Hatchability of Chicken Embryos Exposed to Acute High Temperature Stress at Various Ages1

Hatchability of Chicken Embryos Exposed to Acute High Temperature Stress at Various Ages1 T. B. ANDE 2 and H. R. WILSON Poultry Science Department, University of Florida, Gainesville, Florida 32611 (Received for publication March 28, 1980)

1981 Poultry Science 60:1561-1566 INTRODUCTION Due t o t h e wide variety of incubators in which t e m p e r a t u r e studies have been c o n d u c t e d , t h e r e is considerable variation in t h e literature concerning o p t i m a l incubation t e m p e r a t u r e s (Penquite, 1 9 3 8 ) . However, t h e optimal tempera t u r e for i n c u b a t i o n in force draft incubators is generally considered t o be 37.5 C (Insko, 1 9 4 9 ; Deuchar, 1 9 5 2 ; Romanoff, 1 9 6 0 ) . Most of t h e early w o r k on t h e effect of t e m p e r a t u r e on embryogenesis was concerned with t h e rate of development, metabolism, a n d / o r abnormalities p r o d u c e d b y variations in t e m p e r a t u r e . High incubation t e m p e r a t u r e s were f o u n d t o accelerate e m b r y o n i c g r o w t h , while low t e m p e r a t u r e s retarded e m b r y o n i c growth (Barrot, 1 9 3 7 ; Romanoff et al., 1 9 3 8 ; Henderson, 1 9 3 9 ) . With advancing e m b r y o n i c development, however, t h e effect of tempera t u r e was lessened, and from a b o u t t h e 1 0 t h day of incubation b o t h high and low temperatures slightly retarded e m b r y o n i c growth (Romanoff et al, 1 9 3 8 ) . Henderson ( 1 9 3 0 )

1 Florida Agricultural Experiment Station Journal Series No. 2294. 2 Present address: Paramount Poultry Hatchery, Orange Park, FL 32073.

concluded t h a t t h e influence of t e m p e r a t u r e o n growth did n o t cease until a r o u n d t h e 1 6 t h day of incubation and t h a t t h e u p p e r limit of t e m p e r a t u r e influence after the 1 5 t h day was b e t w e e n 38.8 C a n d 4 0 . 5 C. Therefore, t h e influence of t e m p e r a t u r e on growth was greatest during t h e earlier stages of i n c u b a t i o n and decreased with successive stages ( R o m a n o f f et al, 1 9 3 8 ; Henderson, 1939). Barrot ( 1 9 3 7 ) stated, however, there was an o p t i m u m rate of growth, and any deviation from this g r o w t h rate p r o d u c e d an inferior chick. Alsop ( 1 9 1 9 ) observed t h a t either excessive heat or a limited a m o u n t of heat applied b e t w e e n 22 and 72 hr of i n c u b a t i o n p r o d u c e d various forms of abnormalities in t h e nervous system. She f o u n d t h a t high t e m p e r a t u r e s ( 4 0 t o 4 2 . 2 C) p r o d u c e d a higher percentage of brain abnormalities and low t e m p e r a t u r e s (34.3 C t o 38.9 C) p r o d u c e d more neutral t u b e abnormalities. By exposing incubating eggs t o an uneven t e m p e r a t u r e gradient, Tazelaar ( 1 9 2 9 ) f o u n d t h a t t h e area vasculosa a n d its b l o o d vessels were t h e most easily affected. Nilsen ( 1 9 6 8 ) r e p o r t e d t h a t exposing 3 t o 4-day e m b r y o s t o elevated incubation t e m p e r a t u r e s (41 C t o 4 2 C) induced various degrees of a n u m b e r of different eye malformations, and Leighton et al. ( 1 9 6 4 ) f o u n d t h a t exposing 11 or 12-day e m b r y o s for 1 week t o i n c u b a t i o n

1561

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ABSTRACT Two experiments were conducted in which chicken embryos of various ages were subjected to acute high temperature stress to determine the effect on embryonic mortality and hatchability. Embryos were incubated at a temperature of 37.5 C (control) until they were 3, 7, 11, 16, or 19 days of age. Embryos of each age were separated into five groups and exposed to an incubation stress temperature of 43.3 C for various lengths of time. After exposure, the embryos were placed back into control incubators until hatching. Exposing 3-day-old embryos for 12 hr to a temperature of 43.3 C decreased hatchabiltiy of fertile eggs to approximately 50% of controls. A similar decrease occurred with 7-day-old embryos after only 4 hr of heat stress and with the 11 and 16-day-old embryos after 7 hr of stress. More than 50% reduction in hatchability occurred after 5 hr of heat stress with 19-day-old embryos. These results indicated that 3-day-old embryos are more resistant to heat stress and 7-day-old embryos less resistant to heat stress than previously indicated in the literature. Chicks that hatched following severe heat stress had wiry down, were usually weak and/or inactive, and often were unable to stand. (Key words: embryo, hyperthermia, hatchability, age, stress)

1562

ANDE AND WILSON

no significant decrease in hatchability when 16-day embryos were exposed for up to 24 hr to a temperature of 40.6 C or for 6 hr to a temperature of 43.3 C. However, 43.3 C for 24 hr was lethal to all embryos. Thompson et al. (1976) found no significant decrease in hatchability when exposing 16-day embryos to 40.6 C for periods up to 48 hr, but exposure for 9 hr to a temperature of 43.3 C killed approximately 50% of all embryos. Exposure to 46.1 C for 2 hr or 48.9 C for 1 hr killed all embryos, and the percent of culls increased with the severity of heat stress in all treatments. The purpose of the present study was to determine the effect of acute high temperature stress (43.3 C) on various aged embryos and to estimate the time required for this temperature to reduce the hatchability of fertile eggs to approximately 50% in each age category. EXPERIMENTAL PROCEDURE

Eggs used in these experiments were from a commercial broiler breeder strain (Cobb Color Sex) and were stored no more than 6 days at approximately 13 C and 85% relative humidity prior to setting. Settings were staggered so that five embryonic age categories (3, 7, 11, 16, and 19 days of age) would be available for stressing at one time. Jamesway 252 incubators were used, with two adjusted to 37.5 C and 62% relative humidity for normal incubation and two adjusted to 43.3 C and 62% relative humidity for heat stressing. Eggs were turned every 2 hr until they were transferred to hatching baskets at 19 days of incubation (19-day embryos were stressed prior to transferring). Fertility was determined prior to stressing by candling at 6 to 10 days of incubation, except for eggs in the 3-day age category. All unhatched eggs were broken out to determine the presence of embryos. A Speedomax W. 24-point potentiometer utilizing type T copper constantan thermocouple wire with quick tip connectors was used to monitor the temperature of each incubator from 1 hr prior to stressing until 2 hr after stressing. Individual stress periods were measured from the time the eggs were placed in the high temperature incubator until they were removed. Approximately 5 min were required for high temperature incubators to return to 43.3 C with the addition of each treatment group. Once stressed, treated eggs were returned to

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temperatures of 32.5 C or 42.5 C caused an alteration in the size of the heart and kidneys. Deuchar (1952) observed that each mesoderm region was most susceptible to high temperatures at the moment when it was invaginating and that this was a likely explanation for most of the abnormalities observed. Byerly (1938) found that hens with poor hatches at a normal incubation temperature tended to give the poorest results when the eggs were incubated under abnormal temperature conditions. In addition, a high incubation temperature of 39.2 C caused higher mortality between the 13 th and 15th day and the 18th to 21st day of embryonic development. Romanoff (1936) found that a wider range of temperatures (34.5 C to 37.5 C) was compatible with hatching during the later stage of incubation (16 to 21 days) and a relatively narrow range (37 C to 38 C) that permitted normal hatching under continuous exposure. A normal curve for hatchability has been reported for continuous incubation temperatures between 35 C and 40.5 C, with both extreme temperatures producing approximately 100% mortality (Romanoff, 1936; Barrot, 1937). Changes in the embryo's response to heat have been reported to occur on the 20th and 21st day (Pembrey et al, 1894), 16th day (Henderson, 1930), 10th day (Romanoff, 1939), and 7th day (Moreng and Shaffner, 1951) of incubation. Others (Henderson and Brody, 1927; Henderson, 1930, 1939; Penquite, 1938) have found that the influence of temperature was greatest during the early stages of incubation. Romanoff et al. (1938) and Romanoff (1939) studied the effects of 24-hr exposure to temperatures ranging from 29 C to 41 C and concluded that short exposures to extreme high temperatures were rriost injurious to the developing embryo during the early stages of incubation (0 to 5 days). Moreng and Shaffner (1951) noted that after 1 day of incubation the embryo became very susceptible to high temperature. They found that only 10 min at 43.3 C was lethal to 4-day embryos, while approximately 8 hr of exposure was lethal for 7 to 21-day embryos. More recently, Morgan and Tucker (1967) reported that 52% of all embryos between the ages of 7 to 10 days died when exposed to an incubation temperature of 41 C for 3 hr. They found that the 7 and 8-day embryos were more resistant to the lethal effects of the raised temperature than the 9 or 10-day embryos. Wilson et al. (1975) reported

EMBRYO HEAT STRESS

for 0, 2, 3, 4, or 5 hr. Embryos 16 and 11 days old were exposed for 0, 6, 7, 8, or 9 hr and 3-day embryos were exposed for 0, 9, 10, 11, or 12 hr. Treatments and replications of each treatment were assigned to incubators as in Experiment 1. RESULTS AND DISCUSSION

The exposure of 3-day-old embryos to 43.3 C for 12 hr produced a 43.4% hatch of fertile eggs, which was significantly lower (P<.05) than the 70.4% hatch observed after 9 hr of heat stress (Table 1). In Experiment 2, exposing 3-day embryos for 9 to 12 hr produced a fairly consistent decrease in hatchability with each additional hour of stress. After 12 hr of exposure, a 52.6% hatch of fertile eggs was obtained. These results were consistent with those obtained in Experiment 1, indicating that the critical time (time required to reduce hatch of fertile eggs to approximately 50%) for 3-day embryos was approximately 12 hr at this temperature. The exposure of 7-day embryos to 43.3 C for 3 hr in Experiment 1 resulted in a reduction of hatchability to 51.6% (Table 1). The critical time for 7-day embryos in Experiment 2 occurred between 3 and 4 hr of exposure, with hatch of fertile eggs reduced to 58.9% and 48.3%, respectively. In addition, 7-day embryos were found to be fairly sensitive to this temperature, as indicated by the significant drop in percent hatch after 3 hr of exposure.

TABLE 1. Hatchability (%) of fertile eggs exposed to 43.3 C for various times at 5 ages Exposure time (hrs) 0 2 3 4 5 6 7 8 9 10 11 12

3-Day

7-Day

11 Day

16 -Day

19-Day

Exp. 1

Exp. 2

Exp. 1

Exp. 2

Exp. 1

Exp. 2

Exp. 1

Exp. 2

Exp. 1

98 3 a

93.4 a

99.2 a

98.5 a

100.0 a

99 2 a

98 9 a

98.5 a

98.6 a

51.6 b

90 9ab 87

•jab

70 4 b 43

4C

59.8 b 69.3 b 51.7 b c 52.6C

90.0 a b 58.9 b c 48.3 C 34.6 C

. 93.4 a

36.9 b c

38.6 b

20.9 C

13.9 b

14.0 C

2.5 b

93 3 a 68 47 19 16

8b 6C 8d 0d

73 3 a 8 9b 3 3b

74.8 b 58.6 a 42.6 a 41.2 a 44.3 a

1.3C

0C

0C

Values within a column having a common superscript are not significantly different (P>.05).

Exp. 2 100.0 a 100.0 a 75.0 a b 78.9 a b 41.0 b

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incubators set at 37.5 C. All eggs were transferred to hatching baskets at 19 days of incubation and placed into a separate incubator (set at 37 C and 65% relative humidity) until the morning of the 22nd day of incubation, at which time chicks, pips, nonpips, and culls were removed and counted. Culling was based on the general strength and appearance of the chicks; however, many chicks with marginal amounts of wiry down, which would have been culled under commercial circumstances, were not culled. Analyses of variance (Snedecor, 1956) and Waller-Duncan's multiple comparison test (Waller and Duncan, 1969, 1972) were used to analyze data following arcsine transformation. Experiment 1. A total of 2,685 fertile eggs in the five embryonic age categories were separated into five treatment groups for each category and exposed to a temperature of 43.3 C for 0, 3, 6, 9, or 12 hr. All treatments from any one age category were assigned to the same general location in the incubator to eliminate within-incubator variation. Replications of each treatment group were assigned opposite locations in separate incubators to reduce between incubator error. Experiment 2. A total of 2,610 fertile eggs in the five embryonic age categories were separated into five treatment groups for each category. Stress times were changed from those used in Experiment 1 to more accurately ascertain the length of time required to reduce the hatchability of fertile eggs to approximately 50%. Embryos 19 and 7 days old were exposed

1563

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ANDE AND WILSON

TABLE 2. Incidence (%) of cull chicks hatched from fertile eggs exposed to 43.3 C for various times at 5 ages Exposure t i m e (hrs)

3 -Day Exp. 1 .8b

0 1 2 3 4 5 6 7 8 9 10 11 12

4 4b

1.7 a

Exp. 2 0a 0a 2 0a 1. l a a 6 .4

Exp. 1

16- Day

Exp. 2

.9a

Od

.8b

6.6a

11.2a

2ab 5ab 9ab

24.4a

29.4a

3.4cd 17.3e 51.4b 75.0a

la

12.5a

50.0a

.

12 11 8 19

Exp. 1 2.3b

19-Day

Exp. 2

Exp. 1

1.5 a

1.5 a

0a

1.2 b

.

8.7b

60.0ab

10 0 . 0 a

6.9a

50.0a 50.0a 0a 56.9a

50.0a

1

1

Values within a column having a common superscript are not significantly different (P>,05).

No chicks hatched at that exposure time.

Exp. 2 ()

a

()a

3.0a

32.6a

' '

Exp. 1

.7b

12.8b

1

11 -Day

7-Da>

Exp. 2

1 5.3 a (5.3 aa 2.6

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regardless of the age of the embryo exposed. Cull chicks produced by heat stress were less alert than normal with a high incidence of wiry and/or matted down, curled toes, weak legs, and general lack of balance. Most of the unhatched embryos of all treatment ages and times did not pip (Table 3). An increase in the percentage pips was found with the embryos exposed at 19 days of age, and, to a lesser degree, with those exposed at 16 days of age. Upon examination of unhatched embryos, it was observed that death usually had occurred soon after heat stressing. The 3-day and 19-day embryos were representative stages of two major normal mortality peaks of chicken embryos (Landauer, 1967) and were expected to be relatively sensitive to all types of stress, including heat stress. However, based on the results of these experiments, 3-day embryos were found to be relatively more resistant to heat stress than previous studies would indicate (Henderson, 1939; Romanoff, 1939; Moreng and Shaffner, 1951) and more resistant than other ages tested. The 19-day embryos, however, were less resistant than all ages tested except the 7-day embryos. The intermediate ages of 7, 11, and 16 days were selected according to the reported times of change in embryonic response to heat and at times which would represent a variety of stages in embryonic heat production and oxygen consumption (Romanoff, 1967). Both of the latter parameters are very low at 3 and 7 days,

Exposing 11-day embryos, in Experiment 1, for 6 hr to 43.3 C reduced hatchability to 38.6%, with a significant decrease (P<.05) occurring between 3 and 6 hr (Table 1). Elevenday embryos, in Experiment 2, showed a significant decrease (P<.05) in percent hatch, from 68.8% to 47.6% between 6 and 7 hr of exposure. The critical time for 11-day embryos was considered to be between 3 and 7 hr. In Experiment 1, a significant decrease in hatch occurred when 16-day embryos were exposed for 9 hr at 43.3 C (Table 1). A similar decline in hatchability was not noted in Experiment 2. Nevertheless, the critical time for 16-day embryos appeared to be between 6 and 9 hr. This time was consistent with an observation made by Thompson (1974) that indicated that 6 hr of exposure at 43.3 C may be the lower end of a critical range for 16-day embryos. Exposing 19-day embryos for 3 hr to 43.3 C produced a significant decrease in hatchability, while 6 hr of exposure produced an even greater (P<.01) decline in hatch of fertile eggs (Table 1) in Experiment 1. Exposing 19-day embryos for 5 hr in Experiment 2 produced a significant decrease in hatchability, suggesting that 5 hr was the critical length of time for this age embryo. These results confirm those of Moreng and Shaffner (1951) who reported 5 hr and 45 min as the lethal exposure time at 43.3 C for 19-day embryos. Increases in exposure times resulted in an increased incidence of cull chicks (Table 2),

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EMBRYO HEAT STRESS

TABLE 3. Incidence (%) of fertile eggs that did not pip following exposure to 43.3 C for various times at 5 ages Exposure time (hrs)

3-Day Exp. 1

Exp. 2 0C

OC

0

1 2 3

7.1

5 6

8.9 b c

bc

.

7-Day Exp. 1 Od

47.5

Ob

C

4

.. .

7 8

27.3 a b

12

4-8.03 a

2 4.4 b 2 5.0 b ab 36 . 3 a 4 2.1

8.4 b 41. l a 50.9 a 63.la

11 -Day

Exp. 1 Ob

Exp. 2 0C

58 gab

78.3ab

80 3 a

85.2*

96 7 a

Exp. 1 Ob

Exp. 2 0a

29.6b 50.8b 76.9 a 79.1 a

21.lb 90.0 a

19-Day ixp. 1

34.3 a 48.5 a 54.4 a 50.0 a

93.3 a

Exp. 2 0a

Ob

10.0 b

4.4b

1 6b

60.6bc

16 -Day

0a 17.9 a 14.0 a 50.0 a

94.9 a 96.2 a 100.0 a

.

' ' ' Values within a column having a common superscript are not significantly different (P>.05).

increasing rapidly at 11 days, plateauing at a moderate level at 16 days and beginning a dramatic increase at the end of 19 days. Susceptibility of embryos to heat stress tended to be related inversely to heat production and oxygen consumption, with the exception of the 7-day embryo. The reason for the heat sensitivity of this stage is not apparent. Since the chorionic and allantoic membranes are anastomosing and growing rapidly along the inner surface of the shell at this age (Romanoff, 1960), the chorioallantois might be very susceptible to acute heat stress and result in embryonic death. The anastomosed membrane would have completed its growth around the inner surface of the shell by the 11th day, which possibly would reduce its susceptibility to heat stress. A possible source of variation between the results of this and previous studies is strain or breed used. Most of the previous work has been with light or medium weight breeds rather than a large commercial meat-type strain. Breed and genetic line differences are known to occur for heat stress susceptibility of immature and adult birds (Hutt, 1938; Yeates et al, 1941; Lee et al, 1945; Wilson et al, 1966, 1975) and would be expected to occur also for embryos. Although these results apply to situations involving failure of temperature control equipment, it should be noted that many accidental occurrences of heat stress to embryos also result from electrical power failures that cause additional disruption of the control, ventilation, and egg turning systems. Thus, under applied

conditions, heat stress may be further complicated by lack of oxygen, excess carbon dioxide, lack of turning, and improper humidity. REFERENCES Alsop, F. M., 1919. The effect of abnormal temperature upon the developing nervous system in the chick embryo. Anat. Rec. 15:307-3 32. Barrot, H. G., 1937. Effect of temperature, humidity and other factors on hatch of hen's eggs and on energy metabolism of chick embryos. US Dept. Agr. Tech. Bull. 553. Byerly, T. C , 1938. Effect of different incubation temperatures on mortality of chick embryos. Poultry Sci. 17:200-205. Deuchar, E. M., 1952. Theeffect of a high temperature shock on early morphogenesis in the chick embryo. J. Anat. 86:443-458. Henderson, E. W., 1930. Growth and development. XVI. The influence of temperature and breeding upon the rate of growth of chick embryos. Missouri Agr. Exp. Sta. Res. Bull. 149. Henderson, E. W., 1939. Influence of temperature on length of artificial incubation period of Gallus domesticus. Pages 180—184 in Proc. 7th World's Poultry Congr. Henderson, E. W., and S. Brody, 1927. V. The effect of temperature on the percentage rate of growth of the chick embryo. Missouri Agr. Exp. Sta. Res. Bull. 99. Hutt, F. B., 1938. Genetics of the fowl. VII. Breed differences in susceptibility to extreme heat. Poultry Sci. 17:454-462. Insko, W. M., Jr., 1949. Fertility and hatchability of chicken and turkey eggs. Lewis W. Taylor, ed., John Wiley and Sons, Inc., New York, NY. Landauer, W., 1967. The hatchability of chicken eggs as influenced by environment and heredity. Monogr. 1 (Rev.), Storrs Agr. Exp. Sta., Storrs, CT.

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9 10 11

Exp. 2

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embryo. John Wiley and Sons, New York, NY. Romanoff, A. L., L. L. Smith, and R. A. Sullivan, 1938. Biochemistry and biophysics of the developing hen's eggs. III. Influence of temperature. Cornell Univ. Agr. Exp. Sta. Memoir 216. Snedecor, G. W., 1956. Statistical methods. 5th ed. The Iowa State College Press, Ames, IA. Tazelaar, M. A., 1929. The effect of a temperature gradient on the early development of the chick. Q. J. Microscop. Sci. 72:419-446. Thompson, J. B., Ill, 1974. Hatchability following high temperature stress of sixteen-day chicken embryos. M. S. thesis, University of Florida, Gainesville, FL. Thompson, J. B., Ill, H. R. Wilson, and R. A. Voitle, 1976. Influence of high temperature stress of 16-day embryos on subsequent hatchability. Poultry Sci. 55:892-894. Waller, R. A., and D. B. Duncan, 1969. A Bayes rule for the symmetric multiple comparison problem. J. Amer. Stat. Assoc. 64:1484-1503. Waller, R. A., and D. B. Duncan, 1972. A Bayes rule for the symmetric multiple comparison problem. Corrigenda. J. Amer. Stat. Ass. 67:253-255. Wilson, H. R., A. E. Armas, I. J. Ross, R. W. Dorminey, and C. J. Wilcox, 1966. Familial differences of Single Comb White Leghorn chickens in tolerance of high ambient temperature. Poultry Sci. 45: 784-788. Wilson, H. R., C. J. Wilcox, R. A. Voitle, C. D. Baird, and R. W. Dorminey, 1975. Characteristics of White Leghorn chickens selected for heat tolerance. Poultry Sci. 54:126-130. Yeates, N.T.M., D.H.K. Lee, and H.J.G. Hines, 1941. Reactions of domestic fowl to hot atmospheres. Proc. Roy. Soc. Queensland 53:105-116.

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Lee, D.H.K., K. W. Robinson, N.T.M. Yeates, and M.I.R. Scott, 1945. Poultry husbandry in hot climates—experimental inquiries. Poultry Sci. 24:195-207. Leighton, J., L. Merkow, and M. Locker, 1964. Alteration in size of the heart of late chick embryos after incubation at varied temperatures. Nature 201(4915): 198-199. Moreng, R. E., and C. S. Shaffner, 1951. Lethal internal temperatures for the chicken, from fertile egg to mature bird. Poultry Sci. 30: 255-256. Morgan, W., and W. L. Tucker, 1967. Influence of thermal incubation stresses. Poultry Sci. 46: 1172-1176. Nilsen, N. O., 1968. Eye malformations in chick embryos exposed to elevated incubation temperatures. Acta Opthalmol. 46:322-328. Pembrey, M. S., M. H. Gordon, and R. Warren, 1894. On the response of the chick, before and after hatching, to changes of external temperature. J. Physiol. 17:331-348. Penquite, R., 1938. Influence of temperature and humidity upon the growth of chick embryos in a mechanically ventilated incubator. Iowa Agr. Exp. Sta. Res. Bull. 232. Romanoff, A. L., 1936. Effects of different temperatures in the incubator on the prenatal and postnatal development of the chick. Poultry Sci. 15:311-315. Romanoff, A. L., 1939. Effect of temperature shock on development of chick embryo. Pages 184—186 in Proc. 7th World's Poultry Congr. Romanoff, A. L., 1960. The avian embryo. The Macmillian Co., New York, NY. Romanoff, A. L., 1967. Biochemistry of the avian