Broiler Incubation. 1. Effect of Elevated Temperature During Late Incubation on Body Weight and Organs of Chicks1

PRODUCTION, MODELING, AND EDUCATION Broiler Incubation. 1. Effect of Elevated Temperature During Late Incubation on Body Weight and Organs of Chicks1 N. Leksrisompong,* H. Romero-Sanchez,† P. W. Plumstead,* K. E. Brannan,* and J. Brake*2 *Department of Poultry Science, North Carolina State University, Raleigh 27695; and †Grupo Grica, Faculty of Agriculture, University of Antioquia, AA 1226, Medellin, Colombia ABSTRACT Three experiments were conducted to investigate the effect of increased egg temperature during the final third of incubation on BW, yolk sac, heart, and digestive organs of broiler chicks at hatching. Egg temperatures were found to be approximately 1.0 to 1.5°C higher than incubator air temperature. Elevated egg temperature (39.5°C) after embryonic day 14 generally accelerated hatching time but decreased the relative weight of the heart in all 3 experiments, whereas BW and relative

weights of the gizzard, proventriculus, and small intestines were significantly smaller in 2 of 3 experiments as compared with the control (∼38.2°C). Relative weights of the yolk sac or liver were significantly larger due to elevated egg temperature in single experiments only. A striking feature of the chicks that developed at an elevated egg temperature was their white color as compared with the yellow color of chicks from eggs incubated at more normal temperatures.

Key words: incubation, egg temperature, embryo development, embryo organ, heart 2007 Poultry Science 86:2685–2691 doi:10.3382/ps.2007-00170

INTRODUCTION It has been well documented that when conditions were optimal, chick embryos developed normally and hatched in approximately 21 d (Yalcin and Siegel, 2003), but turning, vital gas exchange, temperature, humidity, and other factors (Landauer, 1967; Lundy, 1969) have been shown to affect embryo growth. However, temperature has been suggested to be the most important factor controlling embryo growth and development (Meijerhof, 2000). Embryo body temperature has been shown to be governed by incubator air temperature, because the chick can not properly regulate its body temperature until the hatching process had been completed (Romijn and Lokhorst, 1955; Wekstein and Zolman, 1967, 1969; Freeman, 1971). Research has shown the optimum incubation temperature for chicken eggs to be from 37.0 to 38.0°C (Insko, 1949; Romanoff, 1960; Landauer, 1967; Lundy, 1969; Wilson, 1991). However, according to French (1997), eggs will absorb heat from the surrounding air during the first half of incubation due to embryo temperature being slightly lower than incubator temperature, but embryos must lose heat during the second half of incubation as their meta-

©2007 Poultry Science Association Inc. Received April 24, 2007. Accepted August 23, 2007. 1 The use of trade names in this publication does not imply endorsement of the products mentioned nor criticism of similar products not mentioned. 2 Corresponding author: [email protected]

bolic rate and heat production increase. Abnormal incubation temperatures have been shown to affect organ development of avian embryos (Shafey, 2004) and posthatch growth of chicks (Romanoff, 1935, 1936; Michels et al., 1974; Decuype`re, 1979; Geers et al., 1982). The objective of the present study was to determine the effects of high temperature during late incubation on hatching BW, yolk sac utilization, and the development of key organs of the broiler chick.

MATERIALS AND METHODS Natureform model NOM-45 and model NMC-2000 incubators (Natureform International, Jacksonville, FL) that held either 5 or 11 trays, respectively, of 180 chicken eggs each were used in this study. The incubators were equipped with an electronic dry bulb temperature controller and an electronic humidistat and controller that automatically adjusted the wet bulb temperature relative to changes in dry bulb temperature to maintain a consistent RH. Proper incubator operation was verified by insertion of an ASTM mercury thermometer (Fisher Scientific, Hampton, NH) and an electronic humidity stick (Testo 605-H1, Testo Inc., Flanders, NJ) each day. To prevent heat loss during egg temperature measurements, a plastic tent was constructed around the doors at the front of the 2 incubators to keep the incubator and external environments equalized during egg temperature measurements in all experiments. The tent was heated with 2 small forced-air electrical resistance heaters to approximately the same temperature as that of the incubators before

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LEKSRISOMPONG ET AL. Table 1. Comparison of egg temperature taken with mercury and infrared thermometers at various incubator air temperatures during infrared thermometer validation trials of experiment 1 Egg temperature Trial1 1

2 Figure 1. Internal egg temperatures as a result of High (triangles) or Normal (squares) incubation air temperature in experiment 2. E = embryonic day. 3

taking egg temperatures; otherwise, egg temperatures were observed to rapidly decrease when an incubator door was opened. The infrared thermometer, as described in experiment 1, was allowed to equilibrate on the floor of an incubator for 15 min before each use. The machine controls were adjusted to maintain the internal egg temperature in the 37.5 to 37.7°C range and 53% RH from setting to embryonic day (E)13 of incubation with egg rotation every 30 min. Thereafter, machine controls were varied to create normal and high internal egg temperatures, as described in the respective experiments.

Experiment 1 Infertile broiler hatching eggs were selected randomly, and a circular hole the diameter of a mercury thermometer was cut into each egg at their equator and a tuberculin syringe was used to withdraw 1 mL of albumen from each egg. A mercury thermometer (model FS 15142C Thermometer, Fisher Scientific) was then inserted into the opening until the entire length of the metal tip was immersed so that the internal liquid temperature could be measured. Some of the extracted albumen was then used to fill any voids, and the mercury thermometer was sealed into the eggshell with a fast-drying caulk. An egg with a mercury thermometer inserted was placed into each of 2 NOM-45 incubators. The incubator air temperatures used during the validation trials ranged from approximately 34.5 to 41.3°C in an incremental manner (Table 1). Egg temperatures were measured simultaneously at each air temperature with both the mercury thermometer and an infrared thermometer (Braun Ear Thermometer Type 6013, The Gillette Company, Boston, MA) by holding the tip in firm contact with the equator of the eggshell after allowing 8 to 12 h for the eggs and thermometers to equilibrate at each successive machine air temperature set point (Meijerhof, 2000). This process was repeated in 4 trials with different eggs in each trial. The eggs were transferred from the setter trays to plastic hatching bas-

4

Incubator air2 34.67 35.78 36.78 37.89 39.06 40.17 41.22 35.50 35.61 36.83 37.94 39.00 40.17 41.28 34.56 35.67 36.89 37.94 39.06 40.11 41.22 34.61 35.83 36.83 37.94 39.00 40.11 41.22

Mercury thermometer2 (°C) 34.60 35.78 36.89 37.78 39.06 40.22 41.33 34.56 35.67 36.89 37.94 39.00 40.11 41.44 35.11 35.67 37.11 37.89 39.22 40.22 41.22 34.56 35.83 36.94 38.17 39.11 40.28 41.17

Infrared thermometer2 34.94 36.11 37.00 37.94 39.06 40.22 41.61 34.83 35.94 36.89 37.94 38.89 40.11 41.33 35.28 35.83 37.22 37.94 39.17 40.17 41.22 34.61 35.83 36.94 38.17 39.11 40.28 41.17

1 Each trial involved a different pair of eggs measured by both methods. 2 The following were very highly significantly (P < 0.0001) correlated: air temperature and mercury thermometer temperature: 0.998, air temperature and infrared thermometer temperature: 0.997, infrared thermometer temperature and mercury thermometer temperature: 0.999.

kets at E20 of incubation and returned to their respective incubators.

Experiments 2 and 3 A 65-wk-old Ross 344 male × 308 broiler breeder flock supplied 150 eggs within a 5-g range (66.5 to 71.5 g) for experiment 2, and a 69-wk-old flock supplied 150 eggs within a 4-g range (67.0 to 71.0 g) for experiment 3. These eggs were divided randomly into 2 groups of 75 each, numbered, and placed in the front rows of each of the 5 trays in the 2 NOM-45 incubators. The remaining positions in the trays were filled with other eggs from the same broiler breeder flocks. Temperature of the 150 eggs was determined at E14, 16, 18, 19, and 20 of incubation. One group of eggs was incubated at normal temperatures and achieved E19 to E20 egg temperatures of 38.2 and 38.4°C in experiments 2 and 3 (Figures 1 and 2), respectively, whereas the high temperature group of eggs was incubated at average E19 to E20 egg temperatures of 40.0°C in experiment 2 (Figure 1) and 40.3°C in experiment 3 (Figure 2).

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Figure 2. Internal egg temperatures as a result of High (triangles) or Normal (squares) incubation air temperature in experiment 3. E = embryonic day.

Figure 3. Internal egg temperatures as a result of High (triangles) or Normal (squares) incubation air temperature in experiment 4. E = embryonic day.

Experiment 4

ferred to a NOM-45 incubator designated to incubate the eggs at a high E19 to E20 average egg temperature of 39.9°C (Figure 3), and the remaining trays were transferred to a NOM-45 incubator designated to incubate the eggs at a normal E19 to E20 egg temperature of 38.0°C (Figure 3). One tray of additional eggs was placed in the lowermost position in each machine to maintain uniform airflow.

Broiler hatching eggs were collected from the same strain of broiler breeder during a 2-d period at 48 wk of age and stored at 18.0°C and 65% RH for 5 d before setting. There were 1,440 eggs set in a Natureform model NMC-2000 incubator to E15 of incubation. There were 8 trays of experimental eggs placed in the machine with 1 additional tray of eggs placed above and 2 trays of eggs placed below the experimental eggs to ensure uniform airflow within the machine. The 15 eggs across the front of each tray (120 eggs total) were numbered consecutively for subsequent temperature determination. The temperatures of the 60 marked eggs on the front rows of the 4 trays in each machine were determined at E15, 16, 17, 18, 19, and 20 of incubation. At E15, four trays were trans-

Chick Evaluation At 21.5 d of incubation, the chicks that had completed the hatching process were removed from the trays, counted, individually identified with neck tags, and sexed using the feather-sexing method. Five chicks from each sex and tray (80 chicks total) were then euthanized with

Table 2. Body weight and relative weights of tissues and organs from broiler chicks on day of hatching in experiment 2 as influenced by incubation temperature, sex, and the incubation temperature × sex interaction Item

BW (g)

Yolk sac

Heart

Liver

Proventriculus

Small intestine

44.6B 47.7A 0.3 0.001

10.75 11.16 0.45 0.502

0.67B 0.81A 0.01 0.001

3.03A 2.80B 0.05 0.001

5.13 5.02 0.08 0.366

0.81 0.84 0.02 0.211

2.75 2.72 0.05 0.714

46.3 45.9 0.3 0.545

11.16 10.75 0.46 0.500

0.75 0.72 0.02 0.405

2.85 2.98 0.05 0.098

5.02 5.14 0.08 0.344

0.79B 0.86A 0.02 0.003

2.72 2.75 0.05 0.605

44.8 44.5 47.8 47.7 0.4 0.761

10.59 10.87 11.71 10.59 0.62 0.266

0.68 0.66 0.81 0.81 0.02 0.663

2.95 3.09 2.76 2.83 0.07 0.581

5.10 5.16 4.95 5.11 0.11 0.658

0.77 0.85 0.80 0.88 0.03 0.925

2.72 2.77 2.71 2.73 0.07 0.747

Incubation temperature1 High Normal SEM Probability Sex Male Female SEM Probability Incubation temperature High High Normal Normal SEM Probability

Gizzard (g/100 g)

Sex Male Female Male Female

Means in columns that possess different superscripts differ significantly (P ≤ 0.01). High incubation eggs were 40.0°C, and Normal incubation eggs were 38.2°C at embryonic days 19 and 20.

A,B 1

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CO2 and necropsied to determine BW and weights of the yolk sac, heart, liver, proventriculus, gizzard, and small intestines from junction with the gizzard to the ileo-cecal junction.

Statistical Analysis Correlations were calculated between air temperature and the 2 egg temperatures in experiment 1. A completely randomized design was utilized taking incubation temperature and sex of chick as the main effects in the 2 × 2 design and chicks as the experimental unit in experiments 2, 3, and 4. An ANOVA using PROC GLM was used to evaluate the data, and when the P-value was significant (P ≤ 0.05) for the interaction term, the LS MEANS procedure was used to partition the means (SAS Institute, 1998). Statements of statistical significance were based upon P < 0.05 unless otherwise stated.

RESULTS Experiment 1 The results of the 4 validation trials of experiment 1 are shown in Table 1. Small differences of <0.5°C between the mercury and infrared thermometers were observed in the first 2 trials before it was learned that an equilibration period was required for the infrared thermometer. The difference was decreased during the third trial when a 15-min equilibration period was allowed, and by the forth trial, there was essentially no difference between the mercury and infrared thermometer readings in the range from 35.8 to 41.2°C when the infrared thermometer was allowed to remain in the machine between each measurement. The very highly significant (P < 0.0001) correlations between air and mercury temperature, air and infrared temperature, and mercury and infrared temperature were 0.998, 0.997, and 0.999, respectively, over the 4 trials.

Experiment 2 The effect of incubation temperature (Figure 1) and sex on BW and relative yolk sac and organ weights on day of hatching in experiment 2 is shown in Table 2. Heart weight and BW were significantly (P < 0.01) smaller, whereas liver weight was significantly larger (P < 0.01) in the high as compared with the normal incubation temperature. The proventriculus was significantly larger in females than in males. The weights of the yolk sac, gizzard, proventriculus, and small intestine were not significantly affected by incubation temperature or sex, and there were no significant incubation temperature × sex interactions.

Experiment 3 The effect of incubation temperature (Figure 2) and sex on BW and relative yolk sac and organ weights on day

of hatching in experiment 3 is shown in Table 3. The weights of the heart, gizzard, proventriculus, and small intestine, as well as BW, were significantly smaller (P < 0.01) due to increased incubation temperature, whereas the weight of the yolk sac was significantly increased (P < 0.01). The proventriculus and small intestines were significantly larger in females than in males. Liver weight was not significantly affected by incubation temperature or sex, and there were no significant incubation temperature × sex interactions.

Experiment 4 The effect of incubation temperature (Figure 3) and sex on BW and relative yolk sac and organs weights on day of hatching in experiment 4 is shown in Table 4. The heart, gizzard, and small intestine weights were significantly (P < 0.01) smaller due to high compared with the normal incubation temperature. The proventriculus was also significantly smaller in the high temperature group, whereas there were no effects on BW or weights of the yolk sac or liver. There were no significant effects due to sex or the incubation temperature × sex interaction.

DISCUSSION The internal egg temperature measured with the infrared thermometer by directly contacting the eggshell was similar to that measured with the mercury thermometer when the infrared thermometer was allowed sufficient time to equilibrate with machine conditions. The infrared thermometer was obviously developed to be used at room temperature and apparently could not be moved from room temperature directly to incubator temperature and immediately function properly. This problem was perceived during the first and second validation trials, and the thermometer was subsequently placed in the incubator for at least 15 min before use. During the final trial, the infrared thermometer remained in the incubator during the entire experiment, only being moved to take the necessary readings. This continuous equilibration procedure presented almost identical readings between the infrared and mercury thermometers. The present discussion concerning high or normal incubation temperatures referred only to the egg temperature and not to the incubator air temperature, because the 2 were not the same and could not be compared in a consistent manner. As demonstrated in Figure 4, when the machine air temperature was 37.3°C on E14, the internal egg temperature reached 38.2°C. To maintain the internal egg temperature at 37.9°C on E19, the machine air temperature had to be reduced to 36.3°C, a difference of about 1.6°C. These findings were in agreement with Meijerhof and van Beek (1993) and Lourens et al. (2005), who showed that air temperature was simply not equal to embryo temperature and that embryo development and hatchability were more likely to be influenced by embryo temperature than by air temperature. Incubation has been not only characterized as 1 of the most critical components

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INCUBATION AT HIGH TEMPERATURE Table 3. Body weight and relative weights of tissues and organs from broiler chicks on day of hatching in experiment 3 as influenced by incubation temperature, sex, and the incubation temperature × sex interaction BW (g)

Yolk sac

Heart

Liver

Gizzard

Incubation temperature1 High Normal SEM Probability

44.3B 46.6A 0.2 0.001

12.07A 9.16B 0.40 0.001

0.63B 0.88A 0.01 0.001

2.93 3.00 0.04 0.267

(g/100 g) 5.08B 5.84A 0.07 0.001

Sex Male Female SEM Probability

45.4 45.6 0.3 0.601

10.36 10.79 0.44 0.414

0.76 0.75 0.02 0.741

2.98 2.95 0.04 0.495

44.0 44.5 46.8 46.5 0.3 0.236

11.38 12.72 9.37 8.97 0.56 0.130

0.62 0.63 0.89 0.87 0.03 0.473

2.96 2.90 3.01 2.99 0.06 0.733

Item

Incubation temperature High High Normal Normal SEM Probability

Proventriculus

Small intestine

0.80B 0.93A 0.02 0.001

2.63B 3.15A 0.05 0.001

5.43 5.51 0.08 0.436

0.84b 0.89a 0.02 0.018

2.81b 2.98a 0.06 0.015

5.03 5.14 5.81 5.86 0.10 0.740

0.77 0.82 0.90 0.96 0.02 0.812

2.53 2.73 3.09 3.21 0.07 0.490

Sex Male Female Male Female

Means in columns that possess different superscripts differ significantly (P ≤ 0.05). Means in columns that possess different superscripts differ significantly (P ≤ 0.01). 1 High incubation eggs were 40.3°C, and Normal incubation eggs were 38.4°C at embryonic days 19 and 20. a,b

A,B

of overall broiler performance but apparently has now become 1 of the more difficult parts of broiler management, because it can no longer be assumed that the internal egg temperature will be the same as the machine air temperature and must be measured independently (Meijerhof, 2000; Lourens et al., 2005). Chick embryos have previously been reported to respond to elevated incubation temperature with accelerated growth and development (Romanoff, 1960; Ricklefs, 1987; Christensen et al., 1999), but an accelerated develop-

ment has been reported to negatively affect hatchling BW (Gladys et al., 2000). Romanoff (1960) previously reported that the size of the chick varied with incubation conditions, because 60-g eggs, when exposed to varying incubation conditions, produced chicks with BW that ranged from 21.9 to 41.4 g. Our data also demonstrated that BW was significantly reduced by high as compared with normal incubation temperatures in experiments 2 and 3 (Tables 2 and 3) but only numerically in experiment 4 (Table 4). The effect in

Table 4. Body weight and relative weights of tissues and organs from broiler chicks on day of hatching in experiment 4 as influenced by incubation temperature, sex, and the incubation temperature × sex interaction Item Incubation temperature1 High Normal SEM Probability Sex Male Female SEM Probability Incubation temperature High High Normal Normal SEM Probability

BW (g)

Yolk sac

Heart

Liver

Gizzard

Proventriculus

Small intestine

43.1 44.2 0.8 0.350

10.06 9.52 0.42 0.375

0.72B 0.86A 0.02 0.001

2.59 2.65 0.05 0.582

(g/100 g) 4.90B 5.41A 0.09 0.002

0.89b 0.96a 0.02 0.035

3.00B 3.33A 0.07 0.004

44.1 43.2 0.8 0.388

9.93 9.66 0.42 0.660

0.81 0.77 0.02 0.262

2.65 2.59 0.05 0.603

5.17 5.13 0.09 0.767

0.92 0.93 0.02 0.602

3.08 3.24 0.07 0.078

43.9 42.4 44.4 43.9 0.8 0.649

10.35 9.77 9.50 9.54 0.60 0.610

0.72 0.73 0.90 0.82 0.03 0.131

2.67 2.51 2.63 2.67 0.05 0.371

4.81 4.98 5.53 5.28 0.13 0.106

0.86 0.92 0.97 0.94 0.03 0.156

2.85 3.14 3.31 3.35 0.09 0.160

Sex Male Female Male Female

Means in columns that possess different superscripts differ significantly (P ≤ 0.05). Means in columns that possess different superscripts differ significantly (P ≤ 0.01). 1 High incubation eggs were 39.9°C, and Normal incubation eggs were 38.0°C at embryonic days 19 and 20. a,b

A,B

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experiment 3 may be explained by a reduction in yolk sac absorption that reduced the nutrients available for embryo development, but this explanation may only be applied to experiment 3, in which the yolk sac was significantly larger. However, inspection of Figure 2 vs. Figures 1 and 3 clearly demonstrated that the difference in incubation temperature treatments was greatest in experiment 4 and also started earlier, because a difference was clearly present by E14. Nevertheless, BW was only numerically decreased by high temperature in experiment 4 (Table 4) as in the other experiments, but the yolk sac was not consistently affected. Thus, it may be concluded that a significant effect on yolk sac weight required that the temperature treatment be applied by E14 and that waiting until E17 (Figure 3) to apply the temperature treatments would negate significant BW effects. Evidently, a critical period exists from E14 to E17 of incubation. The most common sign of chicks having been incubated at a high temperature was their white color, probably due to poor absorption of the yolk sac pigments, which might be used to easily identify possible poor-quality chicks and higher-than-optimum incubation temperature. Other chick quality problems observed were similar to those reported by others and included excessive blood inside the eggshell, some blood on the down and feathers, short feathers, red hocks, unhealed navels, externalized yolk sac remnants (black buttons), cross beaks, ectopic viscera, weakness, an unsteady gait, lack of alertness, matted and coarse down, clubbed down, and a general abnormal and unthrifty appearance (Thompson et al., 1976; Wilson, 1991; Lourens et al., 2005). It was of interest to note that Romanoff and Faber (1933) reported that a decrease in incubation temperature of 2 to 3°C toward the end of incubation resulted in an improved embryo growth rate and metabolism while improving chick quality at hatching, in agreement with the present data. The organ most consistently affected by high vs. normal incubation temperature was the heart. The heart was the only organ that showed significant differences at hatching of up to 29% in all experiments in which temperatures

from 39.7 to 39.9°C were experienced (Tables 2, 3, 4). Wineland et al. (2000) also found that with high setter and hatcher temperatures, the heart was smaller. Normal embryological development of the heart has been shown to begin as early as the 2-somite stage (Graper, 1907), and the heart has been shown to continue to be mitotically active up until 10 d after hatching (Romanoff, 1960). Studies with 3 incubation temperatures (34.5, 36.5, and 39.5°C) have shown temperature to significantly affect the number of mitotically active myocytes in the heart. The weight of the heart of embryos in eggs exposed to the high incubation temperature differed significantly in a positive manner before E7 of incubation; however, the trend changed after E9 to result in an increasing negative association between incubation temperature and cardiac cell division that allowed the lower-temperature embryos to exhibit a greater heart weight late in incubation. Many common broiler problems such as sudden death syndrome and ascites may be related to problems with cardiovascular development and function. The gizzard, proventriculus, and small intestine of chicks incubated in high vs. normal temperatures were smaller in experiment 3 (Table 3) and in experiment 4 (Table 4) but not in experiment 2 (Table 2). The rate of cell division in these organs might also have been affected by excessive egg temperature, but the inconsistency among experiments was difficult to explain and suggested that there may be some other factor(s) that remain not accounted for or other incubation periods that need to be investigated in a similar manner. The liver was the only organ to be larger when chicks were incubated in high temperatures, as was the case in experiment 2 (Table 2). The liver has been shown to undergo its most rapid rate of mitotic growth before E12 of incubation (Schmalhusen, 1926; Dumm and Leavy, 1949), and machine air temperature did not increase until E14 in these experiments, but the lack of consistency again suggested that there may be some factor(s) that remain not accounted for. However, inspection of the overall data (Tables 2, 3, 4) revealed an inverse relationship between yolk sac and liver weight that suggested some interdependency such as transfer of materials from the yolk sac to the liver. Some organs showed significant effects due to sex in the presence of high vs. normal incubation temperature. The proventriculus of males was smaller than that of females (Tables 2 and 3) in experiments 2 and 3. The small intestine was also smaller in males than in females in experiments 2 (Table 2) and 3 (Table 3). As for BW and yolk sac weight, these data also suggested that a critical period of sensitivity to high temperatures for certain organs and tissues existed from E14 to E17 of incubation, whereas the heart exhibited sensitivity from E14 to E21. This differential sensitivity could be helpful in diagnosis of incubation problems.

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Michels, H., R. Geers, and S. Muambi. 1974. The effect of incubation temperature on pre- and posthatching development in chickens. Br. Poult. Sci. 15:517–523. Ricklefs, R. E. 1987. A comparative analysis of avian embryonic growth. J. Exp. Zool. 51:309–324. Romanoff, A. L. 1935. Influence of incubation temperature on the hatchability of eggs, post- natal growth and survival of turkeys. J. Agric. Sci. 25:318–325. Romanoff, A. L. 1936. Effects of different temperatures in the incubator on the prenatal and postnatal development of the chick. Poult. Sci. 15:311–315. Romanoff, A. L. 1960. The Avian Embryo. The Macmillan Co., New York, NY. Romanoff, A. L., and H. A. Faber. 1933. Effect of temperature on the growth, fat and calcium metabolism and mortality of the chick embryo during the latter part of incubation. J. Cell. Comp. Physiol. 2:457–466. Romijn, C., and W. Lokhorst. 1955. Chemical heat regulation in the chick embryo. Poult. Sci. 34:649–654. SAS Institute. 1998. SAS™ User’s Guide. Version 8.2 ed. SAS Inst. Inc., Cary, NC. Schmalhusen, I. I. 1926. Mem. Acad. Sci. Ukraine, Classe Sci. Phys. Math. 2:302–360. Shafey, T. M. 2004. Effect of lighted incubation on embryonic growth and hatchability performance of two strains of layer breeder eggs. Br. Poult. Sci. 45:223–229. Thompson, J. B., III, H. R. Wilson, and R. A. Voitle. 1976. Influence of high temperature stress of 16-day embryos on subsequent hatchability. Poult. Sci. 55:892–894. Wekstein, D. R., and J. F. Zolman. 1967. Homeothermic development of the young chick. Proc. Soc. Exp. Biol. Med. 125:294–297. Wekstein, D. R., and J. F. Zolman. 1969. Ontogeny of heat production in chicks. Fed. Proc. 28:1023–1028. Wilson, H. R. 1991. Inter-relationships of egg size, chick size, posthatching growth and hatchability. World’s Poult. Sci. J. 47:5–20. Wineland, M. J., K. M. Mann, B. D. Fairchild, and V. L. Christensen. 2000. Effect of different setter and hatcher temperature upon the broiler embryo. Poult. Sci. 79(Suppl. 1):123. (Abstr.) Yalcin, S., and P. B. Siegel. 2003. Exposure to cold and heat during incubation on developmental stability of broiler embryos. Poult. Sci. 82:1388–1392.