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Correlation of body weight with hatchling blood glucose concentration and its relationship to embryonic survival1
Correlation of Body Weight with Hatchling Blood Glucose Concentration and Its Relationship to Embryonic Survival1 V. L. Christensen,2,* J. L. Grimes,* W. E. Donaldson,* and S. Lerner† *Department of Poultry Science, College of Agriculture and Life Sciences, Box 7608, North Carolina State University, Raleigh, North Carolina 27695-7608; †British United Turkeys, PO Box 727, Route 60 West, Lewisburg, West Virginia 24901 that Low had longer incubation periods. High embryos grew faster than Low embryos with elevated organ glycogen concentrations. Organic acid analysis indicated elevated plasma α-ketoglutarate, urate, and β-hydroxy butyrate concentrations, suggesting a greater reliance on gluconeogenesis for the High group. Posthatch growth was significantly positively correlated with hatchling blood glucose concentrations in toms but not in hens. Tom poults hatching with elevated glucose were heavier than low glucose hatch mates until 22 wk of age, but hen poults displayed no differences until 16 wk when High hens weighed less than Low hens. These data suggest that the negative correlation between rapid growth and embryonic survival is related to eggshell conductance constants and embryonic energy metabolism.
(Key words: embryonic livability, turkeys, growth, hatchability, blood glucose) 2000 Poultry Science 79:1817–1822
INTRODUCTION Embryonic survival is a function of many variables; however, many previous researchers have noted a significant negative correlation between selection for increased growth and livability (Nestor and Noble, 1995). An increased rate of growth posthatching in potential breeders adversely affects embryonic survival of their offspring. Growth is a function of many variables. Embryonic growth is highly conserved, genetically, but has been shown to be influenced principally by two factors: egg weight and length of the incubation period (Ricklefs, 1987). Researchers have suggested that the decline in viability as rates of growth increased may be due not only to egg weight but may also be mediated by functional eggshell qualities that are altered by selection for in-
Received for publication February 8, 2000. Accepted for publication July 26, 2000. 1 The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products mentioned, nor criticism of similar products not mentioned. 2 To whom correspondence should be addressed: vern_christensen@ ncsu.edu.
creased growth (Christensen and Nestor, 1994). This alteration might have resulted in an embryonic metabolism or growth rate that may be incompatible with the ability of the eggshell to conduct vital gases. A consequence of the alteration is incubation periods that may be too short or too long to result in viable hatchlings with maturity uncharacteristic of the species (Christensen et al., 1999). Incubation periods that are either shortened or lengthened can affect poult quality at hatching because the developmental period is determined by the average time of incubation and not adjusted for only a few eggs. Poults hatching early tend to be dehydrated and usually do not thrive after placement. Similarly, late-hatching poults are not mature, and early removal causes them to perform poorly. If the principles determining hatching could be elucidated, poult quality may be improved. Tissue glycogen or metabolites of carbohydrate metabolism may also be associated with depressed embryo survival (Christensen et al., 1993; 1999). Carbohydrate is es-
Abbreviation Key: High = High blood glucose concentration; Low = low blood glucose concentration; L:D = hours of light/h of darkness per day.
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ABSTRACT The negative correlation between selection for rapid growth and embryonic survival was investigated. Embryonic growth was assessed with hatchling weights of a closed population of commercial turkey breeders. Hatchling weights were highly significantly (P < 0.0001) and positively correlated with blood glucose concentrations at hatching. This relationship existed for both tom and hen poults. Significant differences among dams for hatchling blood glucose were observed. Further experiments examined dams selected for producing poults hatching with high (High) or low (Low) blood glucose concentrations. The High embryos were in larger-sized eggs with the same eggshell conductance but with significantly lower conductance constants than the Low embryos, suggesting
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sential to embryonic viability at the final stages of incubation because of the hypoxia from inadequate egg shell conductance (Freeman, 1965; Christensen et al., 1999). These observations infer that the negative relationship of embryonic survival with increased growth may have basis in altered carbohydrate metabolism. The hypothesis proposed by the present study was that turkey embryos with increased growth rates and blood glucose concentrations would be more viable than those with lower growth rates and depressed glucose levels.
MATERIALS AND METHODS
3 Model 252 incubator. Jamesway Incubator Company, Ltd., Cambridge, ON, Canada N1R 7L3.
RESULTS Correlation Analysis Highly significant (P ≤ 0.0001) correlation coefficients were observed for blood glucose concentration at hatching and the hatchling weight for both families (Table 1). Tom and hen poults showed highly significant correlations. The correlation coefficient for one family was greater than the other, but both were highly significant. Body weight at hatching was also highly significantly correlated with egg weight for both families (data not shown). Correlation coefficients of the blood glucose concentration at hatching with posthatching growth showed no consistent trends, although a few small correlation coefficients were significant early in growth.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 23, 2015
The hypothesis was tested by establishing two families as subpopulations of a commercial flock of turkey breeders. The hens from a flock completing its first laying period were selected by individual blood samples taken from the entire flock that were subjected to Southern blot analysis comparing three DNA probes to determine homology of banding of the approximately 400 hens and 50 toms (Grimes et al., 1996). Fifty hens were selected to form two families to represent the individuals with minimal band sharing among the genotypes. Two sires were identified from among 50 to have the least band sharing with the selected hens. The selected hens were recycled for a second laying period with procedures approved by the Institutional Animal Care and Use Committee of North Carolina State University. Only manipulation of day length (L = hours of light; D = hours of darkness; 6L:18D) was used without feed or water restriction to effect the loss and regrowth of feathers and to synchronize the photo sexual response. The toms were maintained on a LD regimen of 14L:10D to maintain semen production throughout the experiment. The hens were stimulated to initiate egg laying by exposing them to 15.5L:8.5D day lengths following 10 wk of short-day lengths (6L:18D). Within each family, single sire artificial inseminations were performed weekly to produce fertile eggs. Each sire was used to inseminate 25 hens. Hens were inseminated with 200 million viable cells in fresh semen. The hens were trap-nested to pedigree and to identify each egg laid by the hens over several 2-wk periods. Within each family, eggs were not used from two individuals who continued to lay outside of the nest. At the end of 2 wk, the eggs were sorted by dam, set into incubators, and incubated using standard conditions prescribed by the manufacturer3 (dry bulb temperature = 37.5 C; RH = 54%). At the 25th d of incubation, the eggs were placed into a separate hatcher operated at 36.8 C and 85% RH. At the time of hatching, the beak was trimmed with surgical scissors, and a drop of blood was obtained from the trimmed beak. The whole blood was analyzed for glucose concentration with the technique described by Donaldson and Christensen (1991). Body weights at hatching were
measured (nearest 0.1 g) at the same time that the blood sample was obtained. Seven replicate hatches were used to determine the relationship between hatchling BW and blood glucose concentration. At the time of setting, eggs from each hen were weighed to determine the mean eggshell conductance of each hen. The eggs were identified with numbers and were weighed again at the completion of 25 d of incubation. The eggshell conductance values were determined with the calibrated egg technique of Tullett (1981). After hatching and blood sampling, the poults were placed in a brooder building. The hens were grown to 16 wk of age and the toms to 22 wk of age using standard practices and diets. Body weights were measured at 4wk intervals to determine a relationship of BW and blood glucose concentration at hatching. A correlation analysis (SAS Institute, 1998) was used to determine the relationship between blood glucose concentrations at hatching and the body weights from 0 to 22 wk posthatching. Data collection for the correlation analysis was completed at the end of seven biweekly hatches (14 wk of egg laying). Because of significant positive correlations observed between hatchling poult weight and the concentration of blood glucose, seven hens that were selected from both families were identified and selected for further study. Seven selected breeder hens that displayed greater hatchling glucose concentrations and BW were compared to seven breeder hens with low hatchling glucose concentrations. Eggs and embryos were sampled from these groups for an additional 10 wk of egg laying to determine eggshell conductance, organ weights, glycogen concentrations, and blood metabolites (Christensen et al., 1993). Embryos were sampled, and plasma organic acids were assayed with the technique of Donaldson and Christensen (1994). Mortality occurring at Weeks 1 and 4 of incubation, as well as at pipping, and hatchability of each group were compared. Data from the selected hens plasma glucose groups were compared using the GLM procedure of SAS Institute (1998). Significance was based on P ≤ 0.05 unless otherwise noted.
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EMBRYO SURVIVAL TABLE 1. Correlation coefficients of blood glucose with BW at hatching and during growth Sire family 503
116
Week of age Sex
0
4
8
12
16
22
Hens P2 Toms P Hens P Toms P
0.25 (157)1 0.001 0.33 (165) 0.0001 0.40 (195) 0.0001 0.25 (199) 0.0004
0.10 (140) NS 0.17 (149) 0.03 0.0 (178) NS 0.11 (177) NS
0.17 (109) 0.08 0.16 (108) 0.09 0.0 (177) NS 0.14 (176) 0.05
0.33 (49) 0.004 0.22 (52) 0.07 0.0 (53) NS 0.0 (162) NS
0.0 (24) NS 0.29 (49) 0.04 0.0 (113) NS 0.34 (115) NS
... ... 0.0 (33) NS ... ... 0.0 (109) NS
1
Numbers in parentheses are sample sizes used in the analysis. P indicates the probability that the correlation coefficient differs from zero.
2
Hens Dam 759 725 625 602 628 909 706 710 860 707 679 903 651 604 681 904 682 798 838 809 800 829 x ± SEM P
n 3 3 8 6 4 8 7 3 17 11 8 13 9 11 12 10 19 5 11 11 11 9
Glucose a
245 236ab 226abc 226abc 220abcd 216abcde 212bcde 211bcde 209bcdef 207bcdef 207bcdef 207bcdef 206bcdef 206bcdef 204bcdef 203bcdef 203bcdef 201bcdef 197cdef 192def 184ef 177f 205 ± 2 0.0001
Toms BW
n
59.0 63.3 62.5 65.8 67.2 58.9 59.4 62.0 63.9 55.5 58.1 62.3 60.1 59.5 60.7 62.3 59.0 55.6 59.9 51.2 55.4 55.3 59.5 ± 0.2 0.0001
3 5 11 5 3 8 7 4 16 7 9 14 10 12 18 10 11 7 19 6 8 7
Glucose abc
226 239a 245a 223abc 190ef 227ab 221abcd 227ab 204bcde 213bcde 206bcde 206bcde 221abcd 201bcde 206bcde 203bcde 205bcde 199cde 211bcde 207bcde 196de 172f 210 ± 2 0.0001
BW 64.5 63.2 65.2 64.0 71.0 59.3 61.3 60.3 63.4 55.9 58.6 61.3 62.2 58.3 57.9 63.6 58.8 56.4 60.4 51.2 56.4 56.4 60.2 ± 0.2 0.0001
Columnar means followed by different superscripts differ significantly (P ≤ 0.0001).
a−f
TABLE 3. Egg measurements from hatched poults selected for high and low blood glucose concentrations Egg measurement
Blood glucose1
Egg weight (g)
Conductance (G) (mg/day per torr)
Constant (k) [G × incubation period (d)]/ egg weight (g)
High Low x ± SEM P
97.5a 89.3b 90.9 ± 0.8 0.0002
19.5 19.4 19.4 ± 0.5 NS
5.4b 6.1a 5.9 ± 0.1 0.02
a,b
Columnar means followed by a different superscript differ significantly. High = Poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations. 1
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TABLE 2. Pedigreed poult blood glucose concentrations and BW at hatching
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TABLE 4. Survival of embryos when hatching with high or low blood glucose concentrations Blood glucose1 High Low x ± SEM P
Viability measurement (%) Hatchability b
68.1 81.6a 74.9 ± 3.1 0.05
Week 1
Week 4
a
9.8 4.1b 6.9 ± 0.2 0.01
7.6 5.0 6.3 ± 2.6 NS
Pipping a
13.6 7.7b 10.6 ± 0.6 0.0009
TABLE 6. Organ glycogen concentration (mg/g) of hatched poults selected for high or low blood glucose concentrations Blood glucose1 High Low x ± SEM P
Organ Heart a
56 44b 50 ± 0.4 0.0001
Liver a
394 283b 335 ± 7.2 0.02
Muscle 72 69 71 ± 1.8 NS
a,b Columnar means followed by a different superscript differ significantly. 1 High = poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations.
Selected Hens
muscle glycogen content was not different between the two groups (Table 6). Blood plasma organic acid concentrations are given in Table 7. The α-ketoglutarate, β-hydroxybutyrate, and urate concentrations of the High group were significantly depressed when compared to those values for the Low group. Pyruvate and lactate concentrations did not differ between the two groups. When growth of the offspring from the selected hens was examined, tom poults from the High glucose group were significantly heavier than the Low group at nearly every age examined (Table 8). There were no differences in performance of the hens poults from 4 to 12 wk of age. However, at 16 wk of age, the opposite response was noted as the hen poults from the High group weighed significantly less than those from the Low group.
Mean blood glucose concentration and hatchling body weight for one family are given in Table 2. The BW and blood glucose concentrations were significantly different among dams from both families, although the data from only one family are shown. When only the seven High and seven Low hens were examined, egg weights of the elevated glucose hens (High) were significantly heavier than those of the low group (Table 3). Mean blood plasma glucose concentrations of the High group was 285 mg/ dL when examined for the final 10 wk of the experiment, which was significantly greater than that for the Low group of hens (251 mg/dL). Eggshell conductance did not differ between the two glucose groups, but conductance constants (k) of the High group were significantly lower than that of the Low group. Hatchability of the High group was significantly lower by 13.5% than that of the Low (Table 4). Additionally, the Low group displayed lower embryonic mortality during Week 1 of incubation as well as during pipping. Mortality did not differ during the fourth week of incubation. The mean BW of High glucose hatchlings was nearly 12 g more than that of the Low group. Similarly, the High glucose group had heavier hearts, livers, and pipping muscles than did the Low group (Table 5), but only the pipping muscles were larger when compared relative to the BW. Organ glycogen concentrations were significantly greater among High glucose poults than Low, but pipping TABLE 5. Organ weights of hatched poults selected for high or low blood glucose concentrations Organ Blood glucose1
Body (g)
Heart (mg)
Liver (mg)
Muscle (mg)
High Low x ± SEM P
64.2a 52.8b 58.2 ± 0.19 0.0001
401a (0.62)2 328b (0.62) 363 ± 1.3 0.0001
1785a (2.98) 1554b (2.96) 1664 ± 7.8 0.0001
441a (0.68a) 293b (0.54b) 364 ± 1.6 0.0001
a,b Columnar means followed by a different superscript differ significantly. 1 High = poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations. 2 Means in parentheses are the values relative to BW.
DISCUSSION In contrast to our hypothesis, breeder hens producing heavier poults with elevated blood glucose concentrations displayed lower hatchability than breeder hens whose progeny hatched with lower weights and glucose concentrations. Embryonic growth is a highly conserved trait across avian species (Ricklefs and Starcks, 1998). It might be expected that within a species or a subpopulation within a species, the same conservation would be observed. In the present study, breeder hens selected to represent a population of commercial turkeys exhibited significant differences in hatchling BW and blood glucose concentrations. Furthermore, when the entire subpopulation was examined, it was clear that among commercial turkey breeder hens a highly significant correlation existed between hatchling BW and blood glucose concentrations. These observations were also related directly to the weight of the egg in which the embryo was growing. It may be concluded from these observations that within the limits of genetic diversity, embryonic growth may not be conserved in all individuals following selection for commercial traits. A diversity exists in flocks of breeding turkeys that allows adaptation to egg size, length of the incubation period, and eggshell conductance (Ar and Rahn, 1978). Because these variables are interdependent, it may be inferred that some embryos; perhaps at the extremes of the normal distribution for BW, egg weight, or metabolic
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a,b Columnar means followed by a different superscript differ significantly. 1 High = poults hatching with elevated blood glucose concentrations: Low = poults hatching with normal blood glucose concentrations.
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EMBRYO SURVIVAL TABLE 7. Plasma organic acid concentrations (µg/mL) of hatched poults selected for high or low blood glucose concentrations Organic acid 1
Blood glucose
Pyruvate
α-ketoglutarate b
High Low x ± SEM P
1.11 1.48a 1.30 ± 0.02 0.006
Lactate
1.47 1.34 1.41 ± 0.03 NS
Butyrate
Urate
b
1.48 1.41 1.41 ± 0.03 NS
9.1b 11.3a 10.2 ± .2 0.05
49.5 58.7a 54.2 ± 0.6 0.005
a,b
Columnar means followed by a different superscript differ significantly. High = poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations. 1
The heavier embryos were growing in heavier eggs with the same gas conductance properties as the smaller eggs; thus, the conductance constants of such eggs were depressed. The vital gas required for the larger tissue mass was not available, and so rapidly growing embryos merely maintained heart and liver tissues but could grow muscle tissue while hatching. The large amount of glycogen available in heart and liver tissue may indicate an inability to metabolize those stores or, alternately, an increased ability to synthesize glycogen to maintain heart and liver function. Because of the decreased plasma concentrations of organic acids in the heavier poults, it is speculated that the gluconeogenetic process in such embryos may be producing less glucose than is needed. Although the blood glucose concentrations were elevated, the depressed organic acid concentrations in the blood indicate that metabolites are being recycled very rapidly, thus not allowing accumulation in the blood but providing it rapidly for heart and liver metabolism (Christensen et al., 1999). The results of the current study indicate that heavier poults at hatching with elevated plasma glucose levels may be related to nonviable embryos. The viability declines because of an inability of such embryos to maintain organ growth and function through the hatching process. The increased hatchling BW in toms persisted through 22 wk of age. A different rate of growth was observed among hatchling hens, as High BW birds weighed less at 16 wk.
TABLE 8. Body weights (g) of poults hatching with high or low blood glucose concentrations Weeks of age Sex
Glucose1
4
8
12
16
22
Toms
High Low x ± SEM P N High Low x ± SEM P N
872 836 853 ± 15 NS 90 750 714 731 ± 13 0.10 97
4,200a 3,879b 4,018 ± 47 0.003 83 3,289 3,172 3,226 ± 48 NS 89
9,355a 8,737b 8,996 ± 79 0.04 67 6,983 6,895 6,929 ± 60 NS 75
14,469 14,155 14,280 ± 120 0.09 60 9,442b 10,177a 9,945 ± 85 0.01 38
20,838a 19,477b 19,991 ± 150 0.007 45 — — — — —
Hens
a,b
Columnar means followed by a different superscript differ significantly. High = poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations. 1
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rate; may be exposed to extensive periods of hypoxia and life-threatening situations at the end of the incubation period (Freeman, 1965). When we select for increased growth or egg weight, the relationship among egg weight, eggshell conductance, and the length of the incubation period may be magnified to produce perilous conditions for poult survival. Because eggshell conductance does not change during incubation, poults in larger eggs need greater eggshell conductance or longer incubation periods to survive. The well-known negative correlation between hatchability and selection for increased growth (Nestor and Noble, 1995) may be related to metabolism late in incubation. Physiological traits measured in the current study may give useful insight into the causes of decreased hatchability in growth-selected lines of turkeys. Those individuals hatching with elevated blood glucose concentrations have siblings that display lower survival rates at Week 1 of incubation as well as during pipping. The growth of pipping muscle relative to the BW was also enhanced. The rapidly growing sibling hatchlings also have elevated concentrations of glycogen in the heart and liver but not in the muscle. These observations were accompanied by depressed plasma α-ketoglutarate, β-hydroxybutyrate, and urate concentrations. These observations taken together show clearly that the negative relationship between growth and embryonic survival is related to the energy metabolism of the rapidly growing embryo.
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REFERENCES
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 23, 2015
Ar, A., and H. Rahn, 1978. Interdependence of gas conductance, incubation length, and weight of the avian egg. Pages 227−236 in: Respiratory Function in Birds, Adult and Embryonic. J. Piper, ed. Springer Verlag, New York, NY. Christensen, V. L., W. E. Donaldson, and K. E. Nestor, 1993. Hatchability and embryonic metabolism in turkey lines selected for egg production or growth. Poultry Sci. 72:829−838. Christensen, V. L., W. E. Donaldson, and K. E. Nestor, 1999. Eggshell conductance constants and length of the incubation period affect survival of lines of turkey embryos. Poultry Sci. 77 (Suppl. 1):78. (Abstr.). Christensen, V. L., and K. E. Nestor, 1994. Changes in functional qualities of turkey eggshells in strains selected for increased egg production and growth. Poultry Sci. 73:1458−1464. Donaldson, W. E., and V. L. Christensen, 1991. Dietary carbohydrate levels and glucose metabolism in turkey poults. Comp. Biochem. Physiol. 98A:347−350. Donaldson, W. E., and V. L. Christensen, 1994. Dietary carbohydrate effects on some organic acids and aspects of glucose
metabolism in turkey poults. Comp. Biochem. Physiol. 109A:423−430. Freeman, B. M., 1965. The importance of glycogen at the termination of the embryonic existence of Gallus domesticus. Comp. Biochem. Physiol. 14:217−222. Grimes, J. L., V. L. Christensen, S. Lerner, and J. Hillel, 1996. Effect of turkey sire selection within a family on subsequent poult growth. Poultry Sci. 75 (Suppl. 1):119. (Abstr.). Nestor, K. E., and D. O. Noble, 1995. Influence of selection for increased egg production, body weight, and shank width on egg composition and the relationship of the egg traits to hatchability. Poultry Sci. 74:427−433. Ricklefs, R. E., 1987. Comparative analysis of avian embryonic growth. J. Exp. Zool. (Suppl 1.) 1:309−323. Ricklefs, R. E., and M. J. Starcks, 1998. Embryonic growth and development. Pages 31−58 in: Avian Growth and Development. Oxford University Press, New York, NY. SAS Institute, 1998. SAS/STAT Guide for Personal Computers. Version 6 Edition. SAS Institute Inc., Cary, NC. Tullett, S. G., 1981. Theoretical and practical aspects of eggshell porosity. Turkeys 29:24−28.
(Key words: embryonic livability, turkeys, growth, hatchability, blood glucose) 2000 Poultry Science 79:1817–1822
INTRODUCTION Embryonic survival is a function of many variables; however, many previous researchers have noted a significant negative correlation between selection for increased growth and livability (Nestor and Noble, 1995). An increased rate of growth posthatching in potential breeders adversely affects embryonic survival of their offspring. Growth is a function of many variables. Embryonic growth is highly conserved, genetically, but has been shown to be influenced principally by two factors: egg weight and length of the incubation period (Ricklefs, 1987). Researchers have suggested that the decline in viability as rates of growth increased may be due not only to egg weight but may also be mediated by functional eggshell qualities that are altered by selection for in-
Received for publication February 8, 2000. Accepted for publication July 26, 2000. 1 The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products mentioned, nor criticism of similar products not mentioned. 2 To whom correspondence should be addressed: vern_christensen@ ncsu.edu.
creased growth (Christensen and Nestor, 1994). This alteration might have resulted in an embryonic metabolism or growth rate that may be incompatible with the ability of the eggshell to conduct vital gases. A consequence of the alteration is incubation periods that may be too short or too long to result in viable hatchlings with maturity uncharacteristic of the species (Christensen et al., 1999). Incubation periods that are either shortened or lengthened can affect poult quality at hatching because the developmental period is determined by the average time of incubation and not adjusted for only a few eggs. Poults hatching early tend to be dehydrated and usually do not thrive after placement. Similarly, late-hatching poults are not mature, and early removal causes them to perform poorly. If the principles determining hatching could be elucidated, poult quality may be improved. Tissue glycogen or metabolites of carbohydrate metabolism may also be associated with depressed embryo survival (Christensen et al., 1993; 1999). Carbohydrate is es-
Abbreviation Key: High = High blood glucose concentration; Low = low blood glucose concentration; L:D = hours of light/h of darkness per day.
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ABSTRACT The negative correlation between selection for rapid growth and embryonic survival was investigated. Embryonic growth was assessed with hatchling weights of a closed population of commercial turkey breeders. Hatchling weights were highly significantly (P < 0.0001) and positively correlated with blood glucose concentrations at hatching. This relationship existed for both tom and hen poults. Significant differences among dams for hatchling blood glucose were observed. Further experiments examined dams selected for producing poults hatching with high (High) or low (Low) blood glucose concentrations. The High embryos were in larger-sized eggs with the same eggshell conductance but with significantly lower conductance constants than the Low embryos, suggesting
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sential to embryonic viability at the final stages of incubation because of the hypoxia from inadequate egg shell conductance (Freeman, 1965; Christensen et al., 1999). These observations infer that the negative relationship of embryonic survival with increased growth may have basis in altered carbohydrate metabolism. The hypothesis proposed by the present study was that turkey embryos with increased growth rates and blood glucose concentrations would be more viable than those with lower growth rates and depressed glucose levels.
MATERIALS AND METHODS
3 Model 252 incubator. Jamesway Incubator Company, Ltd., Cambridge, ON, Canada N1R 7L3.
RESULTS Correlation Analysis Highly significant (P ≤ 0.0001) correlation coefficients were observed for blood glucose concentration at hatching and the hatchling weight for both families (Table 1). Tom and hen poults showed highly significant correlations. The correlation coefficient for one family was greater than the other, but both were highly significant. Body weight at hatching was also highly significantly correlated with egg weight for both families (data not shown). Correlation coefficients of the blood glucose concentration at hatching with posthatching growth showed no consistent trends, although a few small correlation coefficients were significant early in growth.
Downloaded from http://ps.oxfordjournals.org/ at University of North Dakota on May 23, 2015
The hypothesis was tested by establishing two families as subpopulations of a commercial flock of turkey breeders. The hens from a flock completing its first laying period were selected by individual blood samples taken from the entire flock that were subjected to Southern blot analysis comparing three DNA probes to determine homology of banding of the approximately 400 hens and 50 toms (Grimes et al., 1996). Fifty hens were selected to form two families to represent the individuals with minimal band sharing among the genotypes. Two sires were identified from among 50 to have the least band sharing with the selected hens. The selected hens were recycled for a second laying period with procedures approved by the Institutional Animal Care and Use Committee of North Carolina State University. Only manipulation of day length (L = hours of light; D = hours of darkness; 6L:18D) was used without feed or water restriction to effect the loss and regrowth of feathers and to synchronize the photo sexual response. The toms were maintained on a LD regimen of 14L:10D to maintain semen production throughout the experiment. The hens were stimulated to initiate egg laying by exposing them to 15.5L:8.5D day lengths following 10 wk of short-day lengths (6L:18D). Within each family, single sire artificial inseminations were performed weekly to produce fertile eggs. Each sire was used to inseminate 25 hens. Hens were inseminated with 200 million viable cells in fresh semen. The hens were trap-nested to pedigree and to identify each egg laid by the hens over several 2-wk periods. Within each family, eggs were not used from two individuals who continued to lay outside of the nest. At the end of 2 wk, the eggs were sorted by dam, set into incubators, and incubated using standard conditions prescribed by the manufacturer3 (dry bulb temperature = 37.5 C; RH = 54%). At the 25th d of incubation, the eggs were placed into a separate hatcher operated at 36.8 C and 85% RH. At the time of hatching, the beak was trimmed with surgical scissors, and a drop of blood was obtained from the trimmed beak. The whole blood was analyzed for glucose concentration with the technique described by Donaldson and Christensen (1991). Body weights at hatching were
measured (nearest 0.1 g) at the same time that the blood sample was obtained. Seven replicate hatches were used to determine the relationship between hatchling BW and blood glucose concentration. At the time of setting, eggs from each hen were weighed to determine the mean eggshell conductance of each hen. The eggs were identified with numbers and were weighed again at the completion of 25 d of incubation. The eggshell conductance values were determined with the calibrated egg technique of Tullett (1981). After hatching and blood sampling, the poults were placed in a brooder building. The hens were grown to 16 wk of age and the toms to 22 wk of age using standard practices and diets. Body weights were measured at 4wk intervals to determine a relationship of BW and blood glucose concentration at hatching. A correlation analysis (SAS Institute, 1998) was used to determine the relationship between blood glucose concentrations at hatching and the body weights from 0 to 22 wk posthatching. Data collection for the correlation analysis was completed at the end of seven biweekly hatches (14 wk of egg laying). Because of significant positive correlations observed between hatchling poult weight and the concentration of blood glucose, seven hens that were selected from both families were identified and selected for further study. Seven selected breeder hens that displayed greater hatchling glucose concentrations and BW were compared to seven breeder hens with low hatchling glucose concentrations. Eggs and embryos were sampled from these groups for an additional 10 wk of egg laying to determine eggshell conductance, organ weights, glycogen concentrations, and blood metabolites (Christensen et al., 1993). Embryos were sampled, and plasma organic acids were assayed with the technique of Donaldson and Christensen (1994). Mortality occurring at Weeks 1 and 4 of incubation, as well as at pipping, and hatchability of each group were compared. Data from the selected hens plasma glucose groups were compared using the GLM procedure of SAS Institute (1998). Significance was based on P ≤ 0.05 unless otherwise noted.
1819
EMBRYO SURVIVAL TABLE 1. Correlation coefficients of blood glucose with BW at hatching and during growth Sire family 503
116
Week of age Sex
0
4
8
12
16
22
Hens P2 Toms P Hens P Toms P
0.25 (157)1 0.001 0.33 (165) 0.0001 0.40 (195) 0.0001 0.25 (199) 0.0004
0.10 (140) NS 0.17 (149) 0.03 0.0 (178) NS 0.11 (177) NS
0.17 (109) 0.08 0.16 (108) 0.09 0.0 (177) NS 0.14 (176) 0.05
0.33 (49) 0.004 0.22 (52) 0.07 0.0 (53) NS 0.0 (162) NS
0.0 (24) NS 0.29 (49) 0.04 0.0 (113) NS 0.34 (115) NS
... ... 0.0 (33) NS ... ... 0.0 (109) NS
1
Numbers in parentheses are sample sizes used in the analysis. P indicates the probability that the correlation coefficient differs from zero.
2
Hens Dam 759 725 625 602 628 909 706 710 860 707 679 903 651 604 681 904 682 798 838 809 800 829 x ± SEM P
n 3 3 8 6 4 8 7 3 17 11 8 13 9 11 12 10 19 5 11 11 11 9
Glucose a
245 236ab 226abc 226abc 220abcd 216abcde 212bcde 211bcde 209bcdef 207bcdef 207bcdef 207bcdef 206bcdef 206bcdef 204bcdef 203bcdef 203bcdef 201bcdef 197cdef 192def 184ef 177f 205 ± 2 0.0001
Toms BW
n
59.0 63.3 62.5 65.8 67.2 58.9 59.4 62.0 63.9 55.5 58.1 62.3 60.1 59.5 60.7 62.3 59.0 55.6 59.9 51.2 55.4 55.3 59.5 ± 0.2 0.0001
3 5 11 5 3 8 7 4 16 7 9 14 10 12 18 10 11 7 19 6 8 7
Glucose abc
226 239a 245a 223abc 190ef 227ab 221abcd 227ab 204bcde 213bcde 206bcde 206bcde 221abcd 201bcde 206bcde 203bcde 205bcde 199cde 211bcde 207bcde 196de 172f 210 ± 2 0.0001
BW 64.5 63.2 65.2 64.0 71.0 59.3 61.3 60.3 63.4 55.9 58.6 61.3 62.2 58.3 57.9 63.6 58.8 56.4 60.4 51.2 56.4 56.4 60.2 ± 0.2 0.0001
Columnar means followed by different superscripts differ significantly (P ≤ 0.0001).
a−f
TABLE 3. Egg measurements from hatched poults selected for high and low blood glucose concentrations Egg measurement
Blood glucose1
Egg weight (g)
Conductance (G) (mg/day per torr)
Constant (k) [G × incubation period (d)]/ egg weight (g)
High Low x ± SEM P
97.5a 89.3b 90.9 ± 0.8 0.0002
19.5 19.4 19.4 ± 0.5 NS
5.4b 6.1a 5.9 ± 0.1 0.02
a,b
Columnar means followed by a different superscript differ significantly. High = Poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations. 1
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TABLE 2. Pedigreed poult blood glucose concentrations and BW at hatching
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CHRISTENSEN ET AL.
TABLE 4. Survival of embryos when hatching with high or low blood glucose concentrations Blood glucose1 High Low x ± SEM P
Viability measurement (%) Hatchability b
68.1 81.6a 74.9 ± 3.1 0.05
Week 1
Week 4
a
9.8 4.1b 6.9 ± 0.2 0.01
7.6 5.0 6.3 ± 2.6 NS
Pipping a
13.6 7.7b 10.6 ± 0.6 0.0009
TABLE 6. Organ glycogen concentration (mg/g) of hatched poults selected for high or low blood glucose concentrations Blood glucose1 High Low x ± SEM P
Organ Heart a
56 44b 50 ± 0.4 0.0001
Liver a
394 283b 335 ± 7.2 0.02
Muscle 72 69 71 ± 1.8 NS
a,b Columnar means followed by a different superscript differ significantly. 1 High = poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations.
Selected Hens
muscle glycogen content was not different between the two groups (Table 6). Blood plasma organic acid concentrations are given in Table 7. The α-ketoglutarate, β-hydroxybutyrate, and urate concentrations of the High group were significantly depressed when compared to those values for the Low group. Pyruvate and lactate concentrations did not differ between the two groups. When growth of the offspring from the selected hens was examined, tom poults from the High glucose group were significantly heavier than the Low group at nearly every age examined (Table 8). There were no differences in performance of the hens poults from 4 to 12 wk of age. However, at 16 wk of age, the opposite response was noted as the hen poults from the High group weighed significantly less than those from the Low group.
Mean blood glucose concentration and hatchling body weight for one family are given in Table 2. The BW and blood glucose concentrations were significantly different among dams from both families, although the data from only one family are shown. When only the seven High and seven Low hens were examined, egg weights of the elevated glucose hens (High) were significantly heavier than those of the low group (Table 3). Mean blood plasma glucose concentrations of the High group was 285 mg/ dL when examined for the final 10 wk of the experiment, which was significantly greater than that for the Low group of hens (251 mg/dL). Eggshell conductance did not differ between the two glucose groups, but conductance constants (k) of the High group were significantly lower than that of the Low group. Hatchability of the High group was significantly lower by 13.5% than that of the Low (Table 4). Additionally, the Low group displayed lower embryonic mortality during Week 1 of incubation as well as during pipping. Mortality did not differ during the fourth week of incubation. The mean BW of High glucose hatchlings was nearly 12 g more than that of the Low group. Similarly, the High glucose group had heavier hearts, livers, and pipping muscles than did the Low group (Table 5), but only the pipping muscles were larger when compared relative to the BW. Organ glycogen concentrations were significantly greater among High glucose poults than Low, but pipping TABLE 5. Organ weights of hatched poults selected for high or low blood glucose concentrations Organ Blood glucose1
Body (g)
Heart (mg)
Liver (mg)
Muscle (mg)
High Low x ± SEM P
64.2a 52.8b 58.2 ± 0.19 0.0001
401a (0.62)2 328b (0.62) 363 ± 1.3 0.0001
1785a (2.98) 1554b (2.96) 1664 ± 7.8 0.0001
441a (0.68a) 293b (0.54b) 364 ± 1.6 0.0001
a,b Columnar means followed by a different superscript differ significantly. 1 High = poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations. 2 Means in parentheses are the values relative to BW.
DISCUSSION In contrast to our hypothesis, breeder hens producing heavier poults with elevated blood glucose concentrations displayed lower hatchability than breeder hens whose progeny hatched with lower weights and glucose concentrations. Embryonic growth is a highly conserved trait across avian species (Ricklefs and Starcks, 1998). It might be expected that within a species or a subpopulation within a species, the same conservation would be observed. In the present study, breeder hens selected to represent a population of commercial turkeys exhibited significant differences in hatchling BW and blood glucose concentrations. Furthermore, when the entire subpopulation was examined, it was clear that among commercial turkey breeder hens a highly significant correlation existed between hatchling BW and blood glucose concentrations. These observations were also related directly to the weight of the egg in which the embryo was growing. It may be concluded from these observations that within the limits of genetic diversity, embryonic growth may not be conserved in all individuals following selection for commercial traits. A diversity exists in flocks of breeding turkeys that allows adaptation to egg size, length of the incubation period, and eggshell conductance (Ar and Rahn, 1978). Because these variables are interdependent, it may be inferred that some embryos; perhaps at the extremes of the normal distribution for BW, egg weight, or metabolic
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a,b Columnar means followed by a different superscript differ significantly. 1 High = poults hatching with elevated blood glucose concentrations: Low = poults hatching with normal blood glucose concentrations.
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EMBRYO SURVIVAL TABLE 7. Plasma organic acid concentrations (µg/mL) of hatched poults selected for high or low blood glucose concentrations Organic acid 1
Blood glucose
Pyruvate
α-ketoglutarate b
High Low x ± SEM P
1.11 1.48a 1.30 ± 0.02 0.006
Lactate
1.47 1.34 1.41 ± 0.03 NS
Butyrate
Urate
b
1.48 1.41 1.41 ± 0.03 NS
9.1b 11.3a 10.2 ± .2 0.05
49.5 58.7a 54.2 ± 0.6 0.005
a,b
Columnar means followed by a different superscript differ significantly. High = poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations. 1
The heavier embryos were growing in heavier eggs with the same gas conductance properties as the smaller eggs; thus, the conductance constants of such eggs were depressed. The vital gas required for the larger tissue mass was not available, and so rapidly growing embryos merely maintained heart and liver tissues but could grow muscle tissue while hatching. The large amount of glycogen available in heart and liver tissue may indicate an inability to metabolize those stores or, alternately, an increased ability to synthesize glycogen to maintain heart and liver function. Because of the decreased plasma concentrations of organic acids in the heavier poults, it is speculated that the gluconeogenetic process in such embryos may be producing less glucose than is needed. Although the blood glucose concentrations were elevated, the depressed organic acid concentrations in the blood indicate that metabolites are being recycled very rapidly, thus not allowing accumulation in the blood but providing it rapidly for heart and liver metabolism (Christensen et al., 1999). The results of the current study indicate that heavier poults at hatching with elevated plasma glucose levels may be related to nonviable embryos. The viability declines because of an inability of such embryos to maintain organ growth and function through the hatching process. The increased hatchling BW in toms persisted through 22 wk of age. A different rate of growth was observed among hatchling hens, as High BW birds weighed less at 16 wk.
TABLE 8. Body weights (g) of poults hatching with high or low blood glucose concentrations Weeks of age Sex
Glucose1
4
8
12
16
22
Toms
High Low x ± SEM P N High Low x ± SEM P N
872 836 853 ± 15 NS 90 750 714 731 ± 13 0.10 97
4,200a 3,879b 4,018 ± 47 0.003 83 3,289 3,172 3,226 ± 48 NS 89
9,355a 8,737b 8,996 ± 79 0.04 67 6,983 6,895 6,929 ± 60 NS 75
14,469 14,155 14,280 ± 120 0.09 60 9,442b 10,177a 9,945 ± 85 0.01 38
20,838a 19,477b 19,991 ± 150 0.007 45 — — — — —
Hens
a,b
Columnar means followed by a different superscript differ significantly. High = poults hatching with elevated blood glucose concentrations; Low = poults hatching with normal blood glucose concentrations. 1
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rate; may be exposed to extensive periods of hypoxia and life-threatening situations at the end of the incubation period (Freeman, 1965). When we select for increased growth or egg weight, the relationship among egg weight, eggshell conductance, and the length of the incubation period may be magnified to produce perilous conditions for poult survival. Because eggshell conductance does not change during incubation, poults in larger eggs need greater eggshell conductance or longer incubation periods to survive. The well-known negative correlation between hatchability and selection for increased growth (Nestor and Noble, 1995) may be related to metabolism late in incubation. Physiological traits measured in the current study may give useful insight into the causes of decreased hatchability in growth-selected lines of turkeys. Those individuals hatching with elevated blood glucose concentrations have siblings that display lower survival rates at Week 1 of incubation as well as during pipping. The growth of pipping muscle relative to the BW was also enhanced. The rapidly growing sibling hatchlings also have elevated concentrations of glycogen in the heart and liver but not in the muscle. These observations were accompanied by depressed plasma α-ketoglutarate, β-hydroxybutyrate, and urate concentrations. These observations taken together show clearly that the negative relationship between growth and embryonic survival is related to the energy metabolism of the rapidly growing embryo.
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REFERENCES
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Ar, A., and H. Rahn, 1978. Interdependence of gas conductance, incubation length, and weight of the avian egg. Pages 227−236 in: Respiratory Function in Birds, Adult and Embryonic. J. Piper, ed. Springer Verlag, New York, NY. Christensen, V. L., W. E. Donaldson, and K. E. Nestor, 1993. Hatchability and embryonic metabolism in turkey lines selected for egg production or growth. Poultry Sci. 72:829−838. Christensen, V. L., W. E. Donaldson, and K. E. Nestor, 1999. Eggshell conductance constants and length of the incubation period affect survival of lines of turkey embryos. Poultry Sci. 77 (Suppl. 1):78. (Abstr.). Christensen, V. L., and K. E. Nestor, 1994. Changes in functional qualities of turkey eggshells in strains selected for increased egg production and growth. Poultry Sci. 73:1458−1464. Donaldson, W. E., and V. L. Christensen, 1991. Dietary carbohydrate levels and glucose metabolism in turkey poults. Comp. Biochem. Physiol. 98A:347−350. Donaldson, W. E., and V. L. Christensen, 1994. Dietary carbohydrate effects on some organic acids and aspects of glucose
metabolism in turkey poults. Comp. Biochem. Physiol. 109A:423−430. Freeman, B. M., 1965. The importance of glycogen at the termination of the embryonic existence of Gallus domesticus. Comp. Biochem. Physiol. 14:217−222. Grimes, J. L., V. L. Christensen, S. Lerner, and J. Hillel, 1996. Effect of turkey sire selection within a family on subsequent poult growth. Poultry Sci. 75 (Suppl. 1):119. (Abstr.). Nestor, K. E., and D. O. Noble, 1995. Influence of selection for increased egg production, body weight, and shank width on egg composition and the relationship of the egg traits to hatchability. Poultry Sci. 74:427−433. Ricklefs, R. E., 1987. Comparative analysis of avian embryonic growth. J. Exp. Zool. (Suppl 1.) 1:309−323. Ricklefs, R. E., and M. J. Starcks, 1998. Embryonic growth and development. Pages 31−58 in: Avian Growth and Development. Oxford University Press, New York, NY. SAS Institute, 1998. SAS/STAT Guide for Personal Computers. Version 6 Edition. SAS Institute Inc., Cary, NC. Tullett, S. G., 1981. Theoretical and practical aspects of eggshell porosity. Turkeys 29:24−28.