survival advantage. REFERENCES Bauman, D. E., and J. L. Capper. 2011. Sustainability and dairy production: challenges and opportunities. Proc. Cornell Nutr. Conf. pp. 136-153. Bauman, D. E., and A. L. Lock. 2006. Animal products and human health: perceptions opportunities and challenges. Proc. Cornell Nutr. Conf. pp. 45-57. Bonthuis, M., M.C.B. Hughes, T.I. Ibiebele, A.C. Green, and J.C. van der Pols. 2010. Dairy consumption and patterns of mortality of Australian adults. European Journal of Clinical Nutrition. 64:569577. CDC. 2011. Million hearts: strategies to reduce the prevalence of leading cardiovascular disease risk factors United States 2011. Centers for Disease Control and Prevention, MMWR Report 60:1248-1251. de Oliveira Otto, M., D. Mozaffarian, D. Kromhout, A. G. Bertoni, C. T. Sibley, D. R. Jacobs Jr., and J. A. Nettleton. 2012. Dietary intake of saturated fat by food source and incident cardiovascular disease: the Multi-Ethnic Study of Atherosclerosis. Am. J. Clin. Sci. 96:397-404. EFSA. 2010. Scientific opinion on dietary reference values for fats, including saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, trans fatty acids, and cholesterol. Panel on Dietetic Products, Nutrition, and Allergies. EFSA J. 8:1461. Available at: http://www.efsa.europa.eu/en/efsajournal/pub/1461.htm. Elwood, P. C., D. I. Givens, A. D. Beswick, A. M. Fehily, J. E. Pickering, and J. Gallacher. 2008. The survival advantage of milk and dairy consumption: an overview of evidence from cohort studies of vascular diseases, diabetes and cancer. J. Am. Coll. Nutr. 27:723S-734S. Elwood, P. C., J. E. Pickering, D. I. Givens, and J. E. Gallacher. 2010. The consumption of milk and dairy foods and the incidence of vascular disease and diabetes: an overview of the evidence. Lipids 45:925-939.

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Ervin, R. B., J. D. Wright, C.-Y. Wang, and J. Kennedy-Stephenson. 2004. Dietary intake of fats and fatty acids for the United States population: 1999-2000. U.S. Department of Health and Human Services, Advanced Data Number 348, November 8, 2004. Gebauer, S.K., J. Chardigny, M. Jakobsen, B. Lamarche, A.L. Lock, S. Proctor, and D. Baer. 2011. Effects of ruminant trans fatty acids on cardiovascular disease and cancer: a comprehensive review of epidemiological, clinical, and mechanistic studies. Adv. Nutr. 2:332354. German, J.B., and C.J. Dillard. 2004. Saturated fats: what dietary intake? Am J Clin Nutr. 80:550559. Givens, D. I., and A.-M. Minihane. 2009. Dairy products: their role in the diet and effects on cardiovascular disease. Pages 163-180 in Fatty Acids in Health Promotion and Disease Causation. R. R. Watson, ed. AOCS Publications, Urbana, IL. Goldbohm, R. A., A. M. Chorus, F. Galindo Garre, L. J. Schouten, and P. A. van den Brandt. 2011. Dairy consumption and 10-y total and cardiovascular mortality; a prospective cohort study in the Netherlands. Am. J. Clin. Nutr. 93:615-627. Grantham, N. M., D. J. Magliano, A. Hodge, J. Jowett, P. Meikle, and J. E. Shaw. 2012. The association between dairy food intake and the incidence of diabetes in Australia: the Australian Diabetes, Obesity and Lifestyle Study. Public Health Nutrition, Available on CJO 2012 doi:10.1017/S1368980012001310. Hegsted, D. M., R. B. McGandy, M. L. Myers, and F. J. Stare. 1965. Quantitative effects of dietary fat on serum cholesterol in man. Am. J. Clin. Nutr. 17:281-295. Hite, A. H., R. D. Feinman, G. E. Guzman, M. Satin, P. A. Schoenfeld and R. J. Wood. 2010. In the face of contradictory evidence: report of the Dietary Guidelines for Americans Committee. Nutr. 26:915-924. Hoenselaar, R. 2012. Saturated fat and cardiovascular disease: the discrepancy between the scientific literature and dietary advice. Nutr. 28:118:123. Huth, P. J., and K. M. Park. 2012. Influence of dairy product and milk fat consumption on cardiovascular disease risk: a review of the evidence. Adv. Nutr. 3:266-285. IOM. 2005. Dietary fats: total fat and fatty acids in dietary reference intakes for energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids (macronutrients). Institute of Medicine. Available at: http://books.nap.edu/openbook.php?record_id=10490&page=422. Keys, A. 1953. Atherosclerosis: a problem in newer public health? In: Recent Adv. Therapy. Mt. Sinai Hosp. 20:118-139. Keys, A. 1980. Seven countries: a multivariate analysis of death and coronary heart disease. Harvard University Press, Cambridge, MA. Kliem, K. E., and D. I. Givens. 2012. Dairy products in the food chain: their impact on health. Ann. Rev. Food Sci. 2:21-36. Korhonen, H. J. 2012. Production and properties of health-promoting proteins and peptides from bovine colostrum and milk. Cell. Mol. Biol. 58:26-38. Kratz, M., T. Baars and S Guyenet. 2012. The relationship between high-fat dairy consumption and obesity, cardiovascular, and metabolic disease. Eur. J. Nutr. DOI 10.1007/s00394012-0418-1, published online: 19 July 2012. Lock, A. L., and D. E. Bauman. 2011. Milk fat and human health separating fats from fiction. Proc. Cornell Nutr. Conf., pp. 126-135. Maijala, K. 2000. Cow milk and human development and well-being. Livestock Prod. 65:1-18.

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Mensink, R. P., P. L. Zock, A. D. M. Kester, and M. B. Katan. 2003. Effects of dietary fatty acids and carbohydrates on the ratio of serum total to HDL cholesterol and on serum lipids and apolipoproteins: a meta-analysis of 60 controlled trials. Am. J. Clin. Nutr. 77:1146-1155. Neumann, C. D. M. Harris, and L. M. Rogers. 2002. Contribution of animal source foods in improving diet quality and function in children in the developing world. Nutr. Res. 22:193-220. ODonnell-Megaro, A. M., D. M. Barbano, and D. E. Bauman. 2011. Survey of fatty acid composition of retail milk in the United States including regional and seasonal variations. J. Dairy Sci. 94:5965. Ordovas, J. M. 2005. Diet-heart hypothesis: will diversity bring reconciliation? Am. J. Clin. Nutr. 82:919-920. Parodi, P. W. 2009. Has the association between saturated fatty acids, serum cholesterol and coronary heart disease been over emphasized? Int. Dairy J. 19:345-361. Randolph, T. F., E. Schelling, D. Grace, C. F. Nicholson, J. L. Leroy, D. C. Cole, M. W. Demment, A. Omore, J. Zinsstag, and M. Ruel. 2007. Invited review: Role of livestock in human nutrition and health for poverty reduction in developing countries. J. Anim. Sci. 85:2788-2800. Ravnskov, U. 2002. A hypothesis out-of-date, the diet-heart idea. J. Clin. Epidemiol. 55:10571063. Roger, V. L., A. S. Go, D. M. Lloyd-Jones et al. 2011. Heart disease and stroke statistics2011 update: a report from the American Heart Association. Circulation 123:e18-209. Sir-Tarino, P. W., Q. Sun, F. B. Hu, and R. M. Krauss. 2010. Meta-analysis of prospective cohort studies evaluating the association of saturated fat with cardiovascular disease. Am. J. Clin. Nutr. 91:535-546. Soedamah-Muthu, S. S., E. L. Ding, W. K. Al-Delaimy, F. B. Hu, M. F. Engberink, W. C. Willett, and J. M. Geleijnse. 2011. Milk and dairy consumption and incidence of cardiovascular diseases and all-cause mortality: dose-response meta-analysis of prospective cohort studies. Am. J. Clin. Nutr. 93:158-171. Soedamah-Muthu, S.S., L.D.M. Verberne, E.L. Ding, M.F. Engberink, and J.M. Geleijnse. 2012. Dairy consumption and incidence of hypertension: a dose-response meta-analysis of prospective cohort studies. Hypertension. 60:1131 1137. Tremblay, A., and J. A. Gilbert. 2009. Milk products, insulin resistance syndrome and type 2 diabetes. J. Am. Coll. Nutr. 28(Suppl 1):91S-102S. Ulbricht, T. L. V., and D. A. T. Southgate. 1991. Coronary heart disease: seven dietary factors. Lancet 338:985-992. USDA/USDHHS. 2010. Dietary Guidelines for Americans 2010. Report of the Dietary Guidelines Advisory Committee, USDA and USDHHS, Washington DC. Available at: http://www.cnpp.usda.gov/DGAs2010-DGACReport.htm. van der Pols, J. C., D. Gunnell, G. M. Williams, J. M. P. Holly, C. Bain, and R. M. Martin. 2009. Childhood dairy and calcium intake and cardiovascular mortality in adulthood: 65-year follow-up of the Boyd-Orr cohort. Heart 19:1600-1606. Weinberg, S.L. 2004. The diet-heart hypothesis: a critique. J. Am Coll. Cardiol. 43:731-733. Willett, W. C. 2012. Dietary fats and coronary heart disease. J. Int. Med. 272:13-24. Yerushalmy, J., and H. E. Hilleboe. 1957. Fat in diet and mortility from heart disease, a methodoligic note. New York State J. Med. 57:2343-2353.

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Use of Probiotics in Dairy Rations During Heat Stress

L. W. Hall and R. J. Collier Department of Animals Sciences The University of Arizona Corresponding author: lwhall@email.arizona.edu SUMMARY Heat stress reduces production in lactating dairy cows. 50% of milk yield losses come from reduced feed intake and the balance is due to consequences of physiological changes and metabolic alterations. The physiological changes and metabolic alterations often lead to acidosis in heat stressed dairy cows. Feeding probiotics to heat stressed dairy cows addresses some of the metabolic issues which occur during heat stress. More research is needed to understand the implications of DFMs before, during and after heat. INTRODUCTION Under ideal [thermoneutral] conditions the rumen is a complex microbiome that maintains pH, temperature and mixing in an anaerobic environment. Metabolic heat is generated within the cow and fermentation has a major contribution to this type of heat. Fermentation occurs mainly in the rumen and also in the hindgut. Any alteration in the rumen environment can lead to changes in microbial populations and rumen health. Hot and humid ambient conditions can impair the cows ability to dissipate heat leading to a cascade of events which alters rumen and hindgut function. Heat stress in dairy cows perturbs homeostasis and elicits physiological responses to reduce heat load which can impair proper rumen function. Examples include panting which leads to salivary bicarbonate loss and respiratory alkalosis and diversion of blood flow to the skin surface to dissipate heat load as well as reduced rumen contraction rate (Beede and Collier, 1986). Nutritional management of lactating dairy cows during thermal stress requires a multifaceted approach with the overall goal to reduce metabolic heat load, minimize damaging effects from physiological responses and maintain production. Probiotics are one of the tools available to provide benefits to heat stressed dairy cows that may reduce production losses, maintain a healthy gut and minimize metabolic heat. Heat Stress Heat stress occurs when the body temperature of a lactating dairy cow is 39.4C. High producing dairy cows are at a greater risk to develop heat stress because of their elevated metabolism and high feed intake relative to maintenance requirements. Elevated respiration rates, increased body temperature, decreased feed intake, losses in reproductive performance, health issues and decreased milk yield are observable in hyperthermic lactating dairy cows 153

(Rhoads et al., 2009). Elevated respiration rate leads to increased salivary bicarbonate loss associated with excessive drooling and respiratory alkalosis as the animal exhales excessive amounts of carbon dioxide. The animal responds to respiratory alkalosis by dumping increased bicarbonate in the urine which subsequently leads to metabolic acidosis because of reduced buffering capacity in the rumen from both salivary and urinary bicarbonate loss. Respiratory alkalosis and metabolic acidosis occur when respiration rate increases (Sanchez et al., 1994). Saliva is lost with panting and sodium bicarbonate is lost with excessive drooling and reduces ruminal buffering, increasing the risk of rumen acidosis. Feed intake drops with heat stress and milk yield also decreases. Of the decrease in milk yield, only about 50% can be accounted for from the drop in feed intake. This was demonstrated by pair-feeding lactating dairy cows under thermoneutral conditions the same amount of feed that the heat stressed group consumed (Rhoads et al., 2007; Baumgard et al., 2011). There is still 50% of milk yield losses that are not directly linked with the drop in DMI. It is still uncertain how to account for all of the losses relative to milk synthesis during hyperthermia. Nutrient partitioning, the reduction in ruminal fermentation products (VFA and protein), and the energy required to dissipate heat during heat stress may account for milk yield losses.

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IMPLICATIONS OF HEAT STRESS Figure 1. Summary of production changes associated with heat stress Feed intake - Accounts for 50% loss in milk yield Respiration rate - Expired CO2 * Renal compensation of bicarbonate (less to buffer rumen) - Drooling * Saliva Loss of buffers Mineral loss Water loss Rumen acidosis - Fermentable carbohydrate to the hindgut = hindgut acidosis - Protozoa and fungi - Rumination - pH * Protonation of NH3 to NH4 * VFA * Streptococcus bovis = lactic acid production (10x strength vs VFAs) Surpass limited buffering capacity Lower pH Lactobacillus - Damage rumen wall and alters absorption * Lactate - lowers blood pH * Bacteria and myotic organisms invade rumen wall - ruminitis * Microbes pass rumen epithelium liver abscesses - Laminitis * Vasoactive substances Cellular response - HSP - Mammary cell transport function Post absorptive energetics - Glucose disposal rates - Glucose for milk synthesis - Insulin - Circulating NEFAs Body and metabolic heat Milk Yield Health issues including mastitis, morbidity and mortality

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Acidosis The excessive loss of CO2 from panting leads to respiratory alkalosis (West 2003) and the kidneys compensate in part by excreting more bicarbonate in the urine which reduces available buffering capacity in the rumen saliva has many important functions in ruminants, many that are impaired by heat stress. When panting occurs salivary buffers, minerals and water are lost from drooling. This can lead to lactic acidosis that can cause ruminitis, metabolic acidosis, laminitis, liver abscesses and sickness (Lean et al., 2000). When rumen pH goes below 6.5, NH3 is converted to NH4 transitioning it from a base to a weak acid. Initial increases in VFA production with heat stress can reduce pH depending on the buffering capacity. Rumen acidosis can occur during heat stress. In one study, the mean ruminal pH dropped from ~6.3 to 5.8 when environmental heat load was increased (from 18.3C to 29.4C, Mishra et al., 1970). There is also a reduction in rumination associated with the loss of essential buffers during heat stress. Acidosis will decrease digestion and increase undigested fermentable carbohydrate concentrations. The availability of nonstructural carbohydrates can alter microbial populations and increase the passage of carbohydrates to the hindgut. Streptococcus bovis, in particular will thrive when large amounts of starch are present and produces lactic acid that is 10x stronger than VFAs (Russell and Hino, 1985). When lactic acid increases the pH drops making the rumen environment more acidic. An increase in acidity promotes growth of some microbes including S. bovis and Lactobacillus which will accelerate their growth rate further decreasing the pH. Protozoa and fungi die in acidotic conditions which reduces the outflow of nutrients from the rumen. In addition, the increase in acidity can damage rumen wall epithelium and alter permeability. Lactate can be absorbed across the rumen wall and lower the pH of blood. Bacteria can invade the rumen epithelium and cause ruminitis or make it to the liver where organisms such as Fusobacterium necrophorum and Archanobacterium sppcan cause abscesses (Bolton and Pass, 1988). The liver is vital to gluconeogenesis and performance can be impaired after the occurrence of heat stress. When the rumen becomes too acidic the composition of rumen microbiota can change. Coupled with the increased permeability of the rumen, vasoactive substances can alter blood flow and can increase laminitis. These substances include lactate, serotonins, histamine and endotoxins (Westwood and Lean, 2001) and can be absorbed through the compromised rumen and large intestine. Endotoxin release results from acidosis killing gram-negative bacteria in the rumen and the large intestine (Dong et al., 2011). Grain induced ruminal acidosis has been shown to increase gram-negative Escherichia coli, a potential source of the endotoxin lipopolysaccharide (LPS) (Khafipour et al., 2009). Endotoxin can generate a nonspecific immune retort called an acute phase response (Ametaj et al., 2010). Infusion of (LPS) in lactating dairy cows decreased DMI and milk yield and resulted in greater numbers of cows with metabolic disorders such as displaced abomasum compared to control cows (Zebeli et al., 2011). Hindgut Acidosis The hindgut fermentation provides 5-10% dietary energy under normal conditions. Decreased rumination and digestion associated with rumen acidosis can increase the passage of 156

fermentable carbohydrates. When excessive fermentable carbohydrates make it the hindgut and are fermented, hindgut acidosis can occur (Gressley, 2011). Loose stool that is foamy with mucous indicates hindgut acidosis and possibly the sloughing of intestinal lining. Environmental stress often causes hindgut acidosis in lactating dairy cows. Heat Shock Heat stress can damage proteins within a cell by causing them to unfold (denature). The upregulation of the cytoprotective heat shock proteins (HSP) also called chaperones during heat stress is essential to cell survival because HSPs repair proteins required for normal cellular function by refolding them (Sharma et al., 2009). The cytoskeleton and transport function within epithelial mammary cells is also impaired (Collier et al., 2008). Even though the HSPs benefit the cell there is a nominal cost to repair misfolded proteins. Sharma et al. (2010) under in vitro conditions determined that it cost about 5 ATPs for HSP 70 to repair one protein. However, large scale production of heat shock proteins within mammary cells during heat stress likely contributes to the decline in protein concentration of milk observed during warm summer months. The protein synthetic factory of the mammary cell is diverted to increased synthesis of protective HSPs leading to reduced production of milk caseins. Metabolism During heat stress, cellular and whole body metabolism shifts. Glucose becomes a key fuel source that heat stressed cows rely on to remain euthermic and milk synthesis becomes less important (Rhoads et al., 2011). Heat stressed dairy cows have a greater glucose disposal rate and the amount of glucose dedicated to milk synthesis is about 400g glucose/day less in heat stressed cows compared to their pair-fed cohorts (Baumgard et al., 2011). Plasma insulin levels increase with heat stress and adipose tissue is not mobilized even though the cow is in a negative energy balance. Currently, research indicates that supplying additional glucose alone is not sufficient to prevent these metabolic changes and does not improve milk yield in heat stressed lactating dairy cows. PROBIOTICS The use of probiotics in lactating dairy cows is a widely accepted method to maintain a healthy rumen. Direct fed microbials (DFM) include active cultures or vegetative forms of bacteria and yeast. DFMs can improve anaerobiosis, maintain pH, and improve feed efficiency. The rumen microbiota is responsible for the majority of energy requirements. Rumen fermentation supplies gluconeogenic precursors, microbial proteins, and other gasses such as methane.

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Figure 2. Saccharomyces cerevisiae strain CNCM I-1077 (Levucell SC2, Lallemand, Toulouse, France) and the changes in pH at thermoneutral conditions. Adapted from Bach et al., (2007).

The effectiveness of a probiotic may depend on the flora from individual cows or dairies. Changes of host microbial populations to environmental/ nutritional stimuli can be host specific. A study out of Lethbridge, AB, Canida looked at changes in ruminal variables in Holstein heifers in their first lactation fed a high concentrate or high forage diet. Each animal was classified as least, intermediate and most acidotic. Variables included changes in pH and bacterial community compositions (BCC). Production responses were similar between groups and not effected by rumen acidosis. Differences in BCC were seen in individuals and not specific to treatment or classification (Mohammed et al., 2012). Enhancing production can be accomplished through different methods and strains of probiotics.

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Figure 3. Targets of DMFs not specific to heat stress Maintain pH in HS rumen and improve feed efficiency Levucell SC (Lallemand) Yeast, strain S. cerevisiae I-1077 Lactic acid-utilizing microbes Lactipro (MS Biotec) Bacteria, strain Megasphaera elsdenii NCIMB 41125 Favorable VFA profile Stimulating activity of beneficial rumen bacteria Yea-Sacc (Alltech) Yeast, Saccharomyces cerevisiae CBS 493.94 Maintain healthy microbes Calsporin (Calpis) Bacterial spore, Bacillus subtilis C-3102 Improve Immune response to OmniGen-AF Yeast + B-complex vitamins Additionally, each product has other claims and was placed in grouping by research, observation or claims. If DFMs can reduce acidosis, promote beneficial microbial flora or elicit a favorable immune response, the impact, severity and duration of hyperthermia may be reduced. Benefits have been documented when DFMs were fed to HS dairy cows. Shwartz et al., (2009) fed two strains of yeast with endogenous enzymes to HS lactating dairy cows. Though there were no production differences, treatment reduced rectal temperature at 1200 and 1800 h. To address the challenges of heat stress, the approach will likely address different aspects of heat stress. Betaine is an organic osmolyte that has been shown to promote favorable bacterial growth under stressed condition (Wdowiak-Wrobel et al., 2013) including fluctuations in pH (Laloknam et al., 2006). Yeast extract that contain oligosaccharide can act as a prebiotic and promote favorable microbial growth. Future research with cocktails/ combinations of probiotics and other promoters may answer broader basic and applied questions. Celmanax contains hydrolyzed yeast, yeast extracts and yeast culture and has a claim to improve milk production and reduce somatic cell count. Research supports the finings that yeast cultures can influence microbial metabolism (Miller-Webster et al., 2002) and can stimulate lactic acid utilizing bacteria (Nisbet and Martin, 1991). Lactating dairy cows were fed 4 x 109 cfu/ head of Lactobacillus acidophilus NP51 and Propionibacterium freudenreichii NP24 for 12 weeks between June and September. No differences were found respiration rates or body temperature. Feeding the DFM increased milk yield by 2.4 kg/day, improved protein yield and improved digestibility (Boyd et al., 2011).

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Huber et al. (1994) reported that the addition of fungal culture increased milk yield and cellulose digestibility in some studies. The fungal cultures also decreased body temperature and respiration rates in hot weather, but not in cool. The use of probiotic Lactobacillus GG (ATCC strain number 53103) in young adult mice colon (YAMC) cells induced heat shock expression. Expression was induced in thermoneutral and heat stress (Tao et al., 2006). Current research at the University of Arizona is measuring the expression of HSP70 and HSP27 in DFM treated lactating cows subjected to heat stressed conditions (R.J Collier lab). Preconditioning animals for heat stress needs to be further studied. DMFs can be fed during non-stressed conditions to prevent the amplification of metabolic disturbances during heat stress. Many of the unknowns (Table 1) do not have sufficient data. Some of the responses to probiotic and heat stress (Table 1) need additional research for replication, strain and combination therapy during heat stress Table 1. Observed responses to heat stress and/ or Probiotic Observed Responses Item Feed intake Feed efficiency Respiration rate Drooling Rumen acidosis Hindgut acidosis Microbe community Body temperature Milk yield Morbidity HSP Heat stress (HS) Reduced Reduced Increased Increased Increased Increased Altered
2

Probiotic (non HS) Increased Increased n/a No change Reduced Reduced Improved Unknown Increased Reduced Increased

Probiotic1 HS Improved Increased Reduced Unknown Reduced Unknown Unknown Reduced Increased Unknown Increased

Increased Reduced Increased Increased

CONCLUSION Some of the metabolic alterations that occur with the insult of heat stress can be addressed using DFMs. Preconditioning cows with DFMs should be examined to measure the effect during and while recovering from heat stress. Replacement dairy heifers may be the extreme example with opportunities to influence both hindgut and rumen microbial populations (Dick et al., 2012). Neonatal calves are functional monogastrics and subject to scours. Later as the rumen develops DFM supplementation can promote favorable microbial growth DMFs have the potential to alter fore and hindgut microbes to improve growth, development and improve future lactations. If a DFM is fed to a postnatal calf it can potentially influence the microbial flora in the large intestine prior to rumen development. As the rumen matures in the first few months of 160

a calfs life, the addition of DFMs may promote a healthy rumen and improve the rumen wall development. Reducing acidosis during HS can minimize production losses and prevent metabolic and health disorders. DMFs offer lactic acid utilizing and promoting microbes and more favorable VFA and nutrient production. Supplying heat stressed cows with improved gluconeogenic precursors can allow the animal to dissipate heat and acclimate to heat stress. Comparisons can be inconsistent due to individual and herd variability and the type of probiotic used. Some commercial products include prebiotics, vitamins, minerals and other growth promoters. DFM feed additives are a promising approach to reducing the impacts of heat stress. References Ametaj, B. N., Q. Zebeli, and S. Iqbal. 2010. Nutrition, microbiota, and endotoxin-related diseases in dairy cows. Revista Brasileira De Zootecnia. 39: 433-444. Bach, A., C. Iglesias, and M. Devant. 2007. Daily rumen pH pattern of loose-housed dairy cattle as affected by feeding pattern and live yeast supplementation. Anim. Feed Sci. Technol. 136(1): 146-153. Baumgard, L. H., J. B. Wheelock, S. R. Sanders, C. E. Moore, H. B. Green, M. R. Waldron et al. 2011. Postabsorptive carbohydrate adaptations to heat stress and monensin supplementation in lactating holstein cows. J. Dairy Sci. 94(11): 5620-5633. Beede, D., and R. Collier. 1986. Potential nutritional strategies for intensively managed cattle during thermal stress. J. Anim. Sci. 62(2): 543-554. Bolton, J. R., and D. A. Pass. 1988. The Alimentary Tract. Pages 99-121 in Clinicopathologic Principles for Veterinary Medicine. Robinson, W. F., and C. R. Huxtable, eds. Cambridge University Press, Cambridge. Boyd, J., J. W. West, and J. K. Bernard. 2011. Effects of the addition of direct-fed microbials and glycerol to the diet of lactating dairy cows on milk yield and apparent efficiency of yield. J. Dairy Sci. 94(9): 4616-4622. Collier, R. J., J. L. Collier, R. P. Rhoads, and L. H. Baumgard. 2008. Invited review: Genes involved in the bovine heat stress response. J. Dairy Sci. 91(2): 445-454. Dick, K. J., G. C. Duff, S. W. Limesand, S. P. Cuneo, D. K. Knudson, C. P. McMurphy, L. W. Hall, J. C. Bernal-Rigoli, and J. A. Marchello. 2012. Effect of Lactobacillus acidophilus and Propionibacterium freudenreichii on digestive tract morphology of neo-natal/transition Holstein bull calves and performance and carcass characteristics of calf-fed Holstein steers. Prof. Anim. Sci. (Submitted). Dong, G., S. Liu, Y. Wu, C. Lei, J. Zhou, and S. Zhang. 2011. Diet-induced bacterial immunogens in the gastrointestinal tract of dairy cows: Impacts on immunity and metabolism. Acta Vet. Scand. 53: 48-0147-53-48. Gressley, T. F., M. B. Hall, and L. E. Armentano. 2011. Ruminant nutrition symposium: Productivity, digestion, and health responses to hindgut acidosis in ruminants. J. Anim. Sci. 89(4): 1120-1130. Huber, J. T., G. Higginbotham, R. A. Gomez-Alarcon, R. B. Taylor, K. H. Chen, S. C. Chan et al. 1994. Heat stress interactions with protein, supplemental fat, and fungal cultures. J. Dairy Sci. 77(7): 2080-2090. 161

Khafipour, E., S. Li, J. C. Plaizier, and D. O. Krause. 2009. Rumen microbiome composition determined using two nutritional models of subacute ruminal acidosis. Appl. Environ. Microbiol. 75(22): 7115-7124. Laloknam, S., K. Tanaka, T. Buaboocha, R. Waditee, A. Incharoensakdi, T. Hibino et al. 2006. Halotolerant cyanobacterium aphanothece halophytica contains a betaine transporter active at alkaline pH and high salinity. Appl. Environ. Microbiol. 72(9): 6018-6026. Lean, I., L. Wade, M. Curtis, and J. Porter. 2000. New approaches to control of ruminal acidosis in dairy cattle. ASIAN AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES. 13: 266-269. Miller-Webster, T., W. H. Hoover, M. Holt, and J. E. Nocek. 2002. Influence of yeast culture on ruminal microbial metabolism in continuous culture. J. Dairy Sci. 85(8): 2009-2014. Mishra, M., F. Martz, R. Stanley, H. Johnson, J. Campbell, and E. Hilderbrand. 1970. Effect of diet and ambient temperature-humidity on ruminal pH, oxidation reduction potential, ammonia and lactic acid in lactating cows. J. Anim. Sci. 30(6): 1023-1028. Mohammed, R., D. M. Stevenson, P. J. Weimer, G. B. Penner, and K. A. Beauchemin. 2012. Individual animal variability in ruminal bacterial communities and ruminal acidosis in primiparous holstein cows during the periparturient period. J. Dairy Sci. 95(11): 67166730. Mohammed, R., D. M. Stevenson, P. J. Weimer, G. B. Penner, and K. A. Beauchemin. 2012. Individual animal variability in ruminal bacterial communities and ruminal acidosis in primiparous holstein cows during the periparturient period. J. Dairy Sci. 95(11): 67166730. Nisbet, D. J., and S. A. Martin. 1991. Effect of a saccharomyces cerevisiae culture on lactate utilization by the ruminal bacterium selenomonas ruminantium. J. Anim. Sci. 69(11): 4628-4633. Rhoads, M. L., R. P. Rhoads, M. J. VanBaale, R. J. Collier, S. R. Sanders, W. J. Weber et al. 2009. Effects of heat stress and plane of nutrition on lactating holstein cows: I. production, metabolism, and aspects of circulating somatotropin. J. Dairy Sci. 92(5): 1986-1997. Rhoads, R. P., J. W. Kim, M. E. Van Amburgh, R. A. Ehrhardt, S. J. Frank, and Y. R. Boisclair. 2007. Effect of nutrition on the GH responsiveness of liver and adipose tissue in dairy cows. J. Endocrinol. 195(1): 49-58. Rhoads, R. P., A. J. La Noce, J. B. Wheelock, and L. H. Baumgard. 2011. Alterations in expression of gluconeogenic genes during heat stress and exogenous bovine somatotropin administration. J. Dairy Sci. 94(4): 1917-1921. Russell, J. R., and T. Hino. 1985. Regulation of lactate production in streptococcus bovis: A spiraling effect that contributes to rumen acidosis. J. Dairy Sci. 68(7): 1712-1721. Sanchez, W. K., M. A. McGuire, and D. K. Beede. 1994. Macromineral nutrition by heat stress interactions in dairy cattle: Review and original research. J. Dairy Sci. 77(7): 20512079. Sharma, S. K., P. Christen, and P. Goloubinoff. 2009. Disaggregating chaperones: An unfolding story. Curr. Protein Pept. Sci. 10(5): 432-446. Sharma, S. K., P. De los Rios, P. Christen, A. Lustig, and P. Goloubinoff. 2010. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase. Nat. Chem. Biol. 6(12): 914-920.

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Shwartz, G., M. L. Rhoads, M. J. VanBaale, R. P. Rhoads, and L. H. Baumgard. 2009. Effects of a supplemental yeast culture on heat-stressed lactating holstein cows. J. Dairy Sci. 92(3): 935-942. Tao, Y., K. A. Drabik, T. S. Waypa, M. W. Musch, J. C. Alverdy, O. Schneewind et al. 2006. Soluble factors from lactobacillus GG activate MAPKs and induce cytoprotective heat shock proteins in intestinal epithelial cells. Am. J. Physiol. Cell. Physiol. 290(4): C1018-30. Wdowiak-Wrobel, S., A. Leszcz, and W. Malek. 2013. Salt tolerance in astragalus cicer microsymbionts: The role of glycine betaine in osmoprotection. Curr. Microbiol. West, J. W. 2003. Effects of heat-stress on production in dairy cattle. J. Dairy Sci. 86(6): 21312144. Westwood, C., and I. Lean. 2001. Nutrition and lameness in pasture-fed dairy cattle. PROCEEDINGS-NEW ZEALAND SOCIETY OF ANIMAL PRODUCTION, Zebeli, Q., S. Sivaraman, S. M. Dunn, and B. N. Ametaj. 2011. Intermittent parenteral administration of endotoxin triggers metabolic and immunological alterations typically associated with displaced abomasum and retained placenta in periparturient dairy cows. J. Dairy Sci. 94(10): 4968-4983. Zimbleman, R.B., Rhoads, R.P. Baumgard, L.H. and Collier, R.J. 2009. Revised temperature humidity index (THI) for high producing dairy cows. J. Dairy Sci. 92: E-Suppl. 1:347.

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ARPAS The American Registry of Professional Animal Scientists (www.arpas.org) Southwest Nutrition & Management Conference February 21 & 22, 2013 Approved for up to 10 CEUs: Pre-Conference Symposium 2 CEUs Thursday Conference Sessions 4 CEUs Friday Conference Sessions 4 CEUs As a registrant of the SWNMC (if you are an ARPAS member) you may include these credits on your annual submission of CEUs in the fall. A CEU reporting form will be included with your dues notice OR you may download a form from their website. Your CEUs can be most efficiently recorded by sending in one form at the time you submit your membership renewal in the fall. ARPAS Business Office 1111 N. Dunlap Ave. Savoy, IL 61874

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