JAS Yhan

Journal of Animal Science
Effect of heat stress on oxidative status and reproductive

performance of sows
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Journal: Journal of Animal Science
Manuscript ID: E-2010-3616
Manuscript Type: Animal Production

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Date Submitted by the

19-Oct-2010
Author:
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Complete List of Authors: Zhao, Yan; North Carolina State University, Animal Science
Flowers, William; North Carolina State University, Animal Science
Saraiva, Alysson; North Carolina State University, Animal Science
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Yeum, Kyung-Jin; Tufts University, Jean Mayer USDA-Human

Nutrition Research Center on Aging
Kim, Sung Woo; North Carolina State University, Department of
Animal Science
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Key Words: sows, oxidative stress, heat stress, gestation, lactation

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ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

Page 1 of 28 Journal of Animal Science
2 Running Head: Oxidative status of sows under heat stress
4 Effect of heat stress on oxidative status and reproductive performance of
5 sows1
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7 Y. Zhao,† W. L. Flowers,† A. Saraiva,†§ K.-J. Yeum,‡ and S. W. Kim†2

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†
9 North Carolina State University, Raleigh, NC 27695;
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10 Universidade federal de Viçosa, Viçosa, MG, Brasil;
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11 Tufts University, Boston, MA
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The authors acknowledge the financial supports from North Carolina Pork Council and North
Carolina Agricultural Foundation.
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Corresponding author: sungwoo_kim@ncsu.edu

Journal of Animal Science Page 2 of 28
13 ABSTRACT Reproductive performance of sows reduces during summer due to heat stress.
14 This study is aimed to determine effects of heat stress on oxidative status, and reproductive
15 performance of sows during gestation and lactation. Twenty eight sows were used in this study.
16 Fourteen of them were under moderate ambient temperature environment (CON: daily range of
17 17.8 ± 1.3oC to 11.8 ± 1.4oC in gestation building and 23.0 ± 0.6oC to 20.0 ± 0.4oC in lactation
18 building) and the other 14 sows were under high ambient temperature environment (HT: daily
19 range of 29.7 ± 1.0oC to 21.3 ± 0.9oC in gestation building and 30.3 ± 0.4oC to 23.3 ± 0.3oC in
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20 lactation building). Sows were fed corn-soybean meal based diets during gestation (2.2 kg/d) and
21 lactation (ad libitum) and used to collect reproductive performance and blood samples on d 35,
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22 60, 90, and 109 of gestation and d 1 and 18 of lactation. Plasma samples were used for
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23 determining concentrations of malonedialdehyde, protein carbonyls, 8-hydroxy-deoxyguanosine,

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24 and immunoglobin G and M. Sows in HT had poor reproductive performance than sows in CON
25 indicated by smaller (P < 0.05) litter size and litter weight at birth and d 18 of lactation as well as
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26 smaller (P < 0.05) litter weight gain than those. Sows in HT had a greater (P < 0.05)
27 concentration of malonedialdehyde on d 90 and 109 of gestation, and d 1 of lactation than CON.

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28 The protein carbonyl concentration of HT was greater (P < 0.05) than CON on d 90, 109 of
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29 gestation and d1 and18 of lactation. Plasma concentrations of 8-hydroxy-deoxyguanosine and
30 protein carbonyls were greater (P < 0.05) on d 108 of gestation than the other days for sows in
31 HT. In conclusion, sows are under elevated oxidative stress during the late gestation and
32 lactation periods when they are housed in a heat stress environment. Increased oxidative damage
33 to lipid, protein and DNA is one of the major contributing factors for reduced reproduction
34 performance of sows under heat stress.
35 Key word: heat stress, gestation, lactation, oxidative stress, sow

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36 INTRODUCTION
37 In general, a thermal comfort zone for a pig decreases when a pig gets older and heavier.
38 The thermal comfort zone for a sow ranges from 12 to 22oC (Black et al., 1993). Environmental
39 temperatures remaining above 20oC would cause heat stress especially with temperature above
40 27oC for an extended period of time may lead to decreased reproductive efficiency of sows
41 (Myer and Bucklin, 2001). Heat stress condition often occurs during summer season or in
42 tropical and subtropical regions. Southeastern coastal and Midwestern areas in the US are where
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43 majority of sow farms are located (National Agricultural Statistics Service. 2008). Without
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44 efficient cooling systems, hot and humid summer climate in these regions can cause heat stress
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45 on sows.
Heat stress would cause a negative effect on reproductive performance of a sow during
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47 gestation and lactation. Studies showed that heat stress diminished hypothalamo-pituitary
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48 gonadal axis to secrete FSH, LH, and delayed puberty in gilts (Flowers et al., 1989; Flowers and
49 Day, 1990). Heat stress may affect early development of embryos (Edwards et al. 1968) causing
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50 small litter size, increased number of stillborn, and reduced birth weights (Omtvdt et al., 1971;
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51 Johnston et al., 1999; Renaudeau and Noblet, 2001). Studies have shown that lactating sows
52 exposed to high temperatures had reduced feed intake and milk production (Schoenherr et al.,
53 1989a,b; Black et al., 1993).
54 Reactive oxygen species (ROS) including hydroxyl radical (•OH), superoxide anion (O2•-
55 ), and hydrogen peroxide (H2O2) are produced during biochemical processes within an animal
56 body (Betteridge, 2000). If the balance between the production of ROS and antioxidant defenses
57 is disturbed, an animal can suffer from oxidative stress. Reactions of ROS with macromolecules
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58 such as proteins, lipids, and nucleic acids can produce chain reaction further reacting with
59 additional macromolecules causing lipid peroxidation, protein damage, and DNA damage
60 (Halliwell et al., 1989; Gutteridge, 1995; Hall et al., 1996). During late-gestation and lactation,
61 sows are under severe catabolic status (Ji et al., 2005; Kim et al., 2009), and the catabolic
62 condition increases the production of reactive oxygen species causing increased oxidative stress
63 (Bernardi et al., 2008). Hyperthermia from heat stress can increase production of reactive
64 oxygen species causing oxidative damages in a cellular model (Mitchell and Russo, 1983) and in
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65 mice (Ozawa et al., 2002; Matsuzuka et al., 2005).

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66 However, currently there is limited information on a status of oxidative stress of sows

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67 during gestation and lactation. It is not determined if sows under heat stress would have
68 increased oxidative stress and if it is related to changes in reproductive performance of sows.

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69 Therefore, it is hypothesized that heat stress may increase oxidative stress during late gestation
and lactation of sows leading to reduced reproductive performance. The objective of this study
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71 was to assess effects of heat stress on oxidative status, and reproductive performance of sows
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72 during gestation and lactation.

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73 MATERIALS AND METHODS
74 Procedures used in this study were reviewed and approved by the North Carolina State
75 University Animal Care and Use Committee.
76 Animals and Experimental Design
77 A total of 28 multiparous sows (initial BW: 240.5 ± 9.3 kg; and average parity: 5.1 ± 0.7)
78 were used in this study under different ambient temperature environments considered as

79 moderate (CON) or high (HT). Fourteen sows were randomly assigned to the moderate ambient
80 temperature environment on d 35 of gestation. Average daily maximum and minimum
81 temperatures were 17.8 ± 1.3oC and 11.8 ± 1.4oC, respectively in gestation building and 23.0 ±
82 0.6oC and 20.0 ± 0.4oC in lactation building. Another group of 14 sows were assigned to the
83 under high ambient temperature environment on d 35 of gestation. Average daily maximum and
84 minimum temperatures were 29.7 ± 1.0oC and 21.3 ± 0.9oC, respectively in gestation building
85 and 30.3 ± 0.4oC and 23.3 ± 0.3oC in lactation building. All sows were housed in the North
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86 Carolina State University Swine Educational Unit (Raleigh, NC).

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87 All pregnant sows were housed in gestation stalls (2 × 0.64 m) and fed 2.2 kg feed daily.
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88 Gestation diet contained 13.3% CP and 3.33 Mcal ME/kg. Water was supplied ad libitum. On d
89 108 of gestation, all sows were weighed and moved to farrowing crates (2.1 × 1.5 m). Three
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90 sows in HT treatment and 2 sows in CON treatment did not maintain their pregnancy and did not
farrow. During lactation, sows were fed ad libitum by electronic feeders (JYGA Technologies,
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92 Saint-Nicolas, Canada) in farrowing crates. Lactation diet contained 14.8 % CP and 3.46 Mcal
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93 ME/kg. Average daily feed intake was recorded. Body weight of sow was measured on d 35 and
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94 109 of gestation, d 1 and 18 of lactation. Backfat thickness of sow was measured on d 1 and 18
95 of lactation using an ultrasound scanner (Veterinary Sales & Service Inc., Stuart, FL). After
96 farrowing, numbers of piglets born alive, dead, or mummified, BW of piglets on d 1 and 18 of
97 lactation, and litter size on d 1 and 18 of lactation were measured.
98 Sampling of Blood, Colostrum, and Milk
99 Blood samples were collected from the jugular vein of sows on d 35, 60, 90, 109 of
100 gestation, and d 1 and 18 of lactation. Blood was drawn into 9 mL MONOVETTE tubes
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101 (Sarstedt, Newton, NC) containing EDTA. Plasma samples were collected by centrifuging (5810
102 R, Eppendorf AG, Hamburg, Germany) at 3,000 g, 15 min, 4°C, and allocated into 1.5 mL
103 microcentrifuge tubes. Plasma samples were stored in liquid nitrogen for 1 h, and stored at -80°C
104 until analysis.
105 Colostrum and milk samples were collected from each sow on d 1 and 18 of lactation.
106 Sows were injected with 1 mL oxytocin before sampling colostrum and milk. Around 30 mL of
107 colostrum and 30 mL milk were collected from the first 3 pairs of mammary gland of each sow
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108 and stored at -80°C.

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109 Analysis of Oxidative Stress Parameters

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110 Plasma samples were used to measure concentrations of malondialdehyde (MDA),

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111 protein carbonyl, and 8-hydroxy-deoxyguanosine (8-OHdG). Concentrations of MDA were
112 measured using the TBARS assay kit (Cell Biolabs, San Diego, CA). Plasma samples and MDA
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113 standards were first incubated and reacted with thiobarbituric acid at 95°C, after centrifuge and
butanol extraction, samples and standard were read at 532 nm with an spectrophotometric plate
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114
115 reader (BioTek, Winooski, VT) and the KC4 data analysis software (BioTek, Winooski, VT).
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116 The detection limit of MDA analysis was 0.98 µM.
117 Protein concentration in plasma samples were measured by the BCA kit (Thrmo Fisher
118 Scientific Inc, Rockford, IL), then all plasma samples were diluted with 1 × phosphate-buffered
119 saline (PBS) to reach protein concentration at10 µg/mL before protein carbonyl assay.
120 Concentrations of protein carbonyl were measured via the protein carbonyl ELISA kit (Cell

121 Biolabs, San Diego, CA). The absorbance was measured at 450 nm. The detection limit for
122 protein carbonyl was 0.375 nmol/mg.
123 Concentrations of 8-OHdG in plasma were measured using the oxidative DNA damage
124 ELISA kit (Cell Biolabs, San Diego, CA) following the manufacture’s instruction. The
125 absorbance was read at 450 nm, and concentrations of 8-OHdG were calculated against its
126 standard curve. The detection limit for 8-OHdG was 0.078 ng/mL.
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127 Immunoglobulin Evaluation
128 Concentrations of immunoglobulin G (IgG) and immunoglobulin M (IgM) in sow’s

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129 colostrums and plasma on d 109 of gestation and d 3 and 18 of lactation were measured by using
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130 ELISA kit (Bethyl, Montgomery, TX). The measurement was conducted by the manufacturer’s
instruction. Goat anti-pig IgG or goat anti-pig IgM were used as capture antibodies to coat wells.
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132 Plasma samples and colostrums were diluted to 1:100,000 for IgG and IgM measurement.
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133 Horseradish peroxidase goat anti-pig IgG or IgM was used as the detection. The plate was read at
134 450 nm. Sample concentration was quantified against the known standard curve. Detection limits
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135 were 7.8 ng/mL for IgG, and 15.6 ng/mL for IgM.
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136 Statistical Analysis
137 Parameters for reproductive performance and oxidative stress status were analyzed as a
138 completely randomized design using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). A
139 sow or a litter was the experimental unit. Treatment effects were considered statistically
140 significant when probability values were less than 0.05, and probability less than 0.1 and equal or
141 greater than 0.05 was considered as a trend.

142 RESULTS
143 Reproductive Performance of Sow
144 There was no difference between treatments for sow’s BW on d 35 and 109 of gestation,
145 and d 3 and 18 of lactation. Parity, back fat loss, and ADFI of sows did not differ between CON
146 and HT (Table 2). Sows in CON had a greater (P < 0.05) number of piglets at born and d 18 of
147 lactation than sows in HT. Litter weight at birth and d 18 of lactation as well as litter weight
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148 gain in HT were smaller (P < 0.05) than those in CON. There were no differences between
149 treatments in piglet BW on d 1 and 18 of lactation (Table 2).

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150 Oxidative Stress
151 Plasma concentration of MDA did not differ between CON and HT on d 35 and 60 of
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152 gestation, and d 18 of lactation. However, sows in HT had a greater (P < 0.05) MDA
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153 concentration on d 90 and 109 of gestation, and d 1 of lactation than CON (Table 3). The percent
154 change of MDA concentration from d 35 of gestation in HT was greater (P < 0.05) than that of
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155 CON on d 109 of gestation (Table 4).

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156 Plasma concentration of protein carbonyl in CON was greater (P < 0.05) than that of HT
157 on d 35, but smaller (P < 0.05) on d 109 of gestation and d 18 of lactation. There were no
158 differences on d 60, 90 of gestation and d 3 of lactation between treatments (Table 3). The
159 percent changes of protein carbonyl concentration from d 35 of gestation in HT was greater (P <
160 0.05) than that of CON on d 90 and 109 of gestation and d1 and 18 of lactation (Table 4).
161 Concentration of 8-OHdG in CON was greater (P < 0.05) than that of HT on d 35, 60, 90,
162 and 109 of gestation, and d 1 and 18 of lactation (Table 3). However, there were no differences
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163 in percentage change of 8-OHdG concentration from d 35 of gestation on d 60, 90, and 109 of
164 gestation and d 3 and 18 of lactation between treatments (Table 4).
165 Plasma concentrations of MDA, protein carbonyl, and 8-OHdG were compared among
166 different days during gestation and lactation in CON and HT, respectively. For sows in CON, the
167 MDA concentration on d 18 of lactation was greater (P < 0.05) than all the other days except for
168 d 60 of gestation (Table 3). Protein carbonyl concentration on d 35 of gestation was the greatest
169 (P < 0.05) compared with all the other days in CON. Protein carbonyl concentration on d 3 of
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170 lactation was greater (P < 0.05) than d 18 of lactation (Table 3). The 8-OhdG concentration on d
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171 35 of gestation and d 18 of lactation was smaller (P < 0.05) than all other day during gestation
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172 and lactation in CON (Table 3). For sows in HT, the concentration of MDA tends to be greater
173 (P = 0.068) on d 109 of gestation and d 3 of lactation than all the other days. Concentration of
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174 protein carbonyl on d 18 of lactation was greater (P < 0.05) than the other days except for d 109
of gestation. Protein carbonyl concentration was the highest (P < 0.05) on d 109 of gestation for
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176 sows in HT (Table 3). Concentrations of 8-OHdG on d 109 of gestation and d 3 of lactation were
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177 greater (P < 0.05) than d 35 of gestation and d 18 of lactation (Table 3).
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178 Immunoglobulin Evaluation
179 There was no significant difference between treatments for plasma concentrations of IgG
180 and IgM on d 109 of gestation and d 18 of lactation (Table 5). Plasma concentration of IgG in
181 HT tended to be greater (P = 0.067) than CON on d 3 of lactation. Concentrations of IgM and
182 IgG in colostrum of HT were greater (P < 0.05) than that of CON (Table 5).
183 DISCUSSION
184 Heat Stress and Sow Performance

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185 In this study, sows in HT had a reduced litter weight gain compared with sows in CON
186 indicating that sows in HT produce less milk during lactation than sows in CON. This may be
187 partly due to a smaller litter size at birth but also due to an increased number of piglets dead
188 during lactation. This is a very typical indication of heat stress responses of sows as consistent
189 with other studies (Black et al., 1993; Renaudeau et al., 2001; Renaudeau and Noblet, 2001;
190 Spencer et al., 2003) with reduced milk production or litter weight gain from sows under heat
191 stress. Previous studies also showed that gilts and sows under heat stress in late pregnancy
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192 farrowed fewer piglets born alive with increased number of stillborns (Omtvdt et al., 1971;
193 Johnston et al., 1999; Renaudeau and Noblet, 2001). These results are consistent with our finding
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194 that sows in HT had a smaller number of piglet born alive whereas the total born did not differ.
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195 Bond et al. (1952) showed that sows lost more weight as environmental temperature increases.
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196 Stansbury et al. (1987) reported that sows exposed to 30oC during lactation lost more weight than
197 control group. However, in our study, there was no difference in BW loss of sows during
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198 lactation between treatments. This may be because sows in HT had a reduced milk production
199 whereas ADFI was not reduced compared with sows in CON.
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200 Heat stress can cause reduced feed intake during lactation affecting milk production and
201 thus litter weight gain (Wheelock et al., 2006; Rhoads et al., 2007). It has been shown that
202 animals under heat stress have a slower rate of digesta passage and longer retention time of
203 digesta in intestine which may be responsible for decreased voluntary feed intake (Warren et al.,
204 1974; Wiestra and Christopherson, 1976; Robertshaw, 1981). However, the feed intake of sows
205 under heat stress was not affected compared with those in CON in the current study. This
206 indicates that feed intake was not the main factor for the reduced reproductive performance in
207 our study. It is further shown that heat stress can decrease nutrient uptake in almost all species
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208 (Collier et al., 2008). Collectively, our data indicate that sows in HT had heat stress responses as
209 shown from reduced milk production and litter size and oxidative stress could be the primary
210 effect for reduced reproduction performance.
211 Oxidative Stress during Gestation and Lactation under Heat Stress
212 In this study, MDA was chosen as an indicator for lipid peroxidation despite of its non-
213 specificity because MDA originates from the oxidative degradation of polyunsaturated fatty
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214 acids and is one of the most widely used markers for lipid peroxidation (Simm et al., 2008).
215 Protein carbonyl derivatives are the most common products of protein oxidation. Protein
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216 carbonyls are generated when side chains of several amino acids mainly Pro, Arg, Lys, and Thr
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217 are oxidized (Poppek and Grune, 2006; Simm et al., 2008). The 8-hydroxy-deoxyguanosine is a
major marker for an oxidative damage of nucleic acid (Bowen, 2010). It is formed when free
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218
219 radicals, especially the hydroxyl radical, act on deoxyguanoxine in DNA (Ravanat et al., 2000).
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220 The •OH adds to guanosine and makes a hydroxyguanosine radical, which can then form 8-
221 OHdG. The 8-hydroxy-deoxyguanosine can be misread when DNA is copied, introducing a
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222 mutation (Gutteridge and Halliwell, 1994). During the repair of damaged DNA by exonucleases,
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223 the resulting 8-OHdG is excreted without further metabolism into urine. It is a ubiquitous
224 marker of oxidative stress.
225 This study clearly showed that sows in HT had an increased oxidative stress during late
226 gestation and lactation when comparing the oxidative stress parameters between different days in
227 gestation and lactation. The result is consistent with our previous study (Berchieri-Ronchi et al.,
228 2010). For sows in CON, only oxidative DNA damage was increased during late gestation and
229 lactation whereas other measurements did not have similar pattern. Sows are under severe
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230 catabolic status during late gestation and lactation especially due to the greatly increased nutrient
231 needs for fetal growth (McPherson et al., 2004; Ji et al., 2005), mammary growth (Kim et al.,
232 1999; Ji et al., 2006), and milk production (Kim et al., 2000) whereas their nutrient intakes are
233 insufficient (Kim and Easter, 2003). Catabolic condition causes tissue mobilization, increased
234 production of free radicals and thus elevated oxidative stress (Bernardi et al., 2008). In addition,
235 antioxidative capacity in a sow body is reduced during late gestation and lactation as the
236 increased use of antioxidants for fetuses and mammary glands increases (Berchieri-Ronchi et al.,
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237 2010). The increased production of free radical and decreased antioxidant availability would
238 together cause increased oxidative stress to sows during late gestation and lactation.
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239 In this study, HT treatment had a greater protein and lipid oxidative stress during late
240 gestation and lactation when comparing with CON treatment. In addition to increased negative
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241 energy balance and decreased antioxidant availability during late gestation and lactation,
oxidative damage can be induced by increased production of free radicals when an animal is
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243 under high ambient temperature environment (Mitchell and Russo, 1983; Ozawa et al., 2002;
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244 Matsuzuka et al., 2005) indicating that oxidative stress could be one of stress responses caused
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245 by heat stress.
246 Oxidative Stress and Reproductive Performance of Sows
247 Oxidative stress can be one of the causes for reduced reproduction performance under
248 heat stress. It was reported that proteins can scavenge the majority (50 to 75%) of free radicals
249 generated (Davies et al., 1999). Reactions with free radicals generate protein carbonyls lead to
250 protein aggregates thereby alter the structure of protein and affect their functions. Cellular
251 proteins with oxidation can aggregate each other by covalent bonds becoming resistant to
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252 proteolysis. Thus cells can have limited amino acids from hydrolysis of these altered proteins for
253 protein synthesis consequently increasing turnover of other proteins (Grune et al., 1995; Grune et
254 al., 1998; Ullrich et al., 1999; Simm et al., 2008). Animals under oxidative stress, therefore,
255 would have increased needs of energy and amino acids for cell maintenance and repair. Under a
256 normal condition, protein turnover takes 9 to 12% of energy used for cell maintenance (Milligan,
257 1971; Baldwin et al., 1980). Besides protein oxidation, oxidative stress induced peroxidation of
258 membrane lipids is critical. It leads to changes in the biological properties of cellular membranes
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259 and therefore alterations of membrane-bound receptors or enzymes activities which may lead to
260 cell death (Slatter et al., 2000; Simm et al., 2008). Furthermore, oxidative stress can cause DNA
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261 damage and the mutation problem. The elevatetd 8-OHdG can alter gene expression by
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262 inhibiting methylation. It can also cause mutation by pairing with adenosine rather than cytosine
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263 during DNA replication, leading to GC to AT conversion (Simm et al., 2008).
Edwards et al. (1968) found that gilts exposed to heat stress from 1 to 15 d post breeding
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265 had fewer viable embryos at d 30 postbreeding than gilts exposed to same stress from d 15 to 30
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266 postbreeding. Omtvedt et al. (1971) reported that gilts exposed to heat stress from d 8 to 16
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267 postbreeding had greatest reduction in the number of viable embryos. These results indicate that
268 embryo is susceptible to heat stress during the implantation period and thus it can be speculated
269 that heat stress during the implantation period may also contributed to reduced litter size for heat
270 stressed sows compared with sows in CON in this study.
271 Collectively, sows with heat stress would have increased protein turnover due to
272 increased oxidative damage to cellular proteins, increased cell death due to increased
273 peroxidation of membrane lipids and increased DNA breakdown which can together interfere
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274 fetal development, mammary gland development, and milk production as shown in reduced
275 number of piglets born alive, and reduced litter weight gain from sows under heat stress.
276 In conclusion, sows receive increased oxidative stress during late gestation and lactation
277 when they are under heat stress environment. Increased oxidative damage to lipid, protein and
278 DNA is one of major contributing factors for reduced reproduction performance of sows under
279 heat stress.

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341 Kim, S. W., W. L. Hurley, I. K. Han, and R. A. Easter. 1999. Changes in tissue composition
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344 Kim, S. W., W. L. Hurley, I. K. Han, and R. A. Easter. 2000. Growth of nursing pigs related to
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349 Matsuzuka, T., M. Ozawa, A. Nakamura, A. Ushitani, M. Hirabayashi, and Y. Kanai. 2005.
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350 Effects of heat stress on the redox status in the oviduct and early embryonic development
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351 in mice. J. Reprod. Dev. 51:281–287.

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352 McPherson, R. L., F. Ji, G. Wu, and S. W. Kim. 2004. Fetal growth and compositional changes
353 of fetal tissues in the pigs. J. Anim. Sci. 82:2534–2540.

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354 Milligan, L. P. 1971. Energy efficiency and metabolic transformations. Fed. Proc. 30:1454–1460.
355 Mitchell, J. B., and A. Russo. 1983. Thiols, thiol depletion, and thermosensitivity. Radiat. Res.
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356 95:471–485.
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357 Myer, R., and R. Bucklin. 2001. Influence of hot-humid environment on growth performance
358 and reproduction of swine. http://edis.ifas.ufl.edu/AN107 Accessed Jul. 9, 2001.
359 Omtvedt, I. T., Nelson, R. E., Ronnie, L. Edwards, and D. F. Stephens. 1971. Influence of heat
360 stress during early, mid and late gestation. J. Anim. Sci. 32:312–317.
361 Ozawa, M., M. Hirabayashi, and Y. Kanai. 2002. Developmental competence and oxidative state
362 of mouse zygotes heat-stressed maternally or in vitro. Reproduction 124:683–689.
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363 Poppek, D., T. Grune. 2006. Proteasomal defense of oxidative protein modifications.
364 Antioxidants and Redox Signaling 8:173–184.
365 Ravanat, J. L., P. D. Mascio, G. R. Martinez, M. H. Medeiros, and J. Cadet. 2000. Singlet
366 oxygen induces oxidation of cellular DNA. J. Biol. Chem. 275:40601–40604.
367 Renaudeau, D., and J. Noblet. 2001. Effects of exposure to high ambient temperature and dietary
368 protein level on sow milk production and performance of piglets. J. Anim. Sci. 79:1540–
369 1548.
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370 Renaudeau, D., N. Quiniou, and J. Noblet. 2001. Effects of exposure to high ambient
371 temperature and dietary protein level on performance of multiparous lactating sows. J.
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372 Anim. Sci. 79:1240–1249.

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373 Robertshaw, D. 1981. Environmental aspects of housing for animal production. Pages 3–17 in
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374 The Environmental Physiology of Animal Production. J. A. Clark, ed. Butterworth-
375 Heinemann Ltd, London.

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376 Stansbury, W. F., J. J. McGlone, and L. F. Tribble. 1987. Effects of season, floor type, air
377 temperature and snout coolers on sow and litter performance. J. Anim. Sci. 65:1507–1513.
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378 Schoenherr, W. D., T. S. Stably, and G. L. Cromwell. 1989a. The effects of dietary fat and fiber
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379 addition on energy and nitrogen digestibility in lactating, primiparous sows housed in a
380 warm or hot environment. J. Anim. Sci. 67:473–481.
381 Schoenherr, W. D., T. S. Stably, and G. L. Cromwell. 1989b. The effects of dietary fat or fiber
382 addition on yield and composition of milk from sows housed in a warm or hot
383 environment. J. Anim. Sci. 67:482–495.
384 Simm, A., and H. J. Brömme. 2005. Reactive oxygen species (ROS) and aging: do we need them,
385 can we measure them, should we block them? Signal Transduction 3:115–125.
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386 Slatter, D. A., C. H. Bolton, and A. J. Bailey. 2000. The importance of lipid-derived
387 malondialdehyde in diabetes mellitus. Diabetologia 43:550–557.
388 Spencer, J. D., R. D. Boyd, R. Cabrera, and G. L. Allee. 2003. Early weaning to reduce tissue
389 mobilization in lactating sows and milk supplementation to enhance pig weaning weight
390 during extreme heat stress. J. Anim. Sci. 81:2041–2052.
391 Ullrich O., T. Reinheckel, N. Sitte, and T. Grune. 1999. Degradation of hypochlorite-damaged
392 glucose-6-phosphate dehydrogenase by the 20S proteasome. Free Radic. Biol. Med. 27:
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393 487–492.
394 USDA. 2008. National Agricultural Statistics Service.

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395 http://usda.mannlib.cornell.edu/usda/current/HogsPigs/HogsPigs-09-26-2008.pdf
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396 Accessed Nov. 16, 2008.

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397 Warren, W. P., F. A. Martz, K. H. Asay, E. S. Hilderbrand, C. G. Payne, and J. R. Vogt. 1974.
398 Digestibility and rate of passage by steers fed tall fescue, alfalfa and orchard grass hay in
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399 18 and 32oC ambient temperature. J. Anim. Sci. 39:93–96.
400 Wiestra, R., and R. J. Christopherson. 1976. Effects of cold on digestibility, retention time
vi
401 digesta, reticulum motility and thyroid hormones in sheep. Can. J. Anim. Sci. 56:699–708.
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19

402 Table 1. Composition of gestation and lactation diets (as-fed basis)
Item Gestation Lactation
Ingredient, %
Corn 81.30 74.00
Soybean meal, 48% CP 13.85 17.60
Poultry fat 1.00 3.99
Biolys®1 0.00 0.25

Fo
L-Thr 0.00 0.01
Limestone 1.11 1.08

r
Dicalcium phosphate 2.05 2.38

Pe
Salt 0.50 0.50

er
Trace mineral permix2 0.15 0.15
Vitamin permix3 0.04 0.04

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Total 100.00 100.00
Calculated composition
vi
DM, % 89.67 90.10

ew
ME, kcal/kg 3,330 3,461
CP, % 13.33 14.75
Lys, % 0.63 0.92
Met, % 0.49 0.51
Trp, % 0.14 0.16
Thr, % 0.49 0.55
20

Ca, % 1.03 1.12
Total P, % 0.69 0.76

1
403 Biolys® (Enovik Degussa, Kennesaw, GA) contains 50.7% of L-Lys.
2
404 The trace mineral premix provided per kilogram of complete diet: 3.96 mg of Mn as
405 manganous oxide; 16.5 mg of Fe as ferrous sulfate; 16.5 mg of Zn as zinc sulfate; 1.65 mg of Cu
406 as copper sulfate; 0.30 mg of I as ethylenediamine dihydroiodide; and 0.30 mg of Se as sodium
407 selenite
Fo
3
408 The vitamin premix provided per kilogram of complete diet: 8,228 IU of vitamin A as
409 vitamin A acetate; 1,173 IU of vitamin D3; 47 IU of vitamin E; 0.03 mg of vitamin B12; 5.88 mg
r
410 of riboflavin; 23.52 mg of D-pantothenic acid as calcium panthonate; 35.27 mg of niacin; 0.24
Pe
411 mg of biotin; 1.76 mg folic acid; 3.88 mg menadione

er
Re
vi
ew
21

412 Table 2. Reproductive performance of sows under moderate or high temperature environment
Item CON1 HT1 SEM P-value
No. of sows2 12 11
Parity 5.2 5.8 1.0 0.515
Body weight of sows, kg
d 35 of gestation 243.2 245.7 14.4 0.864

Fo
d 109 of gestation 289.8 280.7 12.9 0.509
d 1 of lactation 275.6 274.8 12.6 0.953

r
d 18 of lactation 260.8 273.3 12.5 0.365

Pe
Body weight changes, kg
Gestation (d 35 to 109) 46.7 35.0 6.5 0.114

er
Lactation (d 1 to 18) -12.7 -4.5 5.1 0.130

Re
Backfat of sows,3 mm
d 1 of lactation 15.8 17.0 1.4 0.402

vi
d 18 of lactation 14.4 16.6 1.4 0.141

ew
Change from d 1 to 18 -1.4 -0.4 0.9 0.333
ADFI of sows, kg 4.6 4.3 0.5 0.478
Litter size, pig
d 1, total born 13.5 12.5 1.1 0.388
d 1, born alive 11.9a 9.5b 1.0 0.044
d 18 10.4a 7.4b 0.7 < 0.001
Litter weight at d 1,4 kg 17.9a 15.0b 1.4 0.049
22

Litter weight at d 18, kg 55.4a 42.1b 4.8 0.013
Litter weight gain from d 1 to 18, kg 37.5a 27.1b 3.8 0.015
Piglet weight at d 1,9 kg 1.52 1.66 0.14 0.331
Piglet weight at d 18, kg 5.42 5.78 0.36 0.351
Piglets ADG from d 1 to 18, g/d 199.5 204.4 18.3 0.794

1
413 CON= moderate ambient temperature environment (daily range of 17.8 ± 1.3oC to 11.8
414 ± 1.4oC in gestation building and 23.0 ± 0.6oC to 20.0 ± 0.4oC in lactation building); HT= high
Fo
415 ambient temperature environment (daily range of 29.7 ± 1.0oC to 21.3 ± 0.9oC in gestation
416 building and 30.3 ± 0.4oC to 23.3 ± 0.3oC in lactation building)

r
2
Initial number of sows was 28. There were 2 sows in CON and 3 sows in HT did not
Pe
417
418 maintain pregnancy and did not farrow which were excluded from the study.
er
3
419 Measured at the P2 position (locate at left side of the10th rib, and 6 cm away from the
420 spine).
Re
4
421 Litter weight at birth includes piglets born alive only.
5
422 Weight of piglets born alive
vi
a,b
423 Means within a row with different superscripts differ (P < 0.05).
ew
424
23

425 Table 3. Oxidative stress indicators of sows under moderate or high ambient temperature
426 environment
Item CON1 HT1 SEM2 P-value3
8-hydroxy-deoxyguanosine, ng/mL
d 35 of gestation* 0.71a 0.32a 0.10 0.002
d 60 of gestation* 1.11b 0.48ab 0.15 0.004
d 90 of gestation* 1.05b 0.46ab 0.13 < 0.001

Fo
d 109 of gestation 1.24b 0.78b 0.16 0.029

r
d 3 of lactation* 1.11b 0.55b 0.13 0.006

Pe
d 18 of lactation* 0.53a 0.25a 0.10 0.015
SEM5 0.27 0.25

er
P-value6 < 0.001 0.015

Re
Malondialdehyde, µM
d 35 of gestation 4.42ab 6.14 1.16 0.161

vi
d 60 of gestation 6.30abc 4.75 1.46 0.305

ew
d 90 of gestation* 3.96ab 7.41 1.29 0.018
d 109 of gestation* 2.44a 9.02 1.39 < 0.001
d 3 of gestation* 4.08ab 9.01 1.82 0.017
d 18 of lactation 6.29c 5.92 0.99 0.712
SEM4 1.78 2.86
P-value5 0.022 0.215
Protein carbonyl, nmol/mg
24

d 35 of gestation* 2.92d 1.41ab 0.34 < 0.001
d 60 of gestation 1.59bc 1.10a 0.32 0.154
d 90 of gestation 0.90ab 1.01a 0.17 0.538
d 109 of gestation* 1.09ab 2.68c 0.26 < 0.001
d 3 of lactation 1.38b 1.42ab 0.15 0.754
d 18 of lactation* 0.66a 1.79b 0.18 < 0.001
SEM4 0.48 0.38

Fo
P-value5 < 0.001 < 0.001

1
r
Pe
er

2
431 SEM is for treatment CON and HT within a same row.
Re
3
432 P-value is for treatment CON and HT within a same row.
4
433 SEM is for different days with a same column.
vi
5
434 P-value is for among days with a same column.
ew
*
435 Means within a row differ (P < 0.05).
a-d
436 Means within a column with different superscripts differ (P < 0.05).
437
438
25

439 Table 4. Percent changes of oxidative stress indicators during gestation and lactation compared
440 with d 35 of gestation in sows under moderate or high ambient temperature environment
8-hydroxy-deoxyguanosine, 3 % changes to d 35 of gestation
d 60 of gestation 0.68 0.57 0.30 0.796
d 90 of gestation 0.55 0.48 0.24 0.830
d 109 of gestation 0.75 1.83 0.46 0.119

Fo
d 3 of lactation 0.66 0.93 0.33 0.561

r
d 18 of lactation -0.20 -0.15 0.19 0.852

Pe
Malondialdehyde,2 % changes to d 35 of gestation
d 60 of gestation 0.35 0.24 0.31 0.809

er
d 90 of gestation 0.03 0.58 0.41 0.368

Re
d 109 of gestation -0.35a 0.77b 0.28 0.013
d 3 of lactation 0.07 0.97 0.41 0.138

vi
d 18 of lactation 0.56 0.23 0.26 0.370

ew
Protein carbonyl, % changes to d 35 of gestation
d 60 of gestation -0.40 0.00 0.21 0.199
d 90 of gestation -0.68a -0.20b 0.08 0.001
d 109 of gestation -0.60a 1.35b 0.34 0.001
d 3 of lactation -0.49a 0.19b 0.16 0.008
d 18 of lactation -0.74a 0.51b 0.22 0.001
26

1

a,b
446
Fo
447
448
r Pe
449
er
450
451
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vi
ew
27

452 Table 5. Immunological parameters of sows under moderate or high ambient temperature
453 environment
Immunoglobulin M, mg/mL
Plasma
d 109 of gestation 3.60 2.42 0.48 0.101

Fo
d 3 of lactation 1.95 1.78 0.22 0.579
d 18 of lactation 2.53 2.33 0.55 0.722

r
Colostrum 1.56a 2.50b 0.29 0.037

Pe
Immunoglobulin G, mg/mL
Plasma
er
d 109 of gestation 11.44 12.18 0.92 0.589

Re
d 3 of lactation 7.77A 9.03B 0.46 0.067
d 18 of lactation 20.07 19.67 2.26 0.862

vi
Colostrum 10.84a 17.61b 1.70 0.010

ew
1

a,b
A,B
459 Means within a row with different superscripts show a tendency to differ (0.05 ≤ P <
460 0.10)
28

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Journal of Animal Science

Effect of heat stress on oxidative status and reproductive

Manuscript ID: E-2010-3616

Manuscript Type: Animal Production

Date Submitted by the

Yeum, Kyung-Jin; Tufts University, Jean Mayer USDA-Human

Key Words: sows, oxidative stress, heat stress, gestation, lactation

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

2 Running Head: Oxidative status of sows under heat stress

4 Effect of heat stress on oxidative status and reproductive performance of

7 Y. Zhao,† W. L. Flowers,† A. Saraiva,†§ K.-J. Yeum,‡ and S. W. Kim†2

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

23 determining concentrations of malonedialdehyde, protein carbonyls, 8-hydroxy-deoxyguanosine,

27 concentration of malonedialdehyde on d 90 and 109 of gestation, and d 1 of lactation than CON.

29 gestation and d1 and18 of lactation. Plasma concentrations of 8-hydroxy-deoxyguanosine and

34 performance of sows under heat stress.

35 Key word: heat stress, gestation, lactation, oxidative stress, sow

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

53 1989a,b; Black et al., 1993).

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

65 mice (Ozawa et al., 2002; Matsuzuka et al., 2005).

66 However, currently there is limited information on a status of oxidative stress of sows

68 increased oxidative stress and if it is related to changes in reproductive performance of sows.

72 during gestation and lactation.

73 MATERIALS AND METHODS

75 University Animal Care and Use Committee.

76 Animals and Experimental Design

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

80 temperature environment on d 35 of gestation. Average daily maximum and minimum

86 Carolina State University Swine Educational Unit (Raleigh, NC).

96 farrowing, numbers of piglets born alive, dead, or mummified, BW of piglets on d 1 and 18 of

97 lactation, and litter size on d 1 and 18 of lactation were measured.

98 Sampling of Blood, Colostrum, and Milk

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

104 until analysis.

108 and stored at -80°C.

109 Analysis of Oxidative Stress Parameters

110 Plasma samples were used to measure concentrations of malondialdehyde (MDA),

111 protein carbonyl, and 8-hydroxy-deoxyguanosine (8-OHdG). Concentrations of MDA were

116 The detection limit of MDA analysis was 0.98 µM.

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

122 protein carbonyl was 0.375 nmol/mg.

128 Concentrations of immunoglobulin G (IgG) and immunoglobulin M (IgM) in sow’s

136 Statistical Analysis

141 greater than 0.05 was considered as a trend.

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

143 Reproductive Performance of Sow

149 treatments in piglet BW on d 1 and 18 of lactation (Table 2).

150 Oxidative Stress

155 CON on d 109 of gestation (Table 4).

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

164 gestation and d 3 and 18 of lactation between treatments (Table 4).

178 Immunoglobulin Evaluation

184 Heat Stress and Sow Performance

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

210 effect for reduced reproduction performance.

224 marker of oxidative stress.

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901

245 by heat stress.

246 Oxidative Stress and Reproductive Performance of Sows

ScholarOne, 375 Greenbrier Drive, Charlottesville, VA, 22901