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Egg Characteristics, Carbohydrate Metabolism, and Thyroid Hormones in Late Chick Embryos from Different Genetic Lines1
Egg Characteristics, Carbohydrate Metabolism, and Thyroid Hormones in Late Chick Embryos from Different Genetic Lines1 V. L. CHRISTENSEN,2 G. B. HAVENSTEIN, and G. S. DAVIS Department of Poultry Science, North Carolina State University, Raleigh, North Carolina 27695-7608
1995 Poultry Science 74:551-562
randombred control lines compared with selected lines suggested that differences Recent investigations with turkeys indi- may exist in the energetics and metabocated that selection for increased egg lism of embryonic development (Ar et al., production or rapid growth has reduced 1987) within a species but of different the eggshell conductance of eggs genetic lines. Specifically, the amount and (Christensen et al., 1993). Increased egg- form of energy placed in eggs may differ shell conductance in turkey eggs from between genetically different lines, thus requiring different mechanisms to complete embryonic development by budgeting maternal investments in eggs (Vleck, Received for publication May 26, 1994. 1991). Accepted for publication October 25, 1994. It has also been suggested that differSalaries and research support provided by state and federal funds appropriated to the North Carolina ences in the energetics and carbohydrate Agricultural Service, North Carolina State University. metabolism of embryos may affect the The use of trade names in this publication does not survivability of developing embryos late imply endorsement by the North Carolina Agricultural Research Service of the products named, nor in the incubation period (Christensen et al., 1993) as well as in the period immediately criticism of similar products not mentioned. 2 To whom correspondence should be addressed. posthatching (Donaldson and Christensen, INTRODUCTION
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ABSTRACT Functional eggshell qualities, thyroid hormones, and carbohydrate metabolism of chick embryos at the end of incubation were compared between a modern (Arbor Acres line) and a randombred control population (Athens-Canadian Randombred). Embryos from the Arbor Acres genetic line developed in larger eggs with more albumen and less yolk than Athens Canadian Randombred lines. Percentage shell and functional eggshell properties measured as eggshell conductance constants did not differ between genetic lines. On a relative basis, hearts were generally smaller and livers heavier in Arbor Acres than in Athens-Canadian Randombred birds. Heart and liver glycogen concentrations were greater in Athens-Canadian Randombred than in Arbor Acres embryos. However, blood glucose was greater in Randombred than in Arbor Acres embryos only at internal pipping, a time of hypoxia and hypercapnia. Blood plasma concentrations of thyroxine did not differ significantly between the modern and Randombred embryos at any stage examined. Modern broiler chick embryos possessed greater concentrations of triiodothyronine as well as greater triiodothyronine to thyroxine ratios than Randombred embryos at external pipping and hatching. It can be inferred from the data that chick embryos differ in their use of carbohydrate during late development between modern and Randombred genetic lines. (Key words: chick embryo, carbohydrate metabolism, genetic line, eggshell, thyroid)
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MATERIALS AND METHODS
Fertile eggs from the ACRBC were obtained from the Southern Regional Poultry Breeding Laboratory, Athens, GA 30602, in December, 1992 and were incubated in a randomly distributed fashion with eggs from an AA commercial line3 of broiler breeders. Approximately 1,500 eggs were set from each line. Eggs were incubated4 at 37.5 C and 54% relative humidity until the 18th d of incubation, when incubator conditions were changed to 36.9 C and 80% relative humidity for the actual hatching process. All eggs were weighed (nearest .01 g) immediately prior to setting and again when the incubator was set at the hatching conditions to determine eggshell conductance values for
3
Arbor Acres, Glastonbury, CT 06033-6501. Natureform Mini NMC 2000, Jacksonville, FL 32202. sSarstedt Inc., Newton, NC 28658-046. 4
each egg using the calibrated egg technique (Tullett, 1981). At hatching all chicks were counted and eggs that did not hatch were broken. Their contents were subsequently examined macroscopically to determine true fertility and estimate the time at death for the nonhatched fertile eggs. Hatchability of fertile eggs was determined by discounting all truly infertile eggs and dividing the number of chicks hatched by the total number of fertile eggs. For determination of the effects of eggshell conductance on embryonic mortality, eggs were sorted into six groups: 1) infertiles, 2) incubation Week 1 deaths, 3) death at the plateau stage in oxygen consumption (18 d of incubation), 4) death at internal pipping (19 d of incubation), 5) death at external pipping (20 d of incubation), and 6) eggs containing chicks that hatched (21 d of incubation). Egg Composition
Fifteen eggs were selected randomly prior to setting from each line of chickens for examination of yolk and albumen weights. Only apparently normal eggs were selected for determination of egg components. Each selected egg was weighed (nearest .01 g) and then broken. The yolk and albumen were separated from the shell and then from each other. Each component was then dried to a constant weight in a drying oven at 65 C. Embryo Sampling
At each of four stages of embryonic development [prepipping (at 17 to 18 d of incubation), internal pipping (19 d of incubation), external pipping (20 d of incubation), and hatching (21 d of incubation)], fertile eggs, as determined by candling, were selected randomly from each of the lines. Descriptions of the morphology of each stage were given by Christensen et al (1982). Ten embryos per genetic line were sampled at each of the four stages of development. The embryo was decapitated and a blood sample was collected into tubes containing 10 mg of EDTA.5 The body with the yolk sac was blotted lightly and weighed immediately. The heart and liver were quickly dissected, blotted on a wet
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1991). Specifically, the perinatal carbohydrate metabolism of some genetic lines seems to be heavily dependent upon gluconeogenesis instead of stored glycogen for survivability during the plateau stage in oxygen consumption (Christensen et al, 1991). The objective of the present study was to compare the eggshell functional qualities and egg components of eggs from a line of modern Arbor Acres broiler breeder hens (AA) selected for growth with those of eggs from the unselected Athens-Canadian Randombred (ACRBC) population of chickens. A secondary objective of the study was to relate embryonic mortality and carbohydrate content of vital tissues (heart, blood, and liver) to the changes in eggshells and egg components from selected and unselected lines of chickens. A third objective was measurement of circulating concentrations of thyroid hormones that may be related to slow or rapid growth (McNabb et al, 1993) that may be available to each embryo for survival of hypoxia. This is part of a larger overall comparison of these two widely different genetic lines (Havenstein et al, 1994a,b; Qureshi and Havenstein, 1994).
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CHICKEN EMBRYO METABOLISM
by modern broiler breeder (AA) chickens rol population (ACRBC)
TABLE 1. Egg components of fertile eggs prodi compared with a randombred Line
Weight
Water
(g) AA ACRBC x ± SEM1 Probability ] n = 15 for
65.8 68.4 44.5 67.7 55.2 ± 3.0 68.1 ± 1.0 .0001 .08 each genetic line.
Solids
Albumen
(%) 31.6 32.2 31.9 ± 1.0 08
6.8 5.9 6.4 ± .7 .004
Yolk
Shell
(% of total solids) 15.5 9.3 16.7 9.6 16.1 ± 1.5 9.4 ± .6 .04 .22
The effects of eggshell conductance on embryonic mortality were assessed using a factorial arrangement of completely random factors (Snedecor and Cochran, 1974). The factors were line of chicken and stage of death. There were two lines of chickens (AA and ACRBC) and four stages of embryonic development (death at the plateau stage, death at internal pipping, death at external pipping, and hatched). Means determined Biochemical Analyses of Tissues to differ significantly were separated by Blood plasma glucose concentrations using the orthogonal contrast procedure were measured using the glucose oxidase comparing all means of nonhatching eggs method described in detail by Donaldson with those of eggs that hatched. Sigand Christensen (1991). Cardiac and hepatic nificance was based on (P < .05) unless glycogen were analyzed using the tech- otherwise stated. nique of Dreiling et al. (1987). Plasma concentrations of thyroxine (T4) and triioRESULTS dothyronine (T3) were analyzed using prepared kits6 in a single assay. The intraassay Egg Quality coefficient of variation was less than 1%. Parallelism was assessed by comparing a Egg weights from the modern-type AA pooled sample of chick embryo blood broiler breeder were almost 50% greater plasma to samples of the same plasma than those from the ACRBC (Table 1). The containing a known amount of T4 or T3 and AA eggs contained significantly more parallelism was identical. solids as albumen and less as yolk than did eggs from ACRBC. No differences were noted between the two genetic lines in the Statistical Analyses percentage of shell on a dried or wet basis. Functional eggshell qualities when exData were analyzed as a completely random design (Snedecor and Cochran, pressed on an egg weight basis (conduc1974) with two treatment groups, i.e., the tance constants) did not differ between the modern and randombred controls. Egg two lines of chickens (Table 2). Eggshell weight data were analyzed as a one-way conductance was greater in AA than analysis of variance using the SAS® soft- ACRBC eggs but when differences in egg ware package for personal computers (SAS weights were accounted for by computing Institute, 1989). Embryo data were sorted conductance constants, no differences were by developmental stage prior to analysis. observed. Embryo Growth diagnostics Products Corp., Los Angeles, CA 90045.
Embryos from both genetic lines reached each stage of development at approxi-
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paper towel, trimmed, and then weighed (nearest .01 mg) on an analytical balance. The tubes containing blood were centrifuged (700 x g) under refrigeration (4 C) for 10 min, the plasma was decanted and frozen (-22 C) until analysis. The tissues were placed in sterile plastic bags and frozen similarly until analyzed.
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TABLE 2. Functional eggshell qualities of eggs produced by modern broiler breeder (AA) chickens compared to randombred controls (ACRBC) gg weight
Weight loss
Conductance
Conductance constant
(g)
(%) 8.2 8.1 8.2 ± 1.3 .02
(mg H 2 0/d/torr) 12.7 9.6 11.2 ± 1.8 .0001
(conductance/ egg weight x 21) 4.0 3.9 4.0 ± .6 .19
E
Line
AA ACRBC x ± SEM1 Probability
66.2 50.7 58.4 ± 3.8 .0001
= 15 for each genetic line.
Tissue Carbohydrate Measurements
Cardiac glycogen concentration was greater in ACRBC than in AA embryos at the plateau and external pipped stages (Table 4). No differences were observed in the glycogen concentration of heart tissue in internally pipped or hatched chicks. Hepatic glycogen was significantly greater in AA than ACRBC embryos at internal pipping (Table 4). No significant differences between lines in hepatic glycogen were observed at the plateau, externally pipped, or hatched stages. Blood plasma glucose concentrations differed only at internal pipping when glucose concentration in ACRBC embryos was reduced compared with that of AA embryos (Table 4). No significant differences in plasma glucose concentrations were seen in the embryos sampled at the
plateau, externally pipped, or hatched stages. Blood Plasma Thyroid Hormone Concentrations
No significant differences were observed in plasma T4 concentrations between lines (Table 5). Plasma T3 concentrations differed significantly only at external pipping. The AA embryos possessed significantly higher levels of T3 in circulation than did ACRBC embryos. Ratios of T3 to T4 differed at external pipping and hatching with AA chick embryos having greater amounts of T3 relative to T4 than did ACRBC chick embryos. Functional Eggshell Quality and Hatchability
The preincubation weight associated with the eggs from the different embryonic death classifications differed significantly (Table 6). Eggs containing embryos dying during external pipping were heavier than eggs that hatched, but no other orthogonal contrasts differed significantly. The interaction of genetic line of chicken with stage at embryonic death for egg weights was not significant. Embryos dying at the plateau stage were from eggs with greater eggshell conductance values than eggs which hatched. Eggshell conductance constants of eggs classified with dead internally pipped embryos was less than for hatched eggs (Table 6). Eggshell conductance values of eggs with embryos from eggs that pipped externally then died were not different from the
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mately the same incubation time and hatched at approximately the same time. The AA embryos weighed more than ACRBC at each of the stages of development examined (Table 3). The weights of heart and liver are presented on both an absolute and relative basis, but because of the observed egg size and BW differences, they will be discussed only on a relative basis. The relative weight of the heart was greater in ACRBC than AA embryos at the plateau stage; however, the reverse was true at the external pipping and hatched stages, with AA embryos having heavier relative heart weights than ACRBC. The AA embryos had heavier relative liver weights than ACRBC at every stage of development examined except the plateau stage.
(mg) 528 436 482 + 130 .06
AA ACRBC x + SEM 1 Probability
*n = 10 for each genetic line.
(mg) 173 148 161 + 27 .02
AA ACRBC x ± SEM 2 Probability
1.22 1.34 1.28 + .23 .17
(%)
.40 .46 .43 + .04 .004
(%)
47.8 34.7 41.0 ± .0001
42.9 32.2 37.6 ± 4.2 .0001
AA ACRBC x ± SEMi Probability
(mg) 684 592 636 ± 80 .01
(mg) 216 168 191 + 27 .0004
5.2
Internal pip
Plateau
Line
1.43 1.71 1.58 ± .12 .0001
(%)
.45 .49 .47 ± .05 .10 Liver
(%)
(mg) 770 578 665 ± 65 .0001
(mg) 221 177 197 ± 27 .001
weights (g) — 46.7 31.8 38.6 ± 3.6 .0001 Heart weights
External pip
Stage of development 1
1 1 1
(%
(%
TABLE 3. Body, heart, and liver weights of embryos from modern-type broiler breeders (AA) Randombred Control (ACRBC) at selected stages of developme
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(mg) 8.5 7.3 7.9 + 6.4 .64
56 86 71.0 ± i68 .25
AA ACRBC x + SEMi
Probability
AA ACRBC x + SEM 1 Probability
AA ACRBC x + SEM 1 Probability
in = 10 for each genetic line.
(mg) 1.3 1.6 1.5 ± .24
.5
Plateau
Line
14.4 15.3 14.9 ± 8.8 .80
(%)
8.0 10.9 9.5 ± 3.7 .04
(%) .5
6.6 8.4 7.6 ± 2.6 .11
(%)
Heart (mg) .7 1.2 1.0 ± .004 : glycogen
giyi_ugt.il
EP
.3
(mg) (%) (mg) 11.4 12.9 9.0 14.6 34.2 20.8 13.2 ± 9.6 24.1 ± 12.7 15.2 ± 8.9 .44 .0006 .005 - Blood plasma glucose concentration (mg/dL 201 203 206 153 203 + 45 177 ± 53 .81 .03
1.4 ± .98
( m g) 1.4 1.4
IP
Stage of development 1
TABLE 4. Tissue glycogen and blood glucose concentration of embryos from modern broiler breeder Randombred Control (ACRBC) stage of development
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CHICKEN EMBRYO METABOLISM
TABLE 5. Blood plasma thyroid hormone concentrations of chick embryos from modern broiler chicks (AA) compared to randombred controls (ACRBC) Stage of development Strain
Plateau
Internal pip
External pip
Hatched
Thyroxine, ng/mL
AA ACRBC x ± SEMi Probability AA ACRBC 5c ± SEMI Probability AA ACRBC x ± SEM1 Probability
1.7 .5 1.1 ± .8 .31 .8 .7 .76 ± .30 .50 .23 .62 .38 ± .3 .10
10.0 7.0 8.6 ± 6.0 .39 1.0 2.2 1.4 ± 1.0 .28 .009 .022 .013 ± .01 .26
9.5 7.8 8.7 ± 4.0 .44 4.8 .7 3.4 ± .4 .03 .041 .010 .03 ± .02 .02
2.3 2.9 2.6 ± 2.5 .66 1.1 1.0 1.1 ± 3.2 .68 .844 .055 .37 ± .1 .05
Triiodothyronine, ng/mL
Ratio
J
n = 15 for each genetic line.
hatched eggs. No genetic line by stage at death interaction for eggshell conductance was observed. Hatchability of the AA broilers was 89% compared with 93.1% for the ACRBC controls but it was not possible to assess these means statistically with repeated measures. Levels of fertility were comparable for the two lines at 95% for AA and 94% for ACRBC. Embryonic mortality during Week 1 of incubation was 5.1% for AA and 4.3% for ACRBC, respectively; during the plateau stage mortality was 2.5 and 2.2% for AA and ACRBC; during internal pipping it was .4% for AA and .6% for ACRBC; and during pipping it was 1.2 and 1.4% for AA and ACRBC, respectively. DISCUSSION Genetic selection for rapid growth may have influenced the energetics of embryonic growth. Vleck (1991) concluded from comparisons between avian species that the fraction of the initial energy that was found in eggs at oviposition that was utilized by embryos for growth is essentially the same across avian species. This was true for both precocial and altricial species even though the maternal investment in altrical species eggs was less. The relationship appears to be true even though species differ widely in their incubation periods and size of hatchlings.
The data from the present study imply that energetics of incubation may differ on an intraspecific basis in chickens when hens deposit more nutrients as yolk and fewer as albumen. Freeman (1962) hypothesized that pipping and hatching responses were due to two separate stimuli. The first was thought to be the "pipping" stimulus and was suggested to be gaseous in nature, whereas the second was a "hatching" stimulus and was thought to be hormonal (or humoral). It has been suggested in the literature that the "pipping" stimulus is either high carbon dioxide (Windle and Barcroft, 1938; Visschedijk, 1968; Erasmus et al., 1970-71; Rahn and Paganelli, 1991), or decreased oxygen (Freeman, 1962). Hormones that may serve as the "hatching" stimulus or effect tissue maturation have been reviewed by Decuypere et al. (1991). Some hormone concentrations that change during hatching are the glucocorticoids (Wentworth and Hussein, 1980) catecholamines (Christensen and Edens, 1989), and thyroid hormones (Balaban and Hill, 1971; McNabb et al, 1993). In the present study we examined only the thyroid hormones. Egg Components The AA broiler breeders invest more albumen and less yolk in eggs than do their
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Variable
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CHRISTENSEN ET AL.
TABLE 6. Relationship of functional eggshell quality to embryonic death in modern broilers (AA) compared to randombred controls (ACRBC) Stage of death 1 Line
Plateau
AA ACRBC
64.3 50.6 57.4
X
AA ACRBC X
Initial egg mass (g) 66.7 54.0 60.4A Conductance constant (Conductance x: 21 4.3 3.8 4.3 3.6 4.3 A 3.7 A Probability Initial egg mass .0001 .0001 38 584 ± 3.8
External pip
Hatched 2
66.2 73.0 50.6 54.9 58.4A 64.0 A d/Initial egg mass) 4.1 4.0 3.9 4.0 4.0 A 4.0 Conductance .19 .05 .44 4.0 ± .6
A
Orthogonal contrasts means of nonhatching eggs with superscripts differed significantly from hatched eggs. !Mean values for embryos dying at each stage of development as determined by egg breakout at 22 d of incubation. 2 Mean values for all eggs that hatched. 3 n = 24 for Plateau; n = 9 for IP; n = 26 for EP; n = 925 for hatched.
ACRBC counterparts. When examining different lines of turkeys over a 7-mo laying period, Nestor et al. (1972) concluded that a significant proportion of the variation in hatchability could be accounted for in negative correlations of hatching rates and measurements of yolk. Thus, the decreased maternal investment of yolk may be favorable to embryonic survival in AA broiler embryos compared with ACRBC embryos. The increased investment as solids in the form of albumen may also confer metabolic advantages such as gluconeogenic amino acids (Watford et al, 1981) for increasing the availability of carbohydrate (Freeman, 1965; 1969). No differences were seen between the two lines in the percentage of dried shell, suggesting that the significant correlation of shell measurements with hatchability seen by Nestor et al. (1972) may have little to do with the differences observed in embryonic physiology of two genetic lines in the present study. This observation would, however, not discount the importance of functional eggshell properties. Examination of the egg components available to the embryo at the beginning of
incubation suggests that AA broiler embryos begin incubation with more protein and less lipid than do their randombred counterparts. These components must match eggshell conductance to allow a hatchling of adequate maturity for posthatching survival that is characteristic of the genetic line (Ar and Rahn, 1978; Vleck, 1991). Incubation periods measured in chicken eggs are significantly shorter than those predicted from their egg energy content, suggesting an abundance of available energy substrates (Vleck, 1991). The differences seen between the two genetic lines in the present study, however, suggest the embryos of the two lines do differ in their abilities to convert available energy substrates in egg components into glycogen for energy during the hypoxia of the plateau stage (Freeman, 1965). Functional Eggshell Qualities Because of the large differences seen in egg weights, conductance values also differed greatly. However, when the differences were adjusted for egg weight and incubation period (Ar and Rahn, 1978) and
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Source of variation Line Stage Line by stage S ± SEM3
Internal pip
CHICKEN EMBRYO METABOLISM
Growth of the Embryo The same differences seen in egg weights were seen in the embryonic BW. Embryo
and hatchling BW are highly correlated with egg weights (Burton and Tullett, 1985). Because of observed differences in BW, heart and liver weights were compared on a relative total rather than on an absolute basis. Relative heart weights were greater during the plateau stage in ACRBC than AA embryos, but after internal pipping heart growth in AA exceeded that of the ACRBC embryos, resulting in significantly heavier relative heart weights in AA hatchling chicks. The data do not support the notion that differences in heart growth may be due to the differences seen in blood thyroid hormone concentrations at all stages of development (Nobikuni et al, 1989). Thyroxine (or possibly T3) is responsible for increasing cardiac glycogen and providing nutrients to chick and poult hearts and muscles for growth (Czarnecki, 1991) but no correlation between thyroid hormones and heart growth were seen in these experiments. Embryo Carbohydrate Measurements Because of the plateau stage of oxygen consumption, embryo metabolism late in incubation is highly dependent upon carbohydrate (Freeman, 1965, 1969; Christensen et al, 1993). Lipid is the primary source of energy for embryonic growth and development (Noble and Connor, 1984); however, when less oxygen is available to the embryo, embryonic metabolism relies less on yolk lipid and more on tissue glycogen because more oxygen is required to metabolize lipids than carbohydrate (Freeman, 1962). Glycogen synthesis occurs throughout incubation via gluconeogenesis in preparation for the hypoxia that occurs at the plateau stage of late incubation (Freeman, 1969). Because of the differences seen in the growth of liver and heart tissues of these lines of chickens, the data for tissue glycogen are reported as milligrams of glycogen per gram of wet tissue. Heart glycogen concentrations were clearly greater in ACRBC than AA broiler chick embryos at most stages of development, suggesting a greater ability in ACRBC than AA embryos to maintain greater tissue glycogen concentrations despite exposure to the hypoxia or hypercapnia of the "pipping" stimulus. The
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were compared as conductance constants (product of eggshell conductance and incubation period divided by initial egg mass), the functional characteristics were nearly identical. This is different than observed with turkey eggs (Christensen et al, 1993). Because the present experiment occurred at a single time in the laying cycle of the hens, the possibility also exists that eggshell functional characteristics would differ between the lines at another stage of lay (Christensen and Nestor, 1994). Despite observing no significant differences between eggshell conductance constants for the lines, eggshell water vapor conductance, and conductance constants both differed similarly among the stages of incubation when embryos of both genetic lines died or hatched. In general, eggs with embryos that died during the plateau stage in oxygen consumption were too permeable compared with eggs that hatched. Eggs with embryos dying during internal pipping had less permeability compared with eggs that hatched. These factors suggest that the conflicting aims of elimination of sufficient metabolic water and carbon dioxide from the eggs as well as providing adequate oxygen for embryonic metabolism may be compromised. Therefore, adequate concentrations of the purported pipping stimuli, excess carbon dioxide or deficient oxygen (Freeman, 1962), may not be attained or excess water may accumulate (Christensen et al, 1993) and result in embryonic death prior to pipping or at internal pipping (Christensen et al, 1993). Alternatively, because of differences in incubation periods (McNabb et al, 1993), the embryonic physiological maturity at the onset of the plateau stage (Rahn et al, 1974) may have been underdeveloped at the time when gases may be needed for physiological stimulation to begin hatching (Windle and Barcroft, 1938; Visschedijk, 1968; Erasmus et al, 1970-71; Rahn and Paganelli, 1991). The lack of physiological maturity or shorter incubation time to synthesize vital tissue glycogen may also contribute to embryonic death at the stages of development observed.
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Thyroid Hormones
Observed values for plasma thyroid hormone concentrations were within the range observed previously (McNabb et al., 1993). Differences seen in blood plasma thyroid hormone concentrations did not correlate well with the observed differences in tissue carbohydrate. Differences in T3 occurred after the observed declines in hepatic and cardiac glycogen. It is difficult to attribute a specific glycolytic (Wittman and Weiss, 1981) or glycogen synthesis function to T3 or T4 because their observed concentration changes occurred after the changes in glycogen. However, it may be important to consider the observed differences in thyroid hormone of embryos of the two types of chickens studied. The increased T3 concentrations in AA broiler embryos at external pipping followed by increased T3 to T4 ratios in hatched chicks is noteworthy and suggests that monodeiodinase enzyme activity is somehow enhanced in AA chicks compared with ACRBC controls. This may be due to differences in circulating levels of growth hormone that may be different between the two strains (Harvey et al, 1991; Darras et al, 1992, 1993). Differences in thyroid hormones did not appear to be related to late embryonic mortality in the present study, but increased T3 did correspond to increased glycogen catabolism (Wittman and Weiss, 1981) in AA compared with ACRBC embryos. Summary and Conclusions
No differences were seen in eggshell conductance constants of eggs from the two genetic lines, which implies that neither AA nor ACRBC embryos would have a greater advantage in the apparently conflicting aims of providing adequate oxygen for metabolism while ventilating carbon dioxide and water vapor through the shell to prevent alkalosis or drowning. Metabolic water from lipid metabolism may be excessive in such eggs, causing them to drown. The ACRBC embryos began incubation with more solids in the form of yolk and less as albumen than AA, suggesting that they may rely more on aerobic metabolism of lipids than anaerobic metabolism of glyco-
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ability to maintain cardiac glycogen concentrations in chicks (Nobikuni et al, 1989) and poults (Czarnecki, 1991) has been suggested to be a function of thyroid hormones as well. Thyroxine may facilitate movement of carbohydrate from synthesis in the liver to storage in the heart because avian heart tissue has little or no gluconeogenic activity (Watford et al, 1981). Because no differences were seen in plasma T4 in the two lines of chickens in the present study, the idea is not supported by the data in the present study. Hepatic glycogen concentrations in ACRBC embryos increased more rapidly than in AA embryos between the plateau and internally pipped stages. Glycogen per gram of wet hepatic tissue doubled in ACRBC embryos whereas the concentration in AA embryos reached a plateau at the same level. In a prior study with turkey embryos it was shown that neither water accompanying glycogen synthesis nor lipid was responsible for the liver growth at these stages (Christensen et al, 1991). Blood plasma glucose concentrations in the ACRBC embryos were also depressed at internal pipping compared with AA embryos. Glucose-6-phosphatase is an induced hepatic gluconeogenic enzyme. Because there is little or no glucose remaining in the egg at this stage of development (van Deth, 1963), low blood glucose concentrations induce glucose-6-phosphatase to recycle lactate to pyruvate as glycogen (or glucose) is catabolized to lactic acid (Watford et al, 1981). Lactate carbon can then be reused for fuel to provide muscular energy for pipping and hatching. Thus, it may be possible that as chick embryos experience the plateau stage in oxygen consumption, stored tissue glycogen is catabolized very rapidly (Freeman, 1969). The ACRBC embryos have an enhanced capacity to regenerate the glycogen perhaps by a gluconeogenic response to low blood glucose concentrations observed during internal pipping (Donaldson and Liou, 1976). It is speculated that low blood glucose concentrations may induce increased hepatic glucose-6-phosphatase activity in ACRBC embryos in order to recycle muscle lactate or the kidneys may be synthesizing glucose from protein or fatty acid substrates at a greater rate in ACRBC than AA broiler embryos as was seen in turkeys (Christensen et al, 1993).
CHICKEN EMBRYO METABOLISM
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Burton, F. G., and S. G. Tullett, 1985. The effects of egg weight and shell porosity on growth and water balance of the chicken embryo. Comp. Biochem. Physiol. 81A:377-385. Christensen, V. L., H. V. Biellier, and J. F. Forward, 1982. Physiology of turkey embryos during pipping and hatching. I. Hematology. Poultry Sci. 61:135-142. Christensen, V. L., W. E. Donaldson, and K. E. Nestor, 1993. Embryonic viability and metabolism in turkey lines selected for egg production or growth. Poultry Sci. 72:829-838. Christensen, V. L., W. E. Donaldson, and J. F. Ort, 1991. The effect ofdietary iodine on hatchability of eggs from two commercial strains of turkeys. Poultry Sci. 70:2529-2537. Christensen, V. L., W. E. Donaldson, J. F. Ort, and J. L. Grimes, 1991. Influence of diet-mediated maternal thyroid alterations on hatchability and metabolism of turkey embryos. Poultry Sci. 70: 1594-1601. Christensen, V. L., and F. W. Edens, 1989. Blood plasma catecholamine concentration of poult embryos during the transition from diffusive to convective respiration. Comp. Biochem. Physiol. 92B:549-553. Christensen, V. L., and K. E. Nestor, 1994. Changes in functional qualities of turkey eggshells in strains selected for increased egg production or growth. Poultry Sci. 73:1458-1464. Czarnecki, C. M., 1991. Influence of exogenous T4 on body weight, feed consumption, T4 levels, and myocardial glycogen in furazolidone-fed turkey poults. Avian Dis. 35:930-936. Darras, V. M., P. Rudas, T. J. Vessir, T. R. Hall, L. M. Huybrechts, A. Vanderpooten, L. R. Berghman, E. Decuypere, and E. R. Kuhn, 1993. Endogenous growth hormone controls high plasma levels of 3,3',5-triiodothyronine (T3) in growing chickens by decreasing the T3-degrading type n i deiodinase activity. Dom. Anim. Endocrinol. 10: 55-65. Darras, V. M., T. J. Vessir, L. R. Berghman, and E. R. Kuhn, 1992. Ontogeny of type I and type in deiodinase activities in embryonic and posthatch chicks: relationship with changes in plasma triiodothyronine and growth hormone levels. Comp. Biochem. Physiol. 103A:131-136. Decuypere, E., E. Dewil, and E. R. Kuhn, 1991. The hatching process and the role of hormones. Pages 239-256 in: Avian Incubation. S. G. Tullet, ed. Butterworth Heinemann, London, England. Donaldson, W. E., and V. L. Christensen, 1991. Dietary carbohydrate level and glucose metabolism in REFERENCES turkey poults. Comp. Biochem. Physiol. 98A: 347-350. Ar, A., B. Arieli, A. Belinsky, and Y. Tom-Tov, 1987. Energy in avian eggs and hatchling: utilization Donaldson, W. E., and G. I. Liou, 1976. Lipogenic and transfer. J. Exp. Zool. 235(Suppl. 1):151-164. enzymes: Parallel responses in liver to glucose consumption by newly hatched chicks. Nutr. Rep. Ar, A., and H. Rahn, 1978. Interdependence of gas Int. 13:471-476. conductance, incubation length, and weight of the avian eggs. Pages 227-236 in: Respiratory Dreiling, C. E., D. E. Brown, L. Casale, and L. Kelly, Function in Birds, Adult and Embryonic. J. Piiper, 1987. Muscle glycogen: comparison of iodine ed. Springer-Verlag, Berlin, Germany. binding and enzyme digestion assays and application to meat samples. Meat Sci. 20:167-177. Balaban, M., and J. Hill, 1971. Effects of thyroxine level and temperature manipulation upon the hatching Erasmus, B. W., B. J. Howell, and H. Rahn, of chick embryos (Gallus domesticus). Dev. Psy1970-71. Ontogeny of acid-base balance in the chobiol. 41(l):17-28. bullfrog and chicken. Respir. Physiol. 11:46-53.
gen for energy during embryonic growth. Carbohydrate metabolism differs among AA and ACRBC embryos, similar to what was observed previously in similarly selected lines of turkeys (Christensen et ah, 1993). However, it could not be related to increased mortality late in the incubation period of either strain. It was observed that the embryonic mortality of both genetic lines was in some unknown way related to the functional eggshell properties because embryos in eggs with reduced eggshell conductances constants, compared with eggs that hatched, died during internal pipping, whereas eggs with embryos dying during the plateau stage had greater eggshell conductance constants than those that hatched. Eggs containing embryos that died during pipping were heavier than eggs that hatched. The AA embryos reduced liver growth and increased heart growth during development, and also processed less glycogen in both of these tissues than did the ACRBC embryos. Thyroid hormones are related to glycogen metabolism, liver growth, and heart growth in avian embryos. Increased concentrations of T3 at external pipping in the AA embryos and greater ratios of T3 to T4 concentrations at the same stage compared with ACRBC embryo concentrations may be related to both increased organ growth and organ decreased glycogen concentration differences. In conclusion, observations of egg components, eggshell conductance, carbohydrate metabolism, and thyroid hormones indicated differences existed between AA and ACRBC embryos. The observed differences could not be related to hatchability differences in the present study.
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CHRISTENSEN ET AL. commercial broiler with a 1957 randombred strain when fed "typical" 1957 and 1991 broiler diets. Poultry Sci. 73:1805-1812. Rahn, H., and C. V. Paganelli, 1991. Energy budget and gas exchange of avian eggs. Pages 175-194 in: Avian Incubation. S. G. Tullett, ed. Butterworth Heinemann, London, England. Rahn, H., C. V. Paganelli, and A. Ar, 1974. The avian egg: air-cell gas tension, metabolism and incubation time. Respir. Physiol. 22:297-309. SAS Institute, 1989. A User's Guide to SAS®89. Sparks Press, Inc., Cary, NC. Snedecor, G. W., and W. G. Cochran, 1974. Statistical Methods. Iowa State University Press, Ames, IA. Tullett, S. G., 1981. Theoretical and practical aspects of eggshell porosity. Turkeys 29:24-28. van Deth, J.H.M.G., 1963. Glucose metabolism of the avian egg. Aust. J. Biol. 41:129-140. Visschedijk, A.H.J., 1968. The air space and embryonic respiration. 1. The pattern of gas exchange in the fertile egg during the closing stages of incubation. Br. Poult. Sci. 9:173-184. Vleck, C. M., 1991. AUometric scaling in avian embryonic development. Pages 39-57 in: Avian Incubation. S. G. Tullett, ed. Butterworth Heinemann, London, England. Watford, M, Y. Hod, Y. Chiao, M. Utter, and R. W. Hanson, 1981. The unique role of the kidney in gluconeogenesis in the chicken. J. Biol. Chem. 256: 10023-10027. Wentworth, B. C, and M. O. Hussein, 1985. Serum corticosterone levels in embryos, newly hatched, and young turkey poults. Poultry Sci. 64: 2195-2196. Windle, W. F., and J. Barcroft, 1938. Some factors governing the initiation of respiration in the chick. Am. J. Physiol. 121:684-691. Wittmann, J., and A. Weiss, 1981. Studies on metabolism of glycogen and adenine nucleotides in embryonic chick liver at the end of incubation. Comp. Biochem. Physiol. 69D1-6.
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Freeman, B. M., 1962. Gaseous metabolism in the domestic chicken, n. Oxygen consumption in the full-term and hatching embryo with a note on a possible cause for "death in the shell." Br. Poult. Sci. 3:63-71. Freeman, B. M., 1965. The importance of glycogen at the termination of the embryonic existence of Gallus domesticus. Comp. Biochem. Physiol. 14: 217-222. Freeman, B. M, 1969. The mobilization of hepatic glycogen in Gallus domesticus at the end of incubation. Comp. Biochem. Physiol. 28: 1169-1176. Harvey, S., R. A. Fraser, and R. W. Lea, 1991. Growth hormone secretion in poultry. Crit. Rev. Poult. Biol. 3:239-282. Havenstein, G. B., P. R. Ferket, S. E. Scheideler, and B. T. Larson, 1994a. Growth, livability, and feed conversion of 1957 vs 1991 broilers when fed "typical" 1957 and 1991 broiler diets. Poultry Sci. 73:1785-1794. Havenstein, G. B., P. R. Ferket, S. E. Scheideler, and D. R. Rives, 1994b. Carcass composition and yield of 1957 vs 1991 broilers when fed "typical" 1957 and 1991 broiler diets. Poultry Sci. 73:1795-1804. McNabb, F.M.A., E. A. Dunnington, P. B. Siegel, and S. Suvarna, 1993. Perinatal thyroid hormones and hepatic 5'deiodinase in relation to hatching time in weight-selected lines of chickens. Poultry Sci. 72:1764-1771. Nestor, K. E., K. I. Brown, and S. P. Touchburn, 1972. Egg quality and poult production in turkeys. 1. Variation during a seven month laying period. Poultry Sci. 51:104-110. Nobikuni, K., K. O. Koga, and H. Nishiyama, 1989. The effects of thyroid hormones on liver glycogen, muscle glycogen and liver lipid in chicks. Jpn. J. Zootech. Sci. 60:346-348. Noble, R. C, and K. Connor, 1984. Lipid metabolism in the chick embryo of domestic fowl (Gallus domesticus). World's Poult. Sci. J. 40:114-120. Qureshi, M. A., and G. B. Havenstein, 1994. A comparison of the immune performance of a 1991
1995 Poultry Science 74:551-562
randombred control lines compared with selected lines suggested that differences Recent investigations with turkeys indi- may exist in the energetics and metabocated that selection for increased egg lism of embryonic development (Ar et al., production or rapid growth has reduced 1987) within a species but of different the eggshell conductance of eggs genetic lines. Specifically, the amount and (Christensen et al., 1993). Increased egg- form of energy placed in eggs may differ shell conductance in turkey eggs from between genetically different lines, thus requiring different mechanisms to complete embryonic development by budgeting maternal investments in eggs (Vleck, Received for publication May 26, 1994. 1991). Accepted for publication October 25, 1994. It has also been suggested that differSalaries and research support provided by state and federal funds appropriated to the North Carolina ences in the energetics and carbohydrate Agricultural Service, North Carolina State University. metabolism of embryos may affect the The use of trade names in this publication does not survivability of developing embryos late imply endorsement by the North Carolina Agricultural Research Service of the products named, nor in the incubation period (Christensen et al., 1993) as well as in the period immediately criticism of similar products not mentioned. 2 To whom correspondence should be addressed. posthatching (Donaldson and Christensen, INTRODUCTION
551
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ABSTRACT Functional eggshell qualities, thyroid hormones, and carbohydrate metabolism of chick embryos at the end of incubation were compared between a modern (Arbor Acres line) and a randombred control population (Athens-Canadian Randombred). Embryos from the Arbor Acres genetic line developed in larger eggs with more albumen and less yolk than Athens Canadian Randombred lines. Percentage shell and functional eggshell properties measured as eggshell conductance constants did not differ between genetic lines. On a relative basis, hearts were generally smaller and livers heavier in Arbor Acres than in Athens-Canadian Randombred birds. Heart and liver glycogen concentrations were greater in Athens-Canadian Randombred than in Arbor Acres embryos. However, blood glucose was greater in Randombred than in Arbor Acres embryos only at internal pipping, a time of hypoxia and hypercapnia. Blood plasma concentrations of thyroxine did not differ significantly between the modern and Randombred embryos at any stage examined. Modern broiler chick embryos possessed greater concentrations of triiodothyronine as well as greater triiodothyronine to thyroxine ratios than Randombred embryos at external pipping and hatching. It can be inferred from the data that chick embryos differ in their use of carbohydrate during late development between modern and Randombred genetic lines. (Key words: chick embryo, carbohydrate metabolism, genetic line, eggshell, thyroid)
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MATERIALS AND METHODS
Fertile eggs from the ACRBC were obtained from the Southern Regional Poultry Breeding Laboratory, Athens, GA 30602, in December, 1992 and were incubated in a randomly distributed fashion with eggs from an AA commercial line3 of broiler breeders. Approximately 1,500 eggs were set from each line. Eggs were incubated4 at 37.5 C and 54% relative humidity until the 18th d of incubation, when incubator conditions were changed to 36.9 C and 80% relative humidity for the actual hatching process. All eggs were weighed (nearest .01 g) immediately prior to setting and again when the incubator was set at the hatching conditions to determine eggshell conductance values for
3
Arbor Acres, Glastonbury, CT 06033-6501. Natureform Mini NMC 2000, Jacksonville, FL 32202. sSarstedt Inc., Newton, NC 28658-046. 4
each egg using the calibrated egg technique (Tullett, 1981). At hatching all chicks were counted and eggs that did not hatch were broken. Their contents were subsequently examined macroscopically to determine true fertility and estimate the time at death for the nonhatched fertile eggs. Hatchability of fertile eggs was determined by discounting all truly infertile eggs and dividing the number of chicks hatched by the total number of fertile eggs. For determination of the effects of eggshell conductance on embryonic mortality, eggs were sorted into six groups: 1) infertiles, 2) incubation Week 1 deaths, 3) death at the plateau stage in oxygen consumption (18 d of incubation), 4) death at internal pipping (19 d of incubation), 5) death at external pipping (20 d of incubation), and 6) eggs containing chicks that hatched (21 d of incubation). Egg Composition
Fifteen eggs were selected randomly prior to setting from each line of chickens for examination of yolk and albumen weights. Only apparently normal eggs were selected for determination of egg components. Each selected egg was weighed (nearest .01 g) and then broken. The yolk and albumen were separated from the shell and then from each other. Each component was then dried to a constant weight in a drying oven at 65 C. Embryo Sampling
At each of four stages of embryonic development [prepipping (at 17 to 18 d of incubation), internal pipping (19 d of incubation), external pipping (20 d of incubation), and hatching (21 d of incubation)], fertile eggs, as determined by candling, were selected randomly from each of the lines. Descriptions of the morphology of each stage were given by Christensen et al (1982). Ten embryos per genetic line were sampled at each of the four stages of development. The embryo was decapitated and a blood sample was collected into tubes containing 10 mg of EDTA.5 The body with the yolk sac was blotted lightly and weighed immediately. The heart and liver were quickly dissected, blotted on a wet
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1991). Specifically, the perinatal carbohydrate metabolism of some genetic lines seems to be heavily dependent upon gluconeogenesis instead of stored glycogen for survivability during the plateau stage in oxygen consumption (Christensen et al, 1991). The objective of the present study was to compare the eggshell functional qualities and egg components of eggs from a line of modern Arbor Acres broiler breeder hens (AA) selected for growth with those of eggs from the unselected Athens-Canadian Randombred (ACRBC) population of chickens. A secondary objective of the study was to relate embryonic mortality and carbohydrate content of vital tissues (heart, blood, and liver) to the changes in eggshells and egg components from selected and unselected lines of chickens. A third objective was measurement of circulating concentrations of thyroid hormones that may be related to slow or rapid growth (McNabb et al, 1993) that may be available to each embryo for survival of hypoxia. This is part of a larger overall comparison of these two widely different genetic lines (Havenstein et al, 1994a,b; Qureshi and Havenstein, 1994).
553
CHICKEN EMBRYO METABOLISM
by modern broiler breeder (AA) chickens rol population (ACRBC)
TABLE 1. Egg components of fertile eggs prodi compared with a randombred Line
Weight
Water
(g) AA ACRBC x ± SEM1 Probability ] n = 15 for
65.8 68.4 44.5 67.7 55.2 ± 3.0 68.1 ± 1.0 .0001 .08 each genetic line.
Solids
Albumen
(%) 31.6 32.2 31.9 ± 1.0 08
6.8 5.9 6.4 ± .7 .004
Yolk
Shell
(% of total solids) 15.5 9.3 16.7 9.6 16.1 ± 1.5 9.4 ± .6 .04 .22
The effects of eggshell conductance on embryonic mortality were assessed using a factorial arrangement of completely random factors (Snedecor and Cochran, 1974). The factors were line of chicken and stage of death. There were two lines of chickens (AA and ACRBC) and four stages of embryonic development (death at the plateau stage, death at internal pipping, death at external pipping, and hatched). Means determined Biochemical Analyses of Tissues to differ significantly were separated by Blood plasma glucose concentrations using the orthogonal contrast procedure were measured using the glucose oxidase comparing all means of nonhatching eggs method described in detail by Donaldson with those of eggs that hatched. Sigand Christensen (1991). Cardiac and hepatic nificance was based on (P < .05) unless glycogen were analyzed using the tech- otherwise stated. nique of Dreiling et al. (1987). Plasma concentrations of thyroxine (T4) and triioRESULTS dothyronine (T3) were analyzed using prepared kits6 in a single assay. The intraassay Egg Quality coefficient of variation was less than 1%. Parallelism was assessed by comparing a Egg weights from the modern-type AA pooled sample of chick embryo blood broiler breeder were almost 50% greater plasma to samples of the same plasma than those from the ACRBC (Table 1). The containing a known amount of T4 or T3 and AA eggs contained significantly more parallelism was identical. solids as albumen and less as yolk than did eggs from ACRBC. No differences were noted between the two genetic lines in the Statistical Analyses percentage of shell on a dried or wet basis. Functional eggshell qualities when exData were analyzed as a completely random design (Snedecor and Cochran, pressed on an egg weight basis (conduc1974) with two treatment groups, i.e., the tance constants) did not differ between the modern and randombred controls. Egg two lines of chickens (Table 2). Eggshell weight data were analyzed as a one-way conductance was greater in AA than analysis of variance using the SAS® soft- ACRBC eggs but when differences in egg ware package for personal computers (SAS weights were accounted for by computing Institute, 1989). Embryo data were sorted conductance constants, no differences were by developmental stage prior to analysis. observed. Embryo Growth diagnostics Products Corp., Los Angeles, CA 90045.
Embryos from both genetic lines reached each stage of development at approxi-
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paper towel, trimmed, and then weighed (nearest .01 mg) on an analytical balance. The tubes containing blood were centrifuged (700 x g) under refrigeration (4 C) for 10 min, the plasma was decanted and frozen (-22 C) until analysis. The tissues were placed in sterile plastic bags and frozen similarly until analyzed.
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CHRISTENSEN ET AL.
TABLE 2. Functional eggshell qualities of eggs produced by modern broiler breeder (AA) chickens compared to randombred controls (ACRBC) gg weight
Weight loss
Conductance
Conductance constant
(g)
(%) 8.2 8.1 8.2 ± 1.3 .02
(mg H 2 0/d/torr) 12.7 9.6 11.2 ± 1.8 .0001
(conductance/ egg weight x 21) 4.0 3.9 4.0 ± .6 .19
E
Line
AA ACRBC x ± SEM1 Probability
66.2 50.7 58.4 ± 3.8 .0001
= 15 for each genetic line.
Tissue Carbohydrate Measurements
Cardiac glycogen concentration was greater in ACRBC than in AA embryos at the plateau and external pipped stages (Table 4). No differences were observed in the glycogen concentration of heart tissue in internally pipped or hatched chicks. Hepatic glycogen was significantly greater in AA than ACRBC embryos at internal pipping (Table 4). No significant differences between lines in hepatic glycogen were observed at the plateau, externally pipped, or hatched stages. Blood plasma glucose concentrations differed only at internal pipping when glucose concentration in ACRBC embryos was reduced compared with that of AA embryos (Table 4). No significant differences in plasma glucose concentrations were seen in the embryos sampled at the
plateau, externally pipped, or hatched stages. Blood Plasma Thyroid Hormone Concentrations
No significant differences were observed in plasma T4 concentrations between lines (Table 5). Plasma T3 concentrations differed significantly only at external pipping. The AA embryos possessed significantly higher levels of T3 in circulation than did ACRBC embryos. Ratios of T3 to T4 differed at external pipping and hatching with AA chick embryos having greater amounts of T3 relative to T4 than did ACRBC chick embryos. Functional Eggshell Quality and Hatchability
The preincubation weight associated with the eggs from the different embryonic death classifications differed significantly (Table 6). Eggs containing embryos dying during external pipping were heavier than eggs that hatched, but no other orthogonal contrasts differed significantly. The interaction of genetic line of chicken with stage at embryonic death for egg weights was not significant. Embryos dying at the plateau stage were from eggs with greater eggshell conductance values than eggs which hatched. Eggshell conductance constants of eggs classified with dead internally pipped embryos was less than for hatched eggs (Table 6). Eggshell conductance values of eggs with embryos from eggs that pipped externally then died were not different from the
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mately the same incubation time and hatched at approximately the same time. The AA embryos weighed more than ACRBC at each of the stages of development examined (Table 3). The weights of heart and liver are presented on both an absolute and relative basis, but because of the observed egg size and BW differences, they will be discussed only on a relative basis. The relative weight of the heart was greater in ACRBC than AA embryos at the plateau stage; however, the reverse was true at the external pipping and hatched stages, with AA embryos having heavier relative heart weights than ACRBC. The AA embryos had heavier relative liver weights than ACRBC at every stage of development examined except the plateau stage.
(mg) 528 436 482 + 130 .06
AA ACRBC x + SEM 1 Probability
*n = 10 for each genetic line.
(mg) 173 148 161 + 27 .02
AA ACRBC x ± SEM 2 Probability
1.22 1.34 1.28 + .23 .17
(%)
.40 .46 .43 + .04 .004
(%)
47.8 34.7 41.0 ± .0001
42.9 32.2 37.6 ± 4.2 .0001
AA ACRBC x ± SEMi Probability
(mg) 684 592 636 ± 80 .01
(mg) 216 168 191 + 27 .0004
5.2
Internal pip
Plateau
Line
1.43 1.71 1.58 ± .12 .0001
(%)
.45 .49 .47 ± .05 .10 Liver
(%)
(mg) 770 578 665 ± 65 .0001
(mg) 221 177 197 ± 27 .001
weights (g) — 46.7 31.8 38.6 ± 3.6 .0001 Heart weights
External pip
Stage of development 1
1 1 1
(%
(%
TABLE 3. Body, heart, and liver weights of embryos from modern-type broiler breeders (AA) Randombred Control (ACRBC) at selected stages of developme
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(mg) 8.5 7.3 7.9 + 6.4 .64
56 86 71.0 ± i68 .25
AA ACRBC x + SEMi
Probability
AA ACRBC x + SEM 1 Probability
AA ACRBC x + SEM 1 Probability
in = 10 for each genetic line.
(mg) 1.3 1.6 1.5 ± .24
.5
Plateau
Line
14.4 15.3 14.9 ± 8.8 .80
(%)
8.0 10.9 9.5 ± 3.7 .04
(%) .5
6.6 8.4 7.6 ± 2.6 .11
(%)
Heart (mg) .7 1.2 1.0 ± .004 : glycogen
giyi_ugt.il
EP
.3
(mg) (%) (mg) 11.4 12.9 9.0 14.6 34.2 20.8 13.2 ± 9.6 24.1 ± 12.7 15.2 ± 8.9 .44 .0006 .005 - Blood plasma glucose concentration (mg/dL 201 203 206 153 203 + 45 177 ± 53 .81 .03
1.4 ± .98
( m g) 1.4 1.4
IP
Stage of development 1
TABLE 4. Tissue glycogen and blood glucose concentration of embryos from modern broiler breeder Randombred Control (ACRBC) stage of development
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CHICKEN EMBRYO METABOLISM
TABLE 5. Blood plasma thyroid hormone concentrations of chick embryos from modern broiler chicks (AA) compared to randombred controls (ACRBC) Stage of development Strain
Plateau
Internal pip
External pip
Hatched
Thyroxine, ng/mL
AA ACRBC x ± SEMi Probability AA ACRBC 5c ± SEMI Probability AA ACRBC x ± SEM1 Probability
1.7 .5 1.1 ± .8 .31 .8 .7 .76 ± .30 .50 .23 .62 .38 ± .3 .10
10.0 7.0 8.6 ± 6.0 .39 1.0 2.2 1.4 ± 1.0 .28 .009 .022 .013 ± .01 .26
9.5 7.8 8.7 ± 4.0 .44 4.8 .7 3.4 ± .4 .03 .041 .010 .03 ± .02 .02
2.3 2.9 2.6 ± 2.5 .66 1.1 1.0 1.1 ± 3.2 .68 .844 .055 .37 ± .1 .05
Triiodothyronine, ng/mL
Ratio
J
n = 15 for each genetic line.
hatched eggs. No genetic line by stage at death interaction for eggshell conductance was observed. Hatchability of the AA broilers was 89% compared with 93.1% for the ACRBC controls but it was not possible to assess these means statistically with repeated measures. Levels of fertility were comparable for the two lines at 95% for AA and 94% for ACRBC. Embryonic mortality during Week 1 of incubation was 5.1% for AA and 4.3% for ACRBC, respectively; during the plateau stage mortality was 2.5 and 2.2% for AA and ACRBC; during internal pipping it was .4% for AA and .6% for ACRBC; and during pipping it was 1.2 and 1.4% for AA and ACRBC, respectively. DISCUSSION Genetic selection for rapid growth may have influenced the energetics of embryonic growth. Vleck (1991) concluded from comparisons between avian species that the fraction of the initial energy that was found in eggs at oviposition that was utilized by embryos for growth is essentially the same across avian species. This was true for both precocial and altricial species even though the maternal investment in altrical species eggs was less. The relationship appears to be true even though species differ widely in their incubation periods and size of hatchlings.
The data from the present study imply that energetics of incubation may differ on an intraspecific basis in chickens when hens deposit more nutrients as yolk and fewer as albumen. Freeman (1962) hypothesized that pipping and hatching responses were due to two separate stimuli. The first was thought to be the "pipping" stimulus and was suggested to be gaseous in nature, whereas the second was a "hatching" stimulus and was thought to be hormonal (or humoral). It has been suggested in the literature that the "pipping" stimulus is either high carbon dioxide (Windle and Barcroft, 1938; Visschedijk, 1968; Erasmus et al., 1970-71; Rahn and Paganelli, 1991), or decreased oxygen (Freeman, 1962). Hormones that may serve as the "hatching" stimulus or effect tissue maturation have been reviewed by Decuypere et al. (1991). Some hormone concentrations that change during hatching are the glucocorticoids (Wentworth and Hussein, 1980) catecholamines (Christensen and Edens, 1989), and thyroid hormones (Balaban and Hill, 1971; McNabb et al, 1993). In the present study we examined only the thyroid hormones. Egg Components The AA broiler breeders invest more albumen and less yolk in eggs than do their
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Variable
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CHRISTENSEN ET AL.
TABLE 6. Relationship of functional eggshell quality to embryonic death in modern broilers (AA) compared to randombred controls (ACRBC) Stage of death 1 Line
Plateau
AA ACRBC
64.3 50.6 57.4
X
AA ACRBC X
Initial egg mass (g) 66.7 54.0 60.4A Conductance constant (Conductance x: 21 4.3 3.8 4.3 3.6 4.3 A 3.7 A Probability Initial egg mass .0001 .0001 38 584 ± 3.8
External pip
Hatched 2
66.2 73.0 50.6 54.9 58.4A 64.0 A d/Initial egg mass) 4.1 4.0 3.9 4.0 4.0 A 4.0 Conductance .19 .05 .44 4.0 ± .6
A
Orthogonal contrasts means of nonhatching eggs with superscripts differed significantly from hatched eggs. !Mean values for embryos dying at each stage of development as determined by egg breakout at 22 d of incubation. 2 Mean values for all eggs that hatched. 3 n = 24 for Plateau; n = 9 for IP; n = 26 for EP; n = 925 for hatched.
ACRBC counterparts. When examining different lines of turkeys over a 7-mo laying period, Nestor et al. (1972) concluded that a significant proportion of the variation in hatchability could be accounted for in negative correlations of hatching rates and measurements of yolk. Thus, the decreased maternal investment of yolk may be favorable to embryonic survival in AA broiler embryos compared with ACRBC embryos. The increased investment as solids in the form of albumen may also confer metabolic advantages such as gluconeogenic amino acids (Watford et al, 1981) for increasing the availability of carbohydrate (Freeman, 1965; 1969). No differences were seen between the two lines in the percentage of dried shell, suggesting that the significant correlation of shell measurements with hatchability seen by Nestor et al. (1972) may have little to do with the differences observed in embryonic physiology of two genetic lines in the present study. This observation would, however, not discount the importance of functional eggshell properties. Examination of the egg components available to the embryo at the beginning of
incubation suggests that AA broiler embryos begin incubation with more protein and less lipid than do their randombred counterparts. These components must match eggshell conductance to allow a hatchling of adequate maturity for posthatching survival that is characteristic of the genetic line (Ar and Rahn, 1978; Vleck, 1991). Incubation periods measured in chicken eggs are significantly shorter than those predicted from their egg energy content, suggesting an abundance of available energy substrates (Vleck, 1991). The differences seen between the two genetic lines in the present study, however, suggest the embryos of the two lines do differ in their abilities to convert available energy substrates in egg components into glycogen for energy during the hypoxia of the plateau stage (Freeman, 1965). Functional Eggshell Qualities Because of the large differences seen in egg weights, conductance values also differed greatly. However, when the differences were adjusted for egg weight and incubation period (Ar and Rahn, 1978) and
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Source of variation Line Stage Line by stage S ± SEM3
Internal pip
CHICKEN EMBRYO METABOLISM
Growth of the Embryo The same differences seen in egg weights were seen in the embryonic BW. Embryo
and hatchling BW are highly correlated with egg weights (Burton and Tullett, 1985). Because of observed differences in BW, heart and liver weights were compared on a relative total rather than on an absolute basis. Relative heart weights were greater during the plateau stage in ACRBC than AA embryos, but after internal pipping heart growth in AA exceeded that of the ACRBC embryos, resulting in significantly heavier relative heart weights in AA hatchling chicks. The data do not support the notion that differences in heart growth may be due to the differences seen in blood thyroid hormone concentrations at all stages of development (Nobikuni et al, 1989). Thyroxine (or possibly T3) is responsible for increasing cardiac glycogen and providing nutrients to chick and poult hearts and muscles for growth (Czarnecki, 1991) but no correlation between thyroid hormones and heart growth were seen in these experiments. Embryo Carbohydrate Measurements Because of the plateau stage of oxygen consumption, embryo metabolism late in incubation is highly dependent upon carbohydrate (Freeman, 1965, 1969; Christensen et al, 1993). Lipid is the primary source of energy for embryonic growth and development (Noble and Connor, 1984); however, when less oxygen is available to the embryo, embryonic metabolism relies less on yolk lipid and more on tissue glycogen because more oxygen is required to metabolize lipids than carbohydrate (Freeman, 1962). Glycogen synthesis occurs throughout incubation via gluconeogenesis in preparation for the hypoxia that occurs at the plateau stage of late incubation (Freeman, 1969). Because of the differences seen in the growth of liver and heart tissues of these lines of chickens, the data for tissue glycogen are reported as milligrams of glycogen per gram of wet tissue. Heart glycogen concentrations were clearly greater in ACRBC than AA broiler chick embryos at most stages of development, suggesting a greater ability in ACRBC than AA embryos to maintain greater tissue glycogen concentrations despite exposure to the hypoxia or hypercapnia of the "pipping" stimulus. The
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were compared as conductance constants (product of eggshell conductance and incubation period divided by initial egg mass), the functional characteristics were nearly identical. This is different than observed with turkey eggs (Christensen et al, 1993). Because the present experiment occurred at a single time in the laying cycle of the hens, the possibility also exists that eggshell functional characteristics would differ between the lines at another stage of lay (Christensen and Nestor, 1994). Despite observing no significant differences between eggshell conductance constants for the lines, eggshell water vapor conductance, and conductance constants both differed similarly among the stages of incubation when embryos of both genetic lines died or hatched. In general, eggs with embryos that died during the plateau stage in oxygen consumption were too permeable compared with eggs that hatched. Eggs with embryos dying during internal pipping had less permeability compared with eggs that hatched. These factors suggest that the conflicting aims of elimination of sufficient metabolic water and carbon dioxide from the eggs as well as providing adequate oxygen for embryonic metabolism may be compromised. Therefore, adequate concentrations of the purported pipping stimuli, excess carbon dioxide or deficient oxygen (Freeman, 1962), may not be attained or excess water may accumulate (Christensen et al, 1993) and result in embryonic death prior to pipping or at internal pipping (Christensen et al, 1993). Alternatively, because of differences in incubation periods (McNabb et al, 1993), the embryonic physiological maturity at the onset of the plateau stage (Rahn et al, 1974) may have been underdeveloped at the time when gases may be needed for physiological stimulation to begin hatching (Windle and Barcroft, 1938; Visschedijk, 1968; Erasmus et al, 1970-71; Rahn and Paganelli, 1991). The lack of physiological maturity or shorter incubation time to synthesize vital tissue glycogen may also contribute to embryonic death at the stages of development observed.
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Thyroid Hormones
Observed values for plasma thyroid hormone concentrations were within the range observed previously (McNabb et al., 1993). Differences seen in blood plasma thyroid hormone concentrations did not correlate well with the observed differences in tissue carbohydrate. Differences in T3 occurred after the observed declines in hepatic and cardiac glycogen. It is difficult to attribute a specific glycolytic (Wittman and Weiss, 1981) or glycogen synthesis function to T3 or T4 because their observed concentration changes occurred after the changes in glycogen. However, it may be important to consider the observed differences in thyroid hormone of embryos of the two types of chickens studied. The increased T3 concentrations in AA broiler embryos at external pipping followed by increased T3 to T4 ratios in hatched chicks is noteworthy and suggests that monodeiodinase enzyme activity is somehow enhanced in AA chicks compared with ACRBC controls. This may be due to differences in circulating levels of growth hormone that may be different between the two strains (Harvey et al, 1991; Darras et al, 1992, 1993). Differences in thyroid hormones did not appear to be related to late embryonic mortality in the present study, but increased T3 did correspond to increased glycogen catabolism (Wittman and Weiss, 1981) in AA compared with ACRBC embryos. Summary and Conclusions
No differences were seen in eggshell conductance constants of eggs from the two genetic lines, which implies that neither AA nor ACRBC embryos would have a greater advantage in the apparently conflicting aims of providing adequate oxygen for metabolism while ventilating carbon dioxide and water vapor through the shell to prevent alkalosis or drowning. Metabolic water from lipid metabolism may be excessive in such eggs, causing them to drown. The ACRBC embryos began incubation with more solids in the form of yolk and less as albumen than AA, suggesting that they may rely more on aerobic metabolism of lipids than anaerobic metabolism of glyco-
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ability to maintain cardiac glycogen concentrations in chicks (Nobikuni et al, 1989) and poults (Czarnecki, 1991) has been suggested to be a function of thyroid hormones as well. Thyroxine may facilitate movement of carbohydrate from synthesis in the liver to storage in the heart because avian heart tissue has little or no gluconeogenic activity (Watford et al, 1981). Because no differences were seen in plasma T4 in the two lines of chickens in the present study, the idea is not supported by the data in the present study. Hepatic glycogen concentrations in ACRBC embryos increased more rapidly than in AA embryos between the plateau and internally pipped stages. Glycogen per gram of wet hepatic tissue doubled in ACRBC embryos whereas the concentration in AA embryos reached a plateau at the same level. In a prior study with turkey embryos it was shown that neither water accompanying glycogen synthesis nor lipid was responsible for the liver growth at these stages (Christensen et al, 1991). Blood plasma glucose concentrations in the ACRBC embryos were also depressed at internal pipping compared with AA embryos. Glucose-6-phosphatase is an induced hepatic gluconeogenic enzyme. Because there is little or no glucose remaining in the egg at this stage of development (van Deth, 1963), low blood glucose concentrations induce glucose-6-phosphatase to recycle lactate to pyruvate as glycogen (or glucose) is catabolized to lactic acid (Watford et al, 1981). Lactate carbon can then be reused for fuel to provide muscular energy for pipping and hatching. Thus, it may be possible that as chick embryos experience the plateau stage in oxygen consumption, stored tissue glycogen is catabolized very rapidly (Freeman, 1969). The ACRBC embryos have an enhanced capacity to regenerate the glycogen perhaps by a gluconeogenic response to low blood glucose concentrations observed during internal pipping (Donaldson and Liou, 1976). It is speculated that low blood glucose concentrations may induce increased hepatic glucose-6-phosphatase activity in ACRBC embryos in order to recycle muscle lactate or the kidneys may be synthesizing glucose from protein or fatty acid substrates at a greater rate in ACRBC than AA broiler embryos as was seen in turkeys (Christensen et al, 1993).
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Burton, F. G., and S. G. Tullett, 1985. The effects of egg weight and shell porosity on growth and water balance of the chicken embryo. Comp. Biochem. Physiol. 81A:377-385. Christensen, V. L., H. V. Biellier, and J. F. Forward, 1982. Physiology of turkey embryos during pipping and hatching. I. Hematology. Poultry Sci. 61:135-142. Christensen, V. L., W. E. Donaldson, and K. E. Nestor, 1993. Embryonic viability and metabolism in turkey lines selected for egg production or growth. Poultry Sci. 72:829-838. Christensen, V. L., W. E. Donaldson, and J. F. Ort, 1991. The effect ofdietary iodine on hatchability of eggs from two commercial strains of turkeys. Poultry Sci. 70:2529-2537. Christensen, V. L., W. E. Donaldson, J. F. Ort, and J. L. Grimes, 1991. Influence of diet-mediated maternal thyroid alterations on hatchability and metabolism of turkey embryos. Poultry Sci. 70: 1594-1601. Christensen, V. L., and F. W. Edens, 1989. Blood plasma catecholamine concentration of poult embryos during the transition from diffusive to convective respiration. Comp. Biochem. Physiol. 92B:549-553. Christensen, V. L., and K. E. Nestor, 1994. Changes in functional qualities of turkey eggshells in strains selected for increased egg production or growth. Poultry Sci. 73:1458-1464. Czarnecki, C. M., 1991. Influence of exogenous T4 on body weight, feed consumption, T4 levels, and myocardial glycogen in furazolidone-fed turkey poults. Avian Dis. 35:930-936. Darras, V. M., P. Rudas, T. J. Vessir, T. R. Hall, L. M. Huybrechts, A. Vanderpooten, L. R. Berghman, E. Decuypere, and E. R. Kuhn, 1993. Endogenous growth hormone controls high plasma levels of 3,3',5-triiodothyronine (T3) in growing chickens by decreasing the T3-degrading type n i deiodinase activity. Dom. Anim. Endocrinol. 10: 55-65. Darras, V. M., T. J. Vessir, L. R. Berghman, and E. R. Kuhn, 1992. Ontogeny of type I and type in deiodinase activities in embryonic and posthatch chicks: relationship with changes in plasma triiodothyronine and growth hormone levels. Comp. Biochem. Physiol. 103A:131-136. Decuypere, E., E. Dewil, and E. R. Kuhn, 1991. The hatching process and the role of hormones. Pages 239-256 in: Avian Incubation. S. G. Tullet, ed. Butterworth Heinemann, London, England. Donaldson, W. E., and V. L. Christensen, 1991. Dietary carbohydrate level and glucose metabolism in REFERENCES turkey poults. Comp. Biochem. Physiol. 98A: 347-350. Ar, A., B. Arieli, A. Belinsky, and Y. Tom-Tov, 1987. Energy in avian eggs and hatchling: utilization Donaldson, W. E., and G. I. Liou, 1976. Lipogenic and transfer. J. Exp. Zool. 235(Suppl. 1):151-164. enzymes: Parallel responses in liver to glucose consumption by newly hatched chicks. Nutr. Rep. Ar, A., and H. Rahn, 1978. Interdependence of gas Int. 13:471-476. conductance, incubation length, and weight of the avian eggs. Pages 227-236 in: Respiratory Dreiling, C. E., D. E. Brown, L. Casale, and L. Kelly, Function in Birds, Adult and Embryonic. J. Piiper, 1987. Muscle glycogen: comparison of iodine ed. Springer-Verlag, Berlin, Germany. binding and enzyme digestion assays and application to meat samples. Meat Sci. 20:167-177. Balaban, M., and J. Hill, 1971. Effects of thyroxine level and temperature manipulation upon the hatching Erasmus, B. W., B. J. Howell, and H. Rahn, of chick embryos (Gallus domesticus). Dev. Psy1970-71. Ontogeny of acid-base balance in the chobiol. 41(l):17-28. bullfrog and chicken. Respir. Physiol. 11:46-53.
gen for energy during embryonic growth. Carbohydrate metabolism differs among AA and ACRBC embryos, similar to what was observed previously in similarly selected lines of turkeys (Christensen et ah, 1993). However, it could not be related to increased mortality late in the incubation period of either strain. It was observed that the embryonic mortality of both genetic lines was in some unknown way related to the functional eggshell properties because embryos in eggs with reduced eggshell conductances constants, compared with eggs that hatched, died during internal pipping, whereas eggs with embryos dying during the plateau stage had greater eggshell conductance constants than those that hatched. Eggs containing embryos that died during pipping were heavier than eggs that hatched. The AA embryos reduced liver growth and increased heart growth during development, and also processed less glycogen in both of these tissues than did the ACRBC embryos. Thyroid hormones are related to glycogen metabolism, liver growth, and heart growth in avian embryos. Increased concentrations of T3 at external pipping in the AA embryos and greater ratios of T3 to T4 concentrations at the same stage compared with ACRBC embryo concentrations may be related to both increased organ growth and organ decreased glycogen concentration differences. In conclusion, observations of egg components, eggshell conductance, carbohydrate metabolism, and thyroid hormones indicated differences existed between AA and ACRBC embryos. The observed differences could not be related to hatchability differences in the present study.
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