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ENVIRONMENT, WELL-BEING, AND BEHAVIOR

Pipping muscle and liver metabolic profile changes and relationships


in broiler embryos on days 15 and 19 of incubation1,2

R. Pulikanti,* E. D. Peebles,*3 R. W. Keirs,† L. W. Bennett,† M. M. Keralapurath,*


and P. D. Gerard‡

*Department of Poultry Science, and †College of Veterinary Medicine, Mississippi State University,
Mississippi State 39762; and ‡Department of Applied Economics and Statistics,
Clemson University, Clemson, SC 29634

ABSTRACT The relative proportions and relationships related with liver glycogen on d 19. In conclusion, in
of pipping muscle and liver nutrients in broiler embryos preparation for hatch between d 15 and 19 of incuba-
on d 15 and 19 of incubation were determined. Ninety tion, weights of the liver and pipping muscle of broiler
hatching eggs obtained from a 30-wk-old broiler breeder embryos increased relative to their BW. This occurred
flock were incubated on 3 replicate tray levels (30 eggs in association with the accumulation of glucose, glyco-
per tray) for 19 d. On 15 and 19 d of incubation, 10 live gen, and protein and with the loss of fat in the pipping
embryos per tray level were necropsied to collect pip- muscle. The carbohydrate stores in the pipping muscle
ping muscle and liver samples. As the broiler embryo were supported by the active metabolism of the liver
developed between d 15 and 19 of incubation, the gly- before 19 d of incubation, which included the transfer
cogen and protein concentrations of the pipping muscle of glucose and fatty acids to the pipping muscle via the
increased, whereas those of the liver decreased, and circulation. Despite the liver’s active supply of these
the fat concentration of the pipping muscle decreased, nutrient subunits for assimilation and oxidation by the
whereas that of the liver increased. Across d 15 and 19, pipping muscle, there was an overall accumulation of
pipping muscle glycogen was negatively correlated with hepatic fat between d 15 and 19 of incubation. These
liver fat, whereas on d 15, pipping muscle glucose was data suggest that the integrated changes in the energy
negatively correlated with liver fat, and pipping muscle profiles of pipping muscle and liver between 15 and 19
glycogen was negatively correlated with liver glucose d of embryogenesis are integral to the broiler embryo’s
and glycogen. Pipping muscle fat was negatively cor- preparation for hatch.
related with liver glucose on d 15 but positively cor-
Key words: broiler, embryo, incubation, nutrient profile, pipping muscle
2010 Poultry Science 89:860–865
doi:10.3382/ps.2009-00531

INTRODUCTION muscle reaches its maximum weight (Pohlman, 1919)


and gains maximum turgidity due to the infiltration
The muscularis complexus (pipping muscle) is the of lymph (Fisher, 1962). Pipping involves a piercing of
primary muscle responsible for the broiler embryo’s the eggshell membranes and shell proper (Moran, 2007)
pipping action toward the end of its embryonic life. and is primarily due to the hydraulic pressure exerted
The other muscles that support pipping activity in- by the turgid pipping muscle rather than the contractile
clude the musculus biventris, musculus spinalis, and force of its muscle fibers alone (Smail, 1964). The pip-
splenius cervicis (Bock and Hikida, 1968). Moreover, ping muscle also serves as a turgid cushion to protect
toward the end of broiler embryonic life, the pipping the embryo during the pipping process (Smail, 1964).
As is any other embryonic tissue or organ, the pipping
muscle is dependent on the yolk sac for its energy and
©2010 Poultry Science Association Inc. nutrient requirements. Complex nutrients including car-
Received October 28, 2009.
Accepted February 9, 2010. bohydrates, proteins, and fats that are mobilized from
1 This is journal no. J-11695 from the Mississippi Agricultural and the yolk sac during embryogenesis must be processed in
Forestry Experiment Station supported by MIS-322210.
2 Use of trade names in this publication does not imply endorsement
the liver and converted into their respective simpler end
by the Mississippi Agricultural and Forestry Experiment Station of
products (glucose, amino acids, and fatty acids) before
these products, nor similar ones not mentioned. being transported through the circulation to various
3 Corresponding author: dpeebles@poultry.msstate.edu
embryonic tissues (De Oliveira et al., 2008).

860
PIPPING MUSCLE METABOLISM 861
Functional relationships among the energetic com- (EMW; [(g of embryo weight/g of set egg weight) ×
ponents of the pipping muscle and liver are of com- 100]) and yolk sac:embryo weight ratio (YER) were de-
mercial importance because of their subsequent effects termined. Whole fresh pipping muscle (PW) and liver
on hatchability, rate of hatch, and posthatch broiler (LW) weights from each embryo were expressed as per-
performance. However, there is a paucity of informa- centages of total fresh embryo BW.
tion concerning the basal energy relationships of the
pipping muscle and liver of broiler embryos during their Statistical Analysis
preparation for hatch. Therefore, the objective in the
current study was to examine these relationships in Tray level was considered as a replicate unit. The ef-
broiler embryos during the later stages of incubation fects of day of incubation (time) on all parameters were
before hatch. analyzed by 1-way ANOVA, and least squares means
were compared in the event of significant global effects
(Steel and Torrie, 1980). Using the MIXED, CORR,
MATERIALS AND METHODS
and GLM procedures of SAS Institute (2003), respec-
General tively, the effect of time on each parameter was evalu-
ated, parameters on each of d 15 and 19 were correlated
Approximately 90 Ross × Ross 308 broiler hatching separately, and partial correlations among parameters
eggs were collected from a common commercial flock across both time periods were computed upon adjusting
(approximately 30 wk of age). The eggs were held for for age effects. Correlations among all tissue parameters
3 d under standard storage conditions and were then were performed on an individual embryo basis. Global
labeled and weighed immediately before being set in effects, differences among least squares means, and cor-
the incubator. The eggs were incubated on 3 replicate relations were considered significant at P ≤ 0.05.
tray levels (30 eggs per tray). The eggs were incubated
under standard commercial conditions and at 28.8 and RESULTS
37.5°C wet and dry bulb temperatures, respectively.
Significant increases were observed for EMW (P ≤
Data Collection and Sample Analyses 0.0001) and PW (P ≤ 0.0001) between d 15 and 19 of
incubation. However, YER significantly decreased (P
Individual egg weights were recorded on 0, 15, and ≤ 0.0001) between d 15 and 19. The mean values for
19 d of incubation. On 15 and 19 d of incubation, 10 the aforementioned parameters at each time period are
embryonated eggs from each tray (i.e., a total of 60 presented in Table 1. Despite the observed changes in
eggs) were broken out for live embryo extraction. From the parameters above, there was no significant differ-
each of the live embryos, the whole liver and pipping ence between the daily relative incubational egg weight
muscle were extracted and weighed. The tissues were loss for eggs sampled on d 15 of incubation (0.62%) and
collected on d 15 and 19 because d 15 is the earliest that of the eggs sampled on d 19 of incubation (0.61%;
time when sufficient amounts of pipping muscle and pooled SEM = 0.025%). Furthermore, LW did not sig-
liver can be obtained for nutrient profile analyses, and nificantly change between d 15 and 19 of incubation.
d 19 is the time at which the hatching process begins Although there was no significant time effect on LW, its
in the broiler embryo and when the pipping muscle is mean values on d 15 and 19 are also presented in Table
fully functional. Immediately after collection, a 0.25-g 1 for reference.
sample was taken from each liver and pipping muscle The mean values for pipping muscle glucose, glyco-
and was stored and preserved in a vial containing 10% gen, protein, and fat concentrations on d 15 and 19
perchloric acid (Bennett et al., 2007). Glucose, glyco- of incubation are presented in Table 2. Between d 15
gen, protein, and fat concentrations were determined in and 19 of incubation, there were significant increases
each of the individual liver and pipping muscle samples in pipping muscle glucose (P ≤ 0.01), pipping muscle
harvested. Tissue glucose and glycogen concentrations glycogen (P ≤ 0.02), and pipping muscle protein (P ≤
were determined using the phenol-sulfuric acid method 0.04) concentrations; however, pipping muscle fat con-
as described by Bennett et al. (2007). Colorimetric pro- centration decreased significantly (P ≤ 0.01) between
tein estimation was accomplished according to Lowry d 15 and 19.
et al. (1951) and colorimetric fat was determined using Between d 15 and 19 of incubation, liver tissue con-
the methodology of Van Handel (1985). Concentrations centrations of protein decreased (P ≤ 0.03) and those of
of the above tissue parameters were expressed as a per- fat increased (P ≤ 0.0006) significantly. No significant
centage of fresh sample weight. differences were observed in liver glucose and glyco-
Relative incubational egg weight loss [(g of egg weight gen concentrations between 15 and 19 d of incubation.
loss/g of set egg weight) × 100] (Peebles et al., 2005) However, differences in liver glycogen content between
and average daily relative incubational egg weight loss d 15 and 19 approached significance (P ≤ 0.07). The
{[g of egg weight loss/(g of set egg weight × d)] × mean values for liver protein, fat, glucose, and glycogen
100} were determined for 0 to 15- and 0 to 19-d inter- concentrations on d 15 and 19 of incubation are pre-
vals of incubation. In addition, relative embryo weight sented in Table 3.
862 PULIKANTI ET AL.
Table 1. Embryo weight as a percentage of set egg weight (EMW), yolk sac:embryo weight ratio
(YER), and pipping muscle (PW) and liver (LW) weights as percentages of embryo weight on d 15
and 19 of incubation1
Item EMW (%) YER PW (%) LW (%)

Day of incubation
15 28.0b 1.36a 0.57b 2.10
19 54.5a 0.34b 1.71a 1.96
Pooled SEM 0.58 0.035 0.045 0.053
P>F 0.0001 0.0001 0.0001 0.10
a,bMean values within a column with no common superscript differ significantly (P ≤ 0.05).
1Tissuesamples from 10 individual embryos on each of 3 replicate incubator trays were used for calculation of
mean values (n = 30).

Across d 15 and 19 of incubation, positive partial liver fat concentration (P ≤ 0.004), whereas on d 19,
correlations existed between PW and liver fat concen- significant negative correlations existed between aver-
tration (P ≤ 0.0003) and between liver glucose and age daily relative incubational egg weight loss and pip-
fat concentrations (P ≤ 0.01), whereas negative par- ping muscle protein concentration (P ≤ 0.04), between
tial correlations existed between EMW and YER (P EMW and LW (P ≤ 0.02), between EMW and YER (P
≤ 0.003), between PW and pipping muscle glycogen ≤ 0.0001), between PW and liver glycogen concentra-
concentration (P ≤ 0.02), and between pipping muscle tion (P ≤ 0.05), and between liver glycogen and fat
glycogen and liver fat concentrations (P ≤ 0.004). The concentrations (P ≤ 0.04). The coefficients for these
coefficients for the aforementioned parameter correla- correlations are presented in Table 6.
tions across d 15 and 19 of incubation are presented in
Table 4. DISCUSSION
For embryos sampled on d 15 of incubation, signifi-
cant positive correlations existed between pipping mus- The embryos investigated experienced normal growth
cle protein and glycogen concentrations (P ≤ 0.03), be- and yolk utilization patterns between d 15 and 19 of
tween pipping muscle fat and glycogen concentrations incubation. The EMW of the commercial broiler (Avia-
(P ≤ 0.01), and between LW and liver protein concen- gen female × Peterson male) embryos examined by Pee-
tration (P ≤ 0.04), whereas significant negative correla- bles et al. (1999) are comparable to those in this study.
tions existed between EMW and YER (P ≤ 0.02), be- Peebles et al. (1999) also showed that relative yolk sac
tween PW and pipping muscle glycogen concentration weight decreased progressively between d 6 and 15 of
(P ≤ 0.03), between pipping muscle glycogen and liver incubation and further suggested that rapid absorption
glucose concentrations (P ≤ 0.01), between pipping and subsequent accumulation of yolk-derived lipids oc-
muscle fat and liver glucose concentrations (P ≤ 0.04), curs in broiler embryo tissues during the last week of
between pipping muscle and liver glycogen concentra- incubation.
tions (P ≤ 0.004), and between pipping muscle glucose Relative LW did not change significantly between d
and liver fat concentrations (P ≤ 0.01). The coefficients 15 and 19 in the current study. Peebles et al. (2001)
for these correlations are presented in Table 5. likewise showed that relative fresh LW in embryos from
For embryos sampled on d 19 of incubation, signifi- 27-wk-old breeder hens did not change significantly be-
cant positive correlations existed between average daily tween d 12 and 18 of incubation. Conversely, relative
relative incubational egg weight loss and pipping mus- PW increased with embryo age between d 15 and 19 of
cle glycogen concentration (P ≤ 0.02), between average incubation in this study. Increased growth of the pip-
daily relative incubational egg weight loss and LW (P ping muscle in proportion to total embryo weight with
≤ 0.02), between pipping muscle fat and liver glyco- embryogenesis after d 15 of incubation has been es-
gen concentrations (P ≤ 0.03), and between PW and tablished in previous research. Maximum incubational

Table 2. Pipping muscle glucose, glycogen, protein, and fat concentrations in broiler embryos on d
15 and 19 of incubation1
Item Glucose (%) Glycogen (%) Protein (%) Fat (%)

Day of incubation
15 0.010b 0.09b 9.5b 2.6a
19 0.022a 0.15a 12.8a 1.1b
Pooled SEM 0.0019 0.011 0.80 0.23
P>F 0.01 0.02 0.04 0.01
a,bMean values within a column with no common superscript differ significantly (P ≤ 0.05).
1Tissuesamples from 10 individual embryos on each of 3 replicate incubator trays were used for calculation of
mean values (n = 30).
PIPPING MUSCLE METABOLISM 863
Table 3. Liver glucose, glycogen, protein, and fat concentrations in broiler embryos on d 15 and 19
of incubation1
Item Glucose (%) Glycogen (%) Protein (%) Fat (%)

Day of incubation
15 0.053 0.13 16.3a 2.8b
19 0.061 0.06 12.5b 9.0a
Pooled SEM 0.0028 0.019 0.80 0.45
P>F 0.10 0.07 0.03 0.0006
a,bMean values within a column with no common superscript differ significantly (P ≤ 0.05).
1Tissuesamples from 10 individual embryos on each of 3 replicate incubator trays were used for calculation of
mean values (n = 30).

PW in Rhode Island Red chicken embryos have been De Oliveira et al. (2008) concluded that there are
recorded on d 20 by Fisher (1958) and on d 21 by Pohl- few studies that relate the structure and metabolism
man (1919). Maximum and d 19 fresh PW as percent- of the hatching muscle to its function. Nevertheless,
ages of embryo BW were reported as 1.44 and 0.46%, the observations concerning liver glycogen concentra-
respectively, by Pohlman (1919) and as 1.93 and 1.57%, tion in this current study are in accordance with those
respectively, by Fisher (1958). The larger d 19 relative of Christensen et al. (2001), who reported a decrease in
PW of the Ross × Ross 308 broiler chicken embryos the liver glycogen concentration of broiler embryos ap-
in the present study (1.71% of BW) in comparison to proaching hatch. In addition, these results suggest that
those reported by Pohlman (1919) and Fisher (1958) although fat mobilization in the liver may be important
suggest a more rapid growth of the pipping muscle rela- for fatty acid oxidation and glycerol gluconeogenesis,
tive to that of the embryo in modern broiler strains there is a relative accumulation of yolk-derived fat in
through d 19 of incubation. the liver in proportion to its other constituents between
The β-oxidation of fatty acids is the primary source of d 15 and 19 of incubation. A relative decrease in the
energy for the developing embryo (Moran, 2007). Fur- percentage of protein in the liver during this time may
thermore, the glycerol component of fats can be used to be related to this relative accumulation of fat.
generate glucose via gluconeogenesis (Klasing, 1998b). The yolk lipids are metabolized within the yolk sac
Gluconeogenesis in the liver is the primary mechanism for membrane to facilitate rapid lipid assimilation by the
the supply of glucose to embryonic tissues immediately growing embryo (Klasing, 1998a). Between mid-incu-
before the start of the hatching process (Moran, 2007) bation and 2 to 3 d before hatch, yolk fat is mobilized
and during pipping (Donaldson et al., 1994). Gluconeo- for the provision of available fatty acids to the rapidly
genesis of protein reserves may also serve as a supplier developing embryo (Moran, 2007). In association with
of energy as carbohydrate reserves become increasingly this process, Peebles et al. (1999) showed that the liver
exhausted toward the end of incubation (Christensen lipid content of broiler embryos increases continuously
et al., 2001). Nevertheless, age-related changes in the between d 12 and 21 of incubation. The significant in-
nutrient biochemical constituents of the liver and pip- crease in the fat content of liver between d 15 and 19
ping muscle determined in the current study suggest of incubation in this study suggests that despite the
an inverse relationship between the protein, glycogen, active supply of fatty acids before hatch, for assimila-
and fat concentrations of the liver and pipping muscle tion and oxidation by the pipping muscle, there is an
as the broiler embryo develops between d 15 and 19 overall accumulation of hepatic fat. However, the signif-
of incubation. More specifically, it was observed that icant decrease in the relative fat content of the pipping
pipping muscle protein and glycogen concentrations in- muscle may be primarily due to significant increases
creased, whereas pipping muscle fat content decreased in its relative concentrations of glucose, glycogen, and
between d 15 and 19. Conversely, protein and glycogen protein along with the influx of lymph to the pipping
concentrations in the liver decreased, whereas liver fat muscle. However, unlike protein, glycogen, and fat, the
content increased significantly between d 15 and 19. age-related changes in glucose concentration of the pip-

Table 4. Coefficients and P-values for partial correlations among embryo weight as a percentage of set egg weight (EMW), yolk
sac:embryo weight ratio (YER), pipping muscle weight (PW), glycogen (PGLY) concentration, and liver glucose (LGLU) and fat
(LFAT) concentrations pooled across d 15 and 19 of incubation1
Item YER PW PGLY LGLU

EMW −0.45 (P ≤ 0.003) — — —


PW — — −0.35 (P ≤ 0.02) —
LFAT — 0.52 (P ≤ 0.0003) −0.43 (P ≤ 0.004) 0.38 (P ≤ 0.01)
1Degrees of freedom = 42 for calculation of each partial correlation coefficient representing 10 individual sample values from each of 3 replicate trays
across d 15 and 19 of incubation (10 individual samples × 3 replicate trays × 2 d of incubation = 60).
864 PULIKANTI ET AL.
Table 5. Coefficients and P-values for correlations among embryo weight as a percentage of set egg weight (EMW); yolk sac:embryo
weight ratio (YER); pipping muscle (PW) and liver (LW) weights as percentages of embryo weight; pipping muscle glucose (PGLU),
glycogen (PGLY), protein (PPRO), and fat (PFAT) concentrations; and liver glucose (LGLU), glycogen (LGLY), protein (LPRO),
and fat (LFAT) concentrations on d 15 of incubation1
Item YER PGLU PGLY PFAT LPRO

EMW −0.42 (P ≤ 0.02) — — — —


PW — — −0.40 (P ≤ 0.03) — —
PPRO — — 0.39 (P ≤ 0.03) — —
PFAT — — 0.46 (P ≤ 0.01) — —
LW — — — — 0.38 (P ≤ 0.04)
LGLU — — −0.46 (P ≤ 0.01) −0.38 (P ≤ 0.04) —
LGLY — — −0.51 (P ≤ 0.004) — —
LFAT — −0.48 (P ≤ 0.01) — — —
1Ten individual sample values from each of 3 replicate trays (n = 30) were used for calculation of each correlation coefficient.

ping muscle were not associated with opposite changes in the liver to allow for an increased fat concentration
in their concentrations in the liver. Although glucose in the liver between d 15 and 19.
concentration in the pipping muscle increased between Specifically, before relative decreases in liver protein
d 15 and 19, along with the accumulation of glycogen concentration in exchange for fat between d 15 and 19,
and protein and the loss of fat, glucose concentration in relative LW was positively correlated with liver protein
the liver appeared to be homeostatically controlled. Fat concentration on d 15. This would indicate that LW
catabolism would reasonably provide an increased pool in d 15 embryos was largely attributed to its protein
of glucose in the inactive pipping muscle of d 19 em- content. However, on d 15 of incubation, liver fat and
bryos, which would then be available for increased gly- pipping muscle glucose were negatively correlated. This
cogen storage in preparation for the pipping process. would further suggest that fat catabolism on d 15 in
Differences in the age-specific changes in the pro- the liver may provide glucose for the preferential ac-
tein, glycogen, and fat contents of the liver and pipping cumulation of glycogen over fat in the pipping muscle
muscle are supported by the observed correlations that by d 19. The negative correlations between liver glucose
were determined within and across the 2 time periods and pipping muscle glycogen, between liver glycogen
selected in this study (d 15 and 19 of incubation). For and pipping muscle glycogen, and between liver glucose
ease of presentation, partial correlations across d 15 and pipping muscle fat in d 15 embryos would addition-
and 19 are separately presented in Table 4, and cor- ally support the occurrence of an interconversion of the
relations within d 15 and 19 are presented in Tables 5 above nutrients as embryonic development approaches
and 6, respectively. Across 15 and 19 d, liver fat was the last phase of incubation.
negatively correlated with pipping muscle glycogen and The aforementioned results suggest that yolk-derived
positively correlated with liver glucose concentration fats transported to the liver are ultimately used for the
and relative PW. This confirms that despite the ac- production and storage of liver glycogen, which after
cumulation of liver fat in association with increased glycogenolysis has occurred can then be transferred in
growth of the pipping muscle between d 15 and 19, fat the form of glucose units to the pipping muscle. The
in the liver was still being catabolized and converted to glucose units assimilated by the pipping muscle can
glucose, which was then transferred to the growing pip- then be subsequently stored in the form of glycogen
ping muscle where the glucose was stored in the form and fat. The age-related catabolism of fat (d 15) and
of glycogen reserves in the embryo’s preparation for then glycogen (d 19) in the liver would be important
hatch. Although the β-oxidation of fatty acids before to the accruement of glycogen stores in the pipping
hatch occurs both in the liver and pipping muscle, fatty muscle by d 19 of incubation. However, the positive
acid β-oxidation may occur to a relatively lesser extent correlation between liver glycogen concentration and

Table 6. Coefficients and P-values for correlations among average daily egg weight loss as a percentage of set egg weight between d
0 and 19 of incubation (DEWL-19); embryo weight as percentage of set egg weight (EMW); yolk sac:embryo weight ratio (YER); pip-
ping muscle (PW) and liver (LW) weights as percentages of embryo weight; pipping muscle glycogen (PGLY), protein (PPRO), and
fat (PFAT) concentrations; and liver glycogen (LGLY) and fat (LFAT) concentrations on d 19 of incubation1
Item DEWL-19 EMW YER PW PFAT LGLY

EMW — — −0.72 (P ≤ 0.0001) — — —


PGLY 0.45 (P ≤ 0.02) — — — — —
PPRO −0.42 (P ≤ 0.04) — — — — —
LW 0.42 (P ≤ 0.02) −0.44 (P ≤ 0.02) — — — —
LGLY — — — −0.37 (P ≤ 0.05) 0.40 (P ≤ 0.03) —
LFAT — — — 0.51 (P ≤ 0.004) — −0.39 (P ≤ 0.04)
1Ten individual sample values from each of 3 replicate trays (n = 30) were used for calculation of each correlation coefficient.
PIPPING MUSCLE METABOLISM 865
pipping muscle fat concentration specifically on d 19 Bock, W. J., and R. S. Hikida. 1968. An analysis of twitch and tonus
supports the above contention that liver glycogenolysis fibers in the hatching muscle. Condor 70:211–222.
Christensen, V. L., M. J. Wineland, G. M. Fasenko, and W. E. Don-
would promote the preferential accumulation of glucose aldson. 2001. Egg storage effects on plasma glucose and supply
and glycogen over that of fat in the pipping muscle on and demand tissue glycogen concentrations of broiler embryos.
d 19. Conversely, in d 19 embryos, liver glycogen con- Poult. Sci. 80:1729–1735.
De Oliveira, J. E., Z. Uni, and P. R. Ferket. 2008. Important meta-
centration was negatively correlated with liver fat and bolic pathways in poultry embryos prior to hatch. World’s Poult.
positively correlated with pipping muscle fat, which is Sci. J. 64:488–499.
suggestive of active gluconeogenic activity in the liver Donaldson, W. E., J. Clark, and V. L. Christensen. 1994. Protein,
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lopavo) poults as affected by post-hatch stressors and holding
correlated with any of the pipping muscle nutrients ei- time. Comp. Biochem. Physiol. 107A:559–562.
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