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Genetic Variation of Neonatal Stress Response to Reduced Temperature Brooding in a Randombred Population of Chickens1
Genetic Variation of Neonatal Stress Response to Reduced Temperature Brooding in a Randombred Population of Chickens1 T. R. SCOTT and K. W. WASHBURN2 Department of Poultry Science, University of Georgia, Athens, Georgia 30602 (Received for publication August 26, 1985)
1986 Poultry Science 6 5 : 8 2 9 - 8 3 6 INTRODUCTION
Circulating levels of corticosteroids have been used to evaluate whether various environmental insults should be termed stressors and to what degree these stressors affect the homeostasis of an animal. Some of these stressors, and the degree to which they affect poultry, have received considerable attention (Freeman, 1971, 1976; Siegel, 1971, 1980). Among the known stressors that activate the nonspecific stress response in poultry is cold exposure, and its effectiveness in eliciting the release of corticosterone in poultry has been demonstrated (Brown and Nestor, 1973, 1974; El Halawani et al, 1973; Buckland et al, 1974; Nir et al, 1975; Nestor and Bacon, 1982). The studies of Brown and Nestor (1973, 1974) have shown that cold exposure can be used to assess individual variation in the adrenal cortical response of young turkeys and therefore, could be used in a selection program for
1 Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia. 2 To whom correspondence should be addressed.
high and low adrenal cortical responsiveness. Reports of variation in selected lines of chickens (Gross and Colmano, 1971; Edens and Siegel, 1975) and Japanese quail (Marks and Siegel, 1980; Satterlee et al, 1982) indicate that the magnitude of the response to other stressors can be increased with genetic selection. The present study is concerned with the neonatal response to cold stress and the genetic basis of this response. Neonatal chicks exhibit an apparent adrenal cortical response to adrenocorticotropic hormone (ACTH) administration (Wise and Frye, 1973; Freeman and Flack, 1981; Kallicharan, 1981). The relatively high levels of circulating corticosterone found in the embryo (Kallicharan and Hall, 1974; Siegel and Gould, 1976; Satterlee et al, 1980; Scott et al, 1981) suggest that this hormone may be very important in the adaptation of chicks to their new environment outside the egg. Also, Scott and Washburn (1985) have shown that brooding young broilers (selected for growth rate) at 26.7 C resulted in elevations of serum corticosterone concentrations primarily during the initial 1 to 2 days following placement in the cooler brooding environment. The objective of this study was to assess genetic variation associated with the adrenal
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ABSTRACT Chicks from a randombred population were assessed for genetic variation in 1-day body weight and serum corticosterone under two brooding temperatures (26.7 and 32.2 C). Brooding at 26.7 C resulted in lower 1-day body weights and higher corticosterone levels. Heritability estimates (h 2 ) for 1-day body weight were moderately high in both temperature groups but were low for gain in both temperatures. The h 2 for corticosterone level from chicks brooded at 26.7 C was moderate, whereas estimates for those brooded at 32.2 C were very low. Statistically significant (but low) negative phenotypic correlations were obtained for corticosterone with body weight and gain in both temperature groups. A significant negative genotypic correlation was observed between corticosterone and body weight for chicks brooded at 26.7 C but not for those brooded at 32.2 C. Corticosterone data from the highest and lowest responding sire families revealed a significant response group by temperature interaction. Chicks from the first of two additional hatches showed significant response group differences in serum corticosterone to the reduced brooding temperature. The neonatal serum corticosterone response to cold stress has a genetic basis; however, because there was a negative genotypic correlation between serum corticosterone and body weight, selection for increased corticosterone levels under reduced temperature brooding would likely result in decreased body size. (Key words: chickens, cold stress, genetics, corticosterone)
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SCOTT AND WASHBURN
cortical response of young chickens to a reduced brooding temperature. MATERIALS AND METHODS
Yij = H + sj + eij where /z is the common mean, sj is the effect of the i t n sire, and e;j is random error. The same model was employed to obtain sire components of variance from data generated on the basis of difference in the response of a sire family tested in the two temperature environments. The individual value of each chick brooded at 26.7 C was subtracted from the corresponding sire family mean for chicks brooded at 32.2 C. Analysis of this new data set, based on the calculated differences, provided an indication of the degree to which additional genetic variation was expressed in the reduced brooding temperature environment. The GLM procedure required use of the RANDOM statement and its Q. option in order to specify random effects and obtain mean squares. Additional use of MANOVA H = statement and its PRINTH and PRINTE options provided hypothesis and error sum of squarescross and product matrices, respectively. These statements and their corresponding options allowed for calculation of variance components for estimation of heritabilities and genetic correlations. Estimates of standard errors for both the heritabilities and genetic correlations were derived according to Becker (1975). Analysis of variance across the two brooding temperature groups was carried out using the following model: Yijk = M + si + tj + stjj + ejj k where pi is the common mean; sj is the effect of ith sire; t; is the effect of the j t n brooding temperature; stjj is the interaction of the it" sire and j t n temperature; ejjk is random error. Further analysis of serum corticosterone from selected high and low response families (HF, LF) was done using the following model: Yijkl = H + fi + fsij + tk + ftJk + fstijk + eijkl where pt is the common mean; fj is the effect of the i r " response group (HF or LF); fsjj is the
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Hatch 1. The Athens-Canadian randombred population (AC) described by Hess (1962) was used. This population has not been selected for growth since its development, and it routinely attains a weight of 700 to 800 g at 7 weeks of age. Twelve-hundred twenty progeny from 26 sire families were distributed within sire family into two brooding temperature environments. Brooding temperatures used were 26.7 C (reduced) and 32.2 C (control). These temperatures were maintained under the heated area of Petersime batteries in which the chicks were housed. Batteries were placed in two light-and temperature-controlled environmental chambers that were allowed to cycle from 12 to 24 C and 24 to 35 C, respectively. Chicks were provided the University of Georgia unmedicated starter diet and water ad libitum in the heated area under the brooder hover. Prior to placement, all chicks were individually indentified by wingbanding. Hatching weights were recorded for later calculation of body weight gain (body weight at samplinghatch weight). After placement, chicks remained in the brooding temperature environments for a full 24 hr. Upon sampling, all chicks were weighed and blood was collected by decapitation into 13- X 100-mm culture tubes. Blood was allowed to clot, and serum was removed, stored frozen, and later assayed for corticosterone using a radioimmunoassay procedure. The radioimmunoassay procedure used was a modification of the Endocrine Sciences radioimmunoassay for corticosterone (Endocrine Sciences, 1972) described by Satterlee et al. (1980). Briefly, the assay involved consecutive extractions of .1-ml serum samples with 1 ml each of 2,2,4-trimethylpentane and dichloromethane. The 2,2,4-trimethlpentane extracts were discarded. Dichloromethane extracts were saved, dried under forced air in 16- X 100-mm culture tubes, and reconstituted with 1 ml of assay buffer. A phosphate buffered saline (pH 7.4) with . 1 % gelatin was used instead of a borate buffer. Reconstituted samples were then carried through the corticosterone radioimmunoassay procedure of Satterlee et al. (1980). The average intraassay variation was 14.3%, and an interassay variation of 23.9% was determined. Extraction efficiency for corticosterone was found to be 88.2%.
Data were analyzed using the General Linear Models (GLM) procedure of the Statistical Analysis System (SAS 79.3) (Barr et al, 1976) computer program. Heritabilities and genetic correlations within each brooding temperature were calculated based on half-sib analysis using the sire components of variance of the following model:
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GENETIC VARIATION TO REDUCED TEMPERATURE BROODING
Yjj = n + f; + qj !J where fi is the common mean; fj is the effect of the i t n response group (HF or LF); ejj is the random error.
RESULTS AND DISCUSSION
Response to Brooding Temperatures (Hatch 1). Brooding at 26.7 C significantly reduced 1-day body weights and gains but significantly elevated serum corticosterone levels of 1-day-old AC chicks (Table 1). Brooding at lower than recommended temperatures has previously been shown to reduce body weights and gains of young broiler-type chickens (Hutson et al, I960; Renwick and Washburn, 1982; Renwick et al, 1985). Although chicks brooded at 26.7 C lost significantly more weight than those brooded at 32.2 C, both groups lost weight during the first 24 hr after placement in both brooding environments. Extremely large coefficients of variation were observed for weight gain in both brooding environments (225 and 819% for the 26.7- and 32.2-C groups); thus, weight gain the first day after hatching is not a satisfactory indicator of genetic variation in growth in this population. Heritability Estimates (Hatch 1). Heritability estimates (h 2 ) for body weight and gain calculated on the basis of actual individual bird values and based on the difference of individuals brooded at 26.7 C from their corresponding sire family mean at 32.2 C are presented in Table 2. The h 2 estimates for body weight were moderately high and similar at 26.7- and 32.2-C brooding temperatures. Since there is a very high correlation between chick weight and egg weight, these estimates for 1-day body weights are probably a reflection of genetic variation in egg size of the dam. The h 2 estimate based on deviation was very low, indicating that additional genetic variation was not expressed when neonates were brooded at 26.7 for 1 day.
TABLE 1. The effect of brooding temperature on body weight, gain, and serum corticosterone of the AthensCanadian Randombred chicks at 24 hours postplacement (Hatch 1) Brooding temperature
Body weight
Gain1
(C) 26.7 32.2
(Mg/100 ml)
(g) 29.0 3 t . 2 29.9 : .2a
b
-1.2 + . l b -0.3 ± . l a
ab Means within a variable with different superscripts are significantly different (P<.01). 1
Gain = 0 to 1-day weight gain.
2
Mean ± SEM.
Serum corticosterone
3.93 : .09 a b 3.25 : .08
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effect of the jth sire within the i t n response group; t]j is the effect of the k t n brooding temperature; ftjk is the interaction of the i t n response group and k t n brooding temperature; fstjjk is the interaction of the k t n brooding temperature and the j t r l sire within the i 1 " response group; ejjki is random error. Hatches 2 and 3. Chicks were obtained from the six sire families exhibiting the highest and six sire families with the lowest serum corticosterone concentrations in response to the 26.7-C brooding temperature in Hatch 1. Hatches 2 and 3 produced 183 and 659 chicks, respectively, which were distributed within HF-LF group and sire family into batteries with the brooding temperature continuously maintained at 26.7 C. Posthatch handling and placement procedures were the same as outlined for Hatch 1, except that in Hatch 3, 120 chicks from both HF and LF response groups were sampled after 24 hr. On Day 7, 80 and 84 additional chicks were sampled from the HF and LF response groups, respectively, and 71 and 86 chicks were sampled from the respective response groups on Day 14. The handling of blood samples in both Hatch 2 and 3 in preparation for the corticosterone radioimmunoassay procedure was the same as that discussed for Hatch 1. Data from both hatches were analyzed by analysis of variance using the following model:
SCOTT AND WASHBURN
832
TABLE 2. The effect of brooding temperature on half-sib heritabilities and additive genetic variance estimated by sib-analysis for an Athens-Canadian Randombred population (Hatch 1) Brooding temperature (C) 26_7
32£
Variable1
h2 ± SE2
%CT| 3
h2 ± SE
% O\
Body weight
.30 + .13 .02 ± .06 .04 + .07 .06 + .08 .22 ± .10 .28 ±.12
7.6 .5 .9 1.5 5.5 7.1
.28 ± .13
7.0
.08 ± .08
1.8
.05 ± .07 .05 ± .07
1.1 1.1
A D Gain A D Corticosterone A D
As pointed out previously, weight gain over this short period of time was not considered an adequate measure of genetic variation in growth for these chicks. The h 2 estimates for weight gain were low in both brooding environments in contrast to the larger estimates reported by Renwick et al. (1985) for 1- and 2-week weight gains of AC chicks brooded at 26.7 and 32.2 C. At this age and over this short period of time, maternal effects and adjustment to the environment may have contributed more to the variance associated with weight gain than the genetic variation in this trait. Serum corticosterone levels of chicks brooded at 26.7 were significantly elevated compared to levels of chicks brooded at 32.2 C (Table 1). This increase is in agreement with other studies (such as Scott and Washburn, 1985), but the magnitude of the increase is less than expected from the stress studies of Wise and Frye (1973), Freeman and Flack (1981), and Killicharan (1981). The h of serum corticosterone levels of 1-day-old chicks maintained at 32.2 C was very low (Table 2). Brooding at 26.7 C increased expression of genetic variance associated with this trait, resulting in moderate h 2 estimates, which indicated that adrenal cortical responsiveness of neonates to a cooler brooding temperature has a genetic basis. This is in agreement with realized heritability estimates for adrenal cortical responsiveness based on circulating levels of corticosterone in older turkeys (Brown and Nestor, 1973, 1974) and
cholesterol in Japanese quail (Marks and Siegel, 1980). The percentage of total variation that was accounted for by the sire components of variance (%a|) was also greater for chicks brooded at 26.7 C, indicating increased genetic expression of the adrenal cortical response at that brooding temperature. Even more of the total variation could be accounted for by the additive genetic variance when calculated on the basis of deviations of individuals in 26.7 C from their sire family means in 32.2 C. Phenotypic and Genotypic Correlations (Hatch 1). There was a negative association of serum corticosterone with 1-day body weight and gain (Table 3). These phenotypic correlations were weak (—.17 to —.27) but statistically significant (P«.001) and similar in magnitude for the two temperature groups. Other studies (Brown and Nestor, 1973; 1974; Nestor and Bacon, 1982; Satterlee et al, 1982) have noted inverse relationships between body weight and adrenal cortical responsiveness in older poultry. Nestor and Bacon (1982) reported a range for phenotypic correlations between body weight at stressing and plasma corticosterone concentrations similar to those observed in this study. Genotypic correlations in the reduced brooding temperature group were higher than those of the 32.2-C group and higher than the associated phenotypic correlations (Table 3). The significant genetic correlation between body weight and serum corticosterone in cooler temperature brooded chicks indicates that selection for increased serum corticosterone
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'Body weight = 1-Day body weight; gain = 0 to 1-day weight gain; corticosterone = serum corticosterone; A = Actual h 2 estimates determined from variance components; D = Difference h2 estimates determined from variance components obtained by comparing individuals at 26.7 to their corresponding sire family mean at 32.2 C. 2 2 h ± SE = Heritability ± standard error of the estimate. 3 %a\ = Percentage of total variation accounted for by the sire variance component.
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GENETIC VARIATION TO REDUCED TEMPERATURE BROODING TABLE 3. The effect of brooding temperature on phenotypic and genotypic correlations within an Athens-Canadian Randombred population (Hatch 1) Brooding temperature (C) 32.2
26.7 Variables1 Cort.:BW Cort.:Gain 1
x t x2 -.20*** -.17***
r
g!g 2 ±SE
- . 6 4 ± .06* - 1 . 3 2 ± .75
rx
x
i a 1 Q* * *
-.27***
r
g. g2 ± SE
- . 4 5 ± .23 - . 2 8 ± .66
BW = 1-day body weight; gain = 0 to 1-day weight gain; Cort. = serum corticosterone.
2r
x , x 2 = Phenotypic correlation; r gjg 2 = genotypic correlation.
***P<.001 means that the phenotypic correlation is significantly different from zero.
levels under cold temperature brooding would likely result in a decreased body size. Brown and Nestor (1973, 1974) emphasized the reduced productivity of their high adrenal response line of turkeys, which made selection for high adrenal reponsiveness appear undesirable. Our study suggests that such selection in neonatal chicks might be beneficial to the adaptability of young chicks to cold temperatures, but it would also be detrimental to early growth. Genotype-Environment Interaction (Hatch 1). By analyzing data across the two brooding temperature environments, it was possible to test for any interaction between sire families and the environments (Table 4). Significant main effects were obtained for all three variables, but only the sire by temperature interaction for serum corticosterone approached significance. Based on these analyses, it was evident that the ranking and degree of difference among sire families was very similar in either
brooding environment for body weight and gain. However, for serum corticosterone, there was a difference in response among sire families between the two environments. To further test the apparent difference in adrenal responsiveness of sire families brooded at 26.7 C, serum corticosterone concentrations of the six families with the highest and six families with the lowest response to reduced temperature brooding were analyzed for significance of response group by temperature interaction. The resulting analysis revealed a significant interaction that reflected the divergence of response of HF and LF groups. This interaction was primarily a result of the HF group exhibiting an adrenal cortical response to the cooler temperature, whereas those members of the LF groups showed essentially no response to the cooler brooding temperature (Fig. 1). Such base population differences in adrenal cortical responsiveness have been the basis for selection programs with poultry in the past
TABLE 4. Analyses of variance for measured variables^ in an Athens-Canadian Randombred population (Hatch 1) Level of significance Variation
d.f'
Body weight
Gain
Corticosterone
Sire (S) Temperature (T) SXT
25 1 25
.0001 .0001 .9755
0001 .0001 .9841
.0012 .0001 .1133
1 2
Body weight = 1-day body weight; gain = 0 to 1-day weight gain; corticosterone = serum corticosterone.
Body weight: error - 1093, total - 1144; gain: error - 1088, total = 1139; corticosterone: error = 1025, total = 1076.
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*P <.001 means that the genotypic correlation is significantly different from zero.
SCOTT. AND WASHBURN
834
(Gross and Colmano, 1971; Brown and Nestor, 1973, 1974; Edensand Siegel, 1975; Marks and Siegel, 1980; Satterlee etal, 1982).
HF LF
Response to Reduced Brooding Temperature (Hatches 2 and 3).
O o
Ui
2
o
26.7 BROODING
TEMPERATURE (C )
FIG. 1. Serum corticosterone responses at 24 hours postplacement of high (HF) and low (LF) response families from Hatch 1 that were brooded at two temperatures.
TABLE 5. The effect of brooding at 26.7 C on body weight, gain, packed red blood cell volume, and serum corticosterone of high and low serum corticosterone families from an Athens-Canadian Randombred population at 24 hours postplacment (Hatch 2) Family group
High Low
Body weight
1
32.6 + .4 33.0 + .4 a
Gain2
a
(g)
Serum corticosterone
PCV2
(Mg/100 ml)
(%) a
-2.5 ± .l -2.3 ± . l a
a
37.7 ± .5 36.7 + .4 a
a' bMeans within a variable with different superscripts are significantly different (P<.05). 'Mean ± SEM. 2 Gain = 0 to 1-day weight gain: PCV = packed cell volume.
5.91 ± .18 a 5.24 ± . 1 8 b
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to o
Although the LF group in Hatch 2 had numerically greater body weights and gains at 1-day of age, the differences were not statistically significant (Table 5). Packed cell volume (PCV) was measured as a result of concern that sire family differences observed for serum corticosterone in Hatch 1 were due to sire family differences in PCV, because Washburn (1967) had reported moderate to high heritability estimates for PCV in an AC population. However, differences in serum corticosterone concentrations between the HF and LF groups were not due to blood concentration of circulating corticosterone (Table 5). None of the variables measured in Hatch 3 had significant HF-LF group effects at any of the ages considered in that study (Table 6). However, the observed trends do agree with the results in Hatches 1 and 2. Throughout, the LF group had numerically heavier body weights and gains, and the HF group, likewise, had numerically greater concentrations of serum corticosterone.
GENETIC VARIATION TO REDUCED TEMPERATURE BROODING
83 5
TABLE 6. The effect of brooding at 26.7 C on body weight, gain, and serum corticosterone of high and low serum corticosterone families from an Athens-Canadian Randombred population at 1, 7. and 14 days postplacement (Hatch 3) Body weight1
Family group
-
Gain1 '•
'•
Serum corticosterone 1 (Mg/100 ml)
w
Day 1 High Low
33.2 ± . 3 3 32.6 ± .3
High Low
58.5 ± 1.0 60.2 ± 1.0
- 2 . 0 ± .1 - 2 . 1 ± .1
5.02 ± .19 4.71 + .16
Day 7 4.19 ± .20 4.12 ± .20
Day 14 114.8 ± 2.2 117.4 ± 1.9
High Low
79.2 + 2.1 80.5 ± 1.9
2.39 ± .20 2.27 ± .17
'Means within the same variable and day are not significantly different (P>.05). 2
Gain = 0 to 1, 0 to 7, and 0 to 14-day weight gain.
3
Mean ± SEM.
ACKNOWLEDGMENTS
The authors would like to express appreciation to H. L. Marks and the United States Department of Agriculture for their contribution in maintaining the Athens-Canadian Randombred population and for assisting us in our use of this population. We would also like to thank Samuel Bowen, Mark Mayfield, Rachel Peavey, and Pamela Stewart for their technical assistance as well as Sherry Davenport for typing the manuscript. REFERENCES Barr, A. J., J. H. Goodnight, J. P. Sail, and T. T. Helwig, 1976. A User's Guide to SAS 76. SAS Inst., Cary, NC. Becker, W. A., 1975. Manual of Quantitative Genetics. 3rd ed. Washington State Univ., Pullman, WA. Brown, K. I., and K. E. Nestor, 1973. Some physiological responses of turkeys selected for high and low adrenal response to cold stress. Poultry Sci. 52:1948-1954. Brown, K. I., and K. E. Nestor, 1974. Implications of selection for high and low adrenal response to stress. Poultry Sci. 53:1297-1306. Buckland, R. B., K. Balgrave, and P. C. Lague, 1974. Competitive protein-binding assay for corticoids in the peripheral plasma of the immature chicken. Poultry Sci. 53:241-245. Edens, F. W., and H. S. Siegel, 1975. Adrenal responses in high and low ACTH response lines of chickens during acute heat stress. Gen. Comp. Endocrinol. 25:64-73. El Halawani, M. E., P. E. Waibel, J. R. Appel, and A. L. Good, 1973. Effects of temperature stress on
catecholamines and corticosterone of male turkeys. Am. J. Physiol. 224:384-388. Endocrine Science, 1972. Plasma corticosterone radioimmunoassay procedure, antiserum B21-42. Tarzana, CA. Freeman, B. M., 1971. Stress and the domestic fowl: a physiological appraisal. World's Poult. Sci. J. 27:263-275. Freeman, B. M., 1976. Stress and the domestic fowl: a physiological reappraisal. World's Poult. Sci. J. 32:249-256. Freeman, B. M., and I. H. Flack, 1981. The sensitivity of the newly hatched fowl to corticotrophin, Comp. Biochem. Physiol. 70A:257-259. Gross, W. B., and G. Colmano, 1971. Effect of infectious agents on chickens selected for plasma corticosterone response to social stress. Poultry Sci. 50:1213-1217. Hess, C. W., 1962. Randombred populations of the southern regional poultry breeding project. World's Poult. Sci. J. 18:147-152. Hutson, H. C , W. F. Kreuger, C. B. Ryan, and J. H. Quisenberry, 1960. Brooder management studies. Poultry Sci. 39:1261. (Abstr.) Kallicharan, R., 1981. The influence of exogenous ACTH on the levels of corticosterone on Cortisol in the plasma of young chicks (Gallus domesticus). Gen. Comp. Endocrinol. 44:249-251. Kallicharan, R., and B. K. Hall, 1974. A developmental study of the levels of progesterone, corticosterone, Cortisol, and cortisone circulating in plasma of chick embryos. Gen. Comp. Endocrinol. 24:364-372. Marks, H. L., and H. S. Siegel, 1980. Divergent selection of Japanese quail for the plasma cholesterol response to ACTH. Poultry Sci. 59:1700-1705. Nestor, K. E., and W. L. Bacon, 1982. Results of cold stress in strains of turkey selected for growth rate and egg production. Poultry Sci. 61:652-654.
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23.1 + 1.0 24.3 ± .9
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SCOTT AND WASHBURN Gildersleeve, 1981. Circulating levels of corticosterone in the serum of developing chick embryos and newly hatched chicks. Poultry Sci. 60:13141320. Scott, T. R., and K. W. Washburn, 1985. Evaluation of growth, hormonal and hematological responses of neonatal chickens to reduced temperature brooding. Poultry Sci. 64:777-784. Siegel, H. S., 1971. Adrenals, stress and the environment. World's Poult. Sci. J. 27:327-349. Siegel, H. S., 1980. Physiology stress in birds. Bioscience 30:529-534. Siegel, H. S., and N. R. Gould, 1976. Chick embryonic plasma proteins and binding capacity for corticosterone. Dev. Biol. 50:510-516. Washburn, K. W., 1967. Heritability of packed red blood cell volume in the domestic fowl. Poultry Sci. 46:1025. Wise, P. M., and B. E. Frye, 1973. Functional development of the hypothalamo-hypophyseal-adrenal cortex axis in the chick embryo, Gallus domesticus. J. Exp. Zool. 185:277-292.
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Nir, I., D. Yam, and M. Perek, 1975. Effects of stress on the corticosterone content of the blood plasma and adrenal gland of intact and bursectomized Gallus domesticus. Poultry Sci. 54:21012110. Renwick, G. M., and K. W. Washburn, 1982. Adaptation of chickens to cool temperature brooding. Poultry Sci. 61:1279-1289. Renwick, G. M., K. W. Washburn, G. M. Lanza, 1985. Genetic variability in growth response of chicks to cold brooding temperatures. Poultry Sci. 64:785-788. Satterlee, D. G., R. B. Abdullah, R. P. Gildersleeve, 1980. Plasma corticosterone radioimmunoassay and levels in the neonate chick. Poultry Sci. 59:900-905. Satterlee, D. G., R. E. Truax, L. A. Jacobs-Perry, W. A. Johnson, 1982. Genetic selection of wild-type Japanese quail for high and low plasma corticosterone response to albino quail intrusion. Poultry Sci. 61:1394-1395. Scott, T. R., W. A. Johnson, D. G. Satterlee, R. P.
1986 Poultry Science 6 5 : 8 2 9 - 8 3 6 INTRODUCTION
Circulating levels of corticosteroids have been used to evaluate whether various environmental insults should be termed stressors and to what degree these stressors affect the homeostasis of an animal. Some of these stressors, and the degree to which they affect poultry, have received considerable attention (Freeman, 1971, 1976; Siegel, 1971, 1980). Among the known stressors that activate the nonspecific stress response in poultry is cold exposure, and its effectiveness in eliciting the release of corticosterone in poultry has been demonstrated (Brown and Nestor, 1973, 1974; El Halawani et al, 1973; Buckland et al, 1974; Nir et al, 1975; Nestor and Bacon, 1982). The studies of Brown and Nestor (1973, 1974) have shown that cold exposure can be used to assess individual variation in the adrenal cortical response of young turkeys and therefore, could be used in a selection program for
1 Supported by State and Hatch funds allocated to the Georgia Agricultural Experiment Stations of the University of Georgia. 2 To whom correspondence should be addressed.
high and low adrenal cortical responsiveness. Reports of variation in selected lines of chickens (Gross and Colmano, 1971; Edens and Siegel, 1975) and Japanese quail (Marks and Siegel, 1980; Satterlee et al, 1982) indicate that the magnitude of the response to other stressors can be increased with genetic selection. The present study is concerned with the neonatal response to cold stress and the genetic basis of this response. Neonatal chicks exhibit an apparent adrenal cortical response to adrenocorticotropic hormone (ACTH) administration (Wise and Frye, 1973; Freeman and Flack, 1981; Kallicharan, 1981). The relatively high levels of circulating corticosterone found in the embryo (Kallicharan and Hall, 1974; Siegel and Gould, 1976; Satterlee et al, 1980; Scott et al, 1981) suggest that this hormone may be very important in the adaptation of chicks to their new environment outside the egg. Also, Scott and Washburn (1985) have shown that brooding young broilers (selected for growth rate) at 26.7 C resulted in elevations of serum corticosterone concentrations primarily during the initial 1 to 2 days following placement in the cooler brooding environment. The objective of this study was to assess genetic variation associated with the adrenal
829
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ABSTRACT Chicks from a randombred population were assessed for genetic variation in 1-day body weight and serum corticosterone under two brooding temperatures (26.7 and 32.2 C). Brooding at 26.7 C resulted in lower 1-day body weights and higher corticosterone levels. Heritability estimates (h 2 ) for 1-day body weight were moderately high in both temperature groups but were low for gain in both temperatures. The h 2 for corticosterone level from chicks brooded at 26.7 C was moderate, whereas estimates for those brooded at 32.2 C were very low. Statistically significant (but low) negative phenotypic correlations were obtained for corticosterone with body weight and gain in both temperature groups. A significant negative genotypic correlation was observed between corticosterone and body weight for chicks brooded at 26.7 C but not for those brooded at 32.2 C. Corticosterone data from the highest and lowest responding sire families revealed a significant response group by temperature interaction. Chicks from the first of two additional hatches showed significant response group differences in serum corticosterone to the reduced brooding temperature. The neonatal serum corticosterone response to cold stress has a genetic basis; however, because there was a negative genotypic correlation between serum corticosterone and body weight, selection for increased corticosterone levels under reduced temperature brooding would likely result in decreased body size. (Key words: chickens, cold stress, genetics, corticosterone)
830
SCOTT AND WASHBURN
cortical response of young chickens to a reduced brooding temperature. MATERIALS AND METHODS
Yij = H + sj + eij where /z is the common mean, sj is the effect of the i t n sire, and e;j is random error. The same model was employed to obtain sire components of variance from data generated on the basis of difference in the response of a sire family tested in the two temperature environments. The individual value of each chick brooded at 26.7 C was subtracted from the corresponding sire family mean for chicks brooded at 32.2 C. Analysis of this new data set, based on the calculated differences, provided an indication of the degree to which additional genetic variation was expressed in the reduced brooding temperature environment. The GLM procedure required use of the RANDOM statement and its Q. option in order to specify random effects and obtain mean squares. Additional use of MANOVA H = statement and its PRINTH and PRINTE options provided hypothesis and error sum of squarescross and product matrices, respectively. These statements and their corresponding options allowed for calculation of variance components for estimation of heritabilities and genetic correlations. Estimates of standard errors for both the heritabilities and genetic correlations were derived according to Becker (1975). Analysis of variance across the two brooding temperature groups was carried out using the following model: Yijk = M + si + tj + stjj + ejj k where pi is the common mean; sj is the effect of ith sire; t; is the effect of the j t n brooding temperature; stjj is the interaction of the it" sire and j t n temperature; ejjk is random error. Further analysis of serum corticosterone from selected high and low response families (HF, LF) was done using the following model: Yijkl = H + fi + fsij + tk + ftJk + fstijk + eijkl where pt is the common mean; fj is the effect of the i r " response group (HF or LF); fsjj is the
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Hatch 1. The Athens-Canadian randombred population (AC) described by Hess (1962) was used. This population has not been selected for growth since its development, and it routinely attains a weight of 700 to 800 g at 7 weeks of age. Twelve-hundred twenty progeny from 26 sire families were distributed within sire family into two brooding temperature environments. Brooding temperatures used were 26.7 C (reduced) and 32.2 C (control). These temperatures were maintained under the heated area of Petersime batteries in which the chicks were housed. Batteries were placed in two light-and temperature-controlled environmental chambers that were allowed to cycle from 12 to 24 C and 24 to 35 C, respectively. Chicks were provided the University of Georgia unmedicated starter diet and water ad libitum in the heated area under the brooder hover. Prior to placement, all chicks were individually indentified by wingbanding. Hatching weights were recorded for later calculation of body weight gain (body weight at samplinghatch weight). After placement, chicks remained in the brooding temperature environments for a full 24 hr. Upon sampling, all chicks were weighed and blood was collected by decapitation into 13- X 100-mm culture tubes. Blood was allowed to clot, and serum was removed, stored frozen, and later assayed for corticosterone using a radioimmunoassay procedure. The radioimmunoassay procedure used was a modification of the Endocrine Sciences radioimmunoassay for corticosterone (Endocrine Sciences, 1972) described by Satterlee et al. (1980). Briefly, the assay involved consecutive extractions of .1-ml serum samples with 1 ml each of 2,2,4-trimethylpentane and dichloromethane. The 2,2,4-trimethlpentane extracts were discarded. Dichloromethane extracts were saved, dried under forced air in 16- X 100-mm culture tubes, and reconstituted with 1 ml of assay buffer. A phosphate buffered saline (pH 7.4) with . 1 % gelatin was used instead of a borate buffer. Reconstituted samples were then carried through the corticosterone radioimmunoassay procedure of Satterlee et al. (1980). The average intraassay variation was 14.3%, and an interassay variation of 23.9% was determined. Extraction efficiency for corticosterone was found to be 88.2%.
Data were analyzed using the General Linear Models (GLM) procedure of the Statistical Analysis System (SAS 79.3) (Barr et al, 1976) computer program. Heritabilities and genetic correlations within each brooding temperature were calculated based on half-sib analysis using the sire components of variance of the following model:
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GENETIC VARIATION TO REDUCED TEMPERATURE BROODING
Yjj = n + f; + qj !J where fi is the common mean; fj is the effect of the i t n response group (HF or LF); ejj is the random error.
RESULTS AND DISCUSSION
Response to Brooding Temperatures (Hatch 1). Brooding at 26.7 C significantly reduced 1-day body weights and gains but significantly elevated serum corticosterone levels of 1-day-old AC chicks (Table 1). Brooding at lower than recommended temperatures has previously been shown to reduce body weights and gains of young broiler-type chickens (Hutson et al, I960; Renwick and Washburn, 1982; Renwick et al, 1985). Although chicks brooded at 26.7 C lost significantly more weight than those brooded at 32.2 C, both groups lost weight during the first 24 hr after placement in both brooding environments. Extremely large coefficients of variation were observed for weight gain in both brooding environments (225 and 819% for the 26.7- and 32.2-C groups); thus, weight gain the first day after hatching is not a satisfactory indicator of genetic variation in growth in this population. Heritability Estimates (Hatch 1). Heritability estimates (h 2 ) for body weight and gain calculated on the basis of actual individual bird values and based on the difference of individuals brooded at 26.7 C from their corresponding sire family mean at 32.2 C are presented in Table 2. The h 2 estimates for body weight were moderately high and similar at 26.7- and 32.2-C brooding temperatures. Since there is a very high correlation between chick weight and egg weight, these estimates for 1-day body weights are probably a reflection of genetic variation in egg size of the dam. The h 2 estimate based on deviation was very low, indicating that additional genetic variation was not expressed when neonates were brooded at 26.7 for 1 day.
TABLE 1. The effect of brooding temperature on body weight, gain, and serum corticosterone of the AthensCanadian Randombred chicks at 24 hours postplacement (Hatch 1) Brooding temperature
Body weight
Gain1
(C) 26.7 32.2
(Mg/100 ml)
(g) 29.0 3 t . 2 29.9 : .2a
b
-1.2 + . l b -0.3 ± . l a
ab Means within a variable with different superscripts are significantly different (P<.01). 1
Gain = 0 to 1-day weight gain.
2
Mean ± SEM.
Serum corticosterone
3.93 : .09 a b 3.25 : .08
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effect of the jth sire within the i t n response group; t]j is the effect of the k t n brooding temperature; ftjk is the interaction of the i t n response group and k t n brooding temperature; fstjjk is the interaction of the k t n brooding temperature and the j t r l sire within the i 1 " response group; ejjki is random error. Hatches 2 and 3. Chicks were obtained from the six sire families exhibiting the highest and six sire families with the lowest serum corticosterone concentrations in response to the 26.7-C brooding temperature in Hatch 1. Hatches 2 and 3 produced 183 and 659 chicks, respectively, which were distributed within HF-LF group and sire family into batteries with the brooding temperature continuously maintained at 26.7 C. Posthatch handling and placement procedures were the same as outlined for Hatch 1, except that in Hatch 3, 120 chicks from both HF and LF response groups were sampled after 24 hr. On Day 7, 80 and 84 additional chicks were sampled from the HF and LF response groups, respectively, and 71 and 86 chicks were sampled from the respective response groups on Day 14. The handling of blood samples in both Hatch 2 and 3 in preparation for the corticosterone radioimmunoassay procedure was the same as that discussed for Hatch 1. Data from both hatches were analyzed by analysis of variance using the following model:
SCOTT AND WASHBURN
832
TABLE 2. The effect of brooding temperature on half-sib heritabilities and additive genetic variance estimated by sib-analysis for an Athens-Canadian Randombred population (Hatch 1) Brooding temperature (C) 26_7
32£
Variable1
h2 ± SE2
%CT| 3
h2 ± SE
% O\
Body weight
.30 + .13 .02 ± .06 .04 + .07 .06 + .08 .22 ± .10 .28 ±.12
7.6 .5 .9 1.5 5.5 7.1
.28 ± .13
7.0
.08 ± .08
1.8
.05 ± .07 .05 ± .07
1.1 1.1
A D Gain A D Corticosterone A D
As pointed out previously, weight gain over this short period of time was not considered an adequate measure of genetic variation in growth for these chicks. The h 2 estimates for weight gain were low in both brooding environments in contrast to the larger estimates reported by Renwick et al. (1985) for 1- and 2-week weight gains of AC chicks brooded at 26.7 and 32.2 C. At this age and over this short period of time, maternal effects and adjustment to the environment may have contributed more to the variance associated with weight gain than the genetic variation in this trait. Serum corticosterone levels of chicks brooded at 26.7 were significantly elevated compared to levels of chicks brooded at 32.2 C (Table 1). This increase is in agreement with other studies (such as Scott and Washburn, 1985), but the magnitude of the increase is less than expected from the stress studies of Wise and Frye (1973), Freeman and Flack (1981), and Killicharan (1981). The h of serum corticosterone levels of 1-day-old chicks maintained at 32.2 C was very low (Table 2). Brooding at 26.7 C increased expression of genetic variance associated with this trait, resulting in moderate h 2 estimates, which indicated that adrenal cortical responsiveness of neonates to a cooler brooding temperature has a genetic basis. This is in agreement with realized heritability estimates for adrenal cortical responsiveness based on circulating levels of corticosterone in older turkeys (Brown and Nestor, 1973, 1974) and
cholesterol in Japanese quail (Marks and Siegel, 1980). The percentage of total variation that was accounted for by the sire components of variance (%a|) was also greater for chicks brooded at 26.7 C, indicating increased genetic expression of the adrenal cortical response at that brooding temperature. Even more of the total variation could be accounted for by the additive genetic variance when calculated on the basis of deviations of individuals in 26.7 C from their sire family means in 32.2 C. Phenotypic and Genotypic Correlations (Hatch 1). There was a negative association of serum corticosterone with 1-day body weight and gain (Table 3). These phenotypic correlations were weak (—.17 to —.27) but statistically significant (P«.001) and similar in magnitude for the two temperature groups. Other studies (Brown and Nestor, 1973; 1974; Nestor and Bacon, 1982; Satterlee et al, 1982) have noted inverse relationships between body weight and adrenal cortical responsiveness in older poultry. Nestor and Bacon (1982) reported a range for phenotypic correlations between body weight at stressing and plasma corticosterone concentrations similar to those observed in this study. Genotypic correlations in the reduced brooding temperature group were higher than those of the 32.2-C group and higher than the associated phenotypic correlations (Table 3). The significant genetic correlation between body weight and serum corticosterone in cooler temperature brooded chicks indicates that selection for increased serum corticosterone
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'Body weight = 1-Day body weight; gain = 0 to 1-day weight gain; corticosterone = serum corticosterone; A = Actual h 2 estimates determined from variance components; D = Difference h2 estimates determined from variance components obtained by comparing individuals at 26.7 to their corresponding sire family mean at 32.2 C. 2 2 h ± SE = Heritability ± standard error of the estimate. 3 %a\ = Percentage of total variation accounted for by the sire variance component.
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GENETIC VARIATION TO REDUCED TEMPERATURE BROODING TABLE 3. The effect of brooding temperature on phenotypic and genotypic correlations within an Athens-Canadian Randombred population (Hatch 1) Brooding temperature (C) 32.2
26.7 Variables1 Cort.:BW Cort.:Gain 1
x t x2 -.20*** -.17***
r
g!g 2 ±SE
- . 6 4 ± .06* - 1 . 3 2 ± .75
rx
x
i a 1 Q* * *
-.27***
r
g. g2 ± SE
- . 4 5 ± .23 - . 2 8 ± .66
BW = 1-day body weight; gain = 0 to 1-day weight gain; Cort. = serum corticosterone.
2r
x , x 2 = Phenotypic correlation; r gjg 2 = genotypic correlation.
***P<.001 means that the phenotypic correlation is significantly different from zero.
levels under cold temperature brooding would likely result in a decreased body size. Brown and Nestor (1973, 1974) emphasized the reduced productivity of their high adrenal response line of turkeys, which made selection for high adrenal reponsiveness appear undesirable. Our study suggests that such selection in neonatal chicks might be beneficial to the adaptability of young chicks to cold temperatures, but it would also be detrimental to early growth. Genotype-Environment Interaction (Hatch 1). By analyzing data across the two brooding temperature environments, it was possible to test for any interaction between sire families and the environments (Table 4). Significant main effects were obtained for all three variables, but only the sire by temperature interaction for serum corticosterone approached significance. Based on these analyses, it was evident that the ranking and degree of difference among sire families was very similar in either
brooding environment for body weight and gain. However, for serum corticosterone, there was a difference in response among sire families between the two environments. To further test the apparent difference in adrenal responsiveness of sire families brooded at 26.7 C, serum corticosterone concentrations of the six families with the highest and six families with the lowest response to reduced temperature brooding were analyzed for significance of response group by temperature interaction. The resulting analysis revealed a significant interaction that reflected the divergence of response of HF and LF groups. This interaction was primarily a result of the HF group exhibiting an adrenal cortical response to the cooler temperature, whereas those members of the LF groups showed essentially no response to the cooler brooding temperature (Fig. 1). Such base population differences in adrenal cortical responsiveness have been the basis for selection programs with poultry in the past
TABLE 4. Analyses of variance for measured variables^ in an Athens-Canadian Randombred population (Hatch 1) Level of significance Variation
d.f'
Body weight
Gain
Corticosterone
Sire (S) Temperature (T) SXT
25 1 25
.0001 .0001 .9755
0001 .0001 .9841
.0012 .0001 .1133
1 2
Body weight = 1-day body weight; gain = 0 to 1-day weight gain; corticosterone = serum corticosterone.
Body weight: error - 1093, total - 1144; gain: error - 1088, total = 1139; corticosterone: error = 1025, total = 1076.
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*P <.001 means that the genotypic correlation is significantly different from zero.
SCOTT. AND WASHBURN
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(Gross and Colmano, 1971; Brown and Nestor, 1973, 1974; Edensand Siegel, 1975; Marks and Siegel, 1980; Satterlee etal, 1982).
HF LF
Response to Reduced Brooding Temperature (Hatches 2 and 3).
O o
Ui
2
o
26.7 BROODING
TEMPERATURE (C )
FIG. 1. Serum corticosterone responses at 24 hours postplacement of high (HF) and low (LF) response families from Hatch 1 that were brooded at two temperatures.
TABLE 5. The effect of brooding at 26.7 C on body weight, gain, packed red blood cell volume, and serum corticosterone of high and low serum corticosterone families from an Athens-Canadian Randombred population at 24 hours postplacment (Hatch 2) Family group
High Low
Body weight
1
32.6 + .4 33.0 + .4 a
Gain2
a
(g)
Serum corticosterone
PCV2
(Mg/100 ml)
(%) a
-2.5 ± .l -2.3 ± . l a
a
37.7 ± .5 36.7 + .4 a
a' bMeans within a variable with different superscripts are significantly different (P<.05). 'Mean ± SEM. 2 Gain = 0 to 1-day weight gain: PCV = packed cell volume.
5.91 ± .18 a 5.24 ± . 1 8 b
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to o
Although the LF group in Hatch 2 had numerically greater body weights and gains at 1-day of age, the differences were not statistically significant (Table 5). Packed cell volume (PCV) was measured as a result of concern that sire family differences observed for serum corticosterone in Hatch 1 were due to sire family differences in PCV, because Washburn (1967) had reported moderate to high heritability estimates for PCV in an AC population. However, differences in serum corticosterone concentrations between the HF and LF groups were not due to blood concentration of circulating corticosterone (Table 5). None of the variables measured in Hatch 3 had significant HF-LF group effects at any of the ages considered in that study (Table 6). However, the observed trends do agree with the results in Hatches 1 and 2. Throughout, the LF group had numerically heavier body weights and gains, and the HF group, likewise, had numerically greater concentrations of serum corticosterone.
GENETIC VARIATION TO REDUCED TEMPERATURE BROODING
83 5
TABLE 6. The effect of brooding at 26.7 C on body weight, gain, and serum corticosterone of high and low serum corticosterone families from an Athens-Canadian Randombred population at 1, 7. and 14 days postplacement (Hatch 3) Body weight1
Family group
-
Gain1 '•
'•
Serum corticosterone 1 (Mg/100 ml)
w
Day 1 High Low
33.2 ± . 3 3 32.6 ± .3
High Low
58.5 ± 1.0 60.2 ± 1.0
- 2 . 0 ± .1 - 2 . 1 ± .1
5.02 ± .19 4.71 + .16
Day 7 4.19 ± .20 4.12 ± .20
Day 14 114.8 ± 2.2 117.4 ± 1.9
High Low
79.2 + 2.1 80.5 ± 1.9
2.39 ± .20 2.27 ± .17
'Means within the same variable and day are not significantly different (P>.05). 2
Gain = 0 to 1, 0 to 7, and 0 to 14-day weight gain.
3
Mean ± SEM.
ACKNOWLEDGMENTS
The authors would like to express appreciation to H. L. Marks and the United States Department of Agriculture for their contribution in maintaining the Athens-Canadian Randombred population and for assisting us in our use of this population. We would also like to thank Samuel Bowen, Mark Mayfield, Rachel Peavey, and Pamela Stewart for their technical assistance as well as Sherry Davenport for typing the manuscript. REFERENCES Barr, A. J., J. H. Goodnight, J. P. Sail, and T. T. Helwig, 1976. A User's Guide to SAS 76. SAS Inst., Cary, NC. Becker, W. A., 1975. Manual of Quantitative Genetics. 3rd ed. Washington State Univ., Pullman, WA. Brown, K. I., and K. E. Nestor, 1973. Some physiological responses of turkeys selected for high and low adrenal response to cold stress. Poultry Sci. 52:1948-1954. Brown, K. I., and K. E. Nestor, 1974. Implications of selection for high and low adrenal response to stress. Poultry Sci. 53:1297-1306. Buckland, R. B., K. Balgrave, and P. C. Lague, 1974. Competitive protein-binding assay for corticoids in the peripheral plasma of the immature chicken. Poultry Sci. 53:241-245. Edens, F. W., and H. S. Siegel, 1975. Adrenal responses in high and low ACTH response lines of chickens during acute heat stress. Gen. Comp. Endocrinol. 25:64-73. El Halawani, M. E., P. E. Waibel, J. R. Appel, and A. L. Good, 1973. Effects of temperature stress on
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23.1 + 1.0 24.3 ± .9
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SCOTT AND WASHBURN Gildersleeve, 1981. Circulating levels of corticosterone in the serum of developing chick embryos and newly hatched chicks. Poultry Sci. 60:13141320. Scott, T. R., and K. W. Washburn, 1985. Evaluation of growth, hormonal and hematological responses of neonatal chickens to reduced temperature brooding. Poultry Sci. 64:777-784. Siegel, H. S., 1971. Adrenals, stress and the environment. World's Poult. Sci. J. 27:327-349. Siegel, H. S., 1980. Physiology stress in birds. Bioscience 30:529-534. Siegel, H. S., and N. R. Gould, 1976. Chick embryonic plasma proteins and binding capacity for corticosterone. Dev. Biol. 50:510-516. Washburn, K. W., 1967. Heritability of packed red blood cell volume in the domestic fowl. Poultry Sci. 46:1025. Wise, P. M., and B. E. Frye, 1973. Functional development of the hypothalamo-hypophyseal-adrenal cortex axis in the chick embryo, Gallus domesticus. J. Exp. Zool. 185:277-292.
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