J. therm. Biol. Vol. 17, No. 6, pp. 339-345, 1992 0306-4565/92 $5.00+0.00 Printed in Great Britain. All rights reserved Copyright © 1992 Pergamon Press Ltd

THE EFFECT OF INCUBATION TEMPERATURE ON OXYGEN CONSUMPTION AND ORGAN GROWTH IN

DOMESTIC-FOWL EMBRYOS

Q. ZHANG and G. C. WmTTOW Department of Physiology, John A. Burns School of Medicine, University of Hawaii,

1960 East West Road, Honolulu, HI 96822, U.S.A.

(Received 10 June 1992; accepted in revised form 10 August 1992)

Abstraet--l. Oxygen consumption and organ growth were measured in domestic-fowl embryos incubated at different temperatures (36, 38 and 40°C).

2. Embryonic oxygen consumption was highest at an incubation temperature of 40°C and lowest at 36°C. These differences were ascribed largely to variations in embryo size at different incubation temperatures.

3. At incubation temperatures of 40 and 38°C, there was a plateau in oxygen consumption late in incubation, but this was not apparent at 36°C.

4. At 36°C, some tissues (e.g. eyeballs) were "spared" the repression of growth that characterized the embryo as a whole, while other tissues (e.g. stomach) incurred a much greater growth reduction. Similarly, at 40°C, stomach growth exceeded that of the embryo as a whole, while the eyeballs were largely spared the enhanced growth.

5. A simple index of tissue age revealed that, in general, there were consensual changes in tissue maturity and growth at different temperatures but that there were some disparities between growth and maturity in individual organs.

Key Word Index: Avian embryos; temperature; organ growth; oxygen consumption; Gallus domesticus

INTRODUCTION

Fertilized eggs of the domestic fowl are usually incubated at a constant temperature of 37.8°C (see e.g. Freeman and Vince, 1974). Incubation at lower or higher temperatures results in differences in the duration of incubation and also in the mass of the embryo (Romanoff, 1960; Freeman, 1984; Kuroda et aL, 1990). At incubation temperatures as low as 34.5°C or as high as 40.5°C the embryos fail to hatch. Using large groups of eggs, Barott (1937) showed that embryonic oxygen consumption was also related to incubation temperature. Thus, variations in incu- bation temperature may have profound effects on embryonic growth and metabolism. The purpose of the present study was to determine if these changes reflected uniform changes in the growth of different organs and tissues in the embryo, or whether there were differential effects of temperature on the various organs. This enquiry was prompted by a previous study (Zhang et aL, 1992) in which variations in growth induced by exposure of embryos to electro- magnetic fields during incubation resulted in con- siderable changes in the growth of some organs while others were "spared" the acceleration or repression of growth experienced by the embryo as a whole.

MATERIALS AND METHODS

Fertilized eggs of White Leghorn hens were ob- tained from a local hatchery. The control group of eggs was incubated at 38°C and the experimental

groups at either 36 or 40°C. The eggs were incubated in forced-draft incubators (CJQF, model 1202). The relative humidity in the incubator was maintained at 60% and the eggs were turned at least twice daily.

Each day, eggs were removed from each group and subjected to the following procedures: first, the oxy- gen consumption of the eggs was measured in a modified Scholander respirometer (Ackerman et al., 1980). The chamber containing the egg was immersed in a water bath at the same temperature as the incubation temperature and the chamber was venti- lated with air for 60 min before measurements began. The oxygen consumption was measured over a 60 rain period by introducing measured volumes of oxygen into the chamber to bring the meniscus of the manometer between the egg chamber and its compen- sating chamber back to zero. Carbon dioxide pro- duced by the embryo was absorbed by Ascarite (Thomas Scientific) placed at the bottom of the chamber. All values of oxygen consumption were corrected to standard temperature and pressure for dry gas (STPD).

After the oxygen consumption had been deter- mined, the egg was weighed to the nearest 0.01 g on an electronic balance (Mettler PM-400). The egg was then opened and the embryo carefully separated from the yolk sac. The wet mass of the embryo was determined on a Mettler H6 balance to the nearest 0.1 mg. The following linear dimensions of the em- bryo were then measured with a dial caliper: (1) whole embryo length--top of the head to the tip of the tail, with the embryo in a relaxed posture, (2)

339

340 Q. ZHANG and G. C. WHITTOW

Table 1. Mean mass of the eggs in the three temperature groups

Temperature (°C) Means __. SD (g) n t* P

36 63.021 + 2.300 34 1.694 >0.05 38 61.911 + 2.818 29 40 61.842 + 2.067 30 1.055 >0.05

*Comparison of the group at 36 or 40"C with the control group (38°C).

n, number of eggs.

culmen length--tip of the beak to the beginning of nasal skin on the upper mandible, (3) wing length--tip of the wing to the shoulder joint, with the wing extended, (4) leg length--tip of the middle toe to the head of the femur, (5) toe length--tip of the nail to the proximal fold in the webbing between the middle and lateral toe, (6) neck length-- from the lower mandible to the thirteenth cervical vertebra.

The embryo was then carefully dissected and the following organs and tissues were weighed, wet, on the Mettler H6 balance: (1) leg muscles--muscles from both legs, (2) pectoral muscles--muscles from both sides, (3) heart, (4) liver, (5) stomach, (6) intestine, (7) lungs--including both lungs, (8) brain, (9) eyeballs--including both eyeballs. After weighing the organ, it was dried in an oven at 60°C until the dry mass was constant. The difference between the wet and dry mass yielded the water content of the tissue which is inversely related to tissue maturity (Sotherland and Rahn, 1987).

As the age of the embryos was known from the first day of incubation, all measured data were related to embryo incubation time.

The data were plotted and curves fitted using the Sigma Plot computer program. The statistical signifi- cance of differences was determined by a t-test and the t-test subjected to Dunnett's test (Dunnett, 1955). The standard deviation (SD) was used as a measure of variation in the data.

RESULTS

Egg mass

Table 1 shows that the egg mass of neither exper- imental group was significantly different from the control group.

Oxygen consumption

The oxygen consumption (70 : ) of the embryos increased with incubation time at all three incubation temperatures (Fig. 1). At incubation temperatures of 40 and 38°C, there was evidence of a plateau in 7 0 : in the final third of incubation, followed by a further increase in pipped eggs. This was not apparent in eggs incubated at 36°C. The 702 was higher in eggs incubated at 40°C than in control eggs incubated at 38°C. Oxygen consumption was lowest in eggs incu- bated at 36°C. The differences in 7 0 : between eggs incubated at the three temperatures tended to in- crease as the embryos' age increased. The mean 7 0 : in eggs incubated at 40°C (522.2 ml/day) was signifi- cantly higher (t =3.80; P <0.001) than that of controls (297.1 ml/day), while the mean 7 0 : (107.2ml/day) of eggs incubated at 36°C was significantly lower than controls (t = 2.32; P < 0.05).

I

2000

1500

1000

500

i

[ ]

[ ]

[ ]

8 ° 8~

v og~v By v

08 °

I I I

10 15 20 25

Duration of incubation (days)

Fig. 1. E m b r y o n i c o x y g e n c o n s u m p t i o n (x~O:) in eggs i n c u b a t e d a t di f ferent t empe ra tu r e s . [ ] , 40°C; ~ , 38°C:

O , 36°C.

Embryo mass

The wet mass of the embryo increased during incubation at all incubation temperatures (Fig. 2). Second-order regression lines were a good fit to the data in the three groups. The correlation coefficients for the 36, 38 and 40°C groups were 0.98, 0.99 and 0.99, respectively. Embryo mass was greatest in the eggs incubated at 40°C and least in the eggs at 36°C. The disparity between the eggs incubated at 36°C and the control eggs increased as incubation proceeded but this was not true of the eggs incubated at 40°C. The mean mass of the eggs incubated at 36°C (4.772g) was significantly (t = 3.28; P <0.01) less than that (12.416 g) of the control eggs. The mean mass of the eggs incubated at 40°C (18.894 g) was significantly greater (t = 2.32; P < 0.05) than that of control eggs. It is apparent from Fig. 2 that the difference between the 36°C group and the control embryos was greater than that between the 40°C group and controls.

5 0

4O

3O

~, 20

0

10 15 20 25 D u r a t i o n o f i n c u b a t i o n (days)

Fig. 2. Whole-embryo mass in eggs incubated at different temperatures. The regression coefficients for the solid lines

are given in the text. S y m b o l s as in Fig. 1.

0.3

0.2

0.1

0.0 5 25

0.5

Embryonic organ growth and temperature

i i i

D [] V

V

10 15 20

[] 0.4

v

E~e ~= 0.3 n ~

0.2

0.1

0.0 I 5 10 15 20 25

Duration of incubation (days)

Fig. 3. Embryonic heart and lung mass in eggs incubated at different temperatures. Notations as in Fig. 2.

v

=o

1.2

1.0

0.8

0.6

i i i

v

341

0.4

0.2

0.0 5 25

v v O

10 15 20

1 , 2 I I [

1.0 ~

® 0 . 8 v o

o.6

~', 13.4

0.2

0.0 t i 5 10 15 20 25

Duration of incubation (days)

Fig. 4. Embryonic brain and eyeball mass in eggs incubated at different temperatures. Notations as in Fig. 2.

Organ mass

Heart. H e a r t m a s s inc reased curv i l inear ly wi th i n c u b a t i o n age a t all t h ree i n c u b a t i o n t e m p e r a t u r e s (Fig. 3). T h e c o r r e l a t i o n coeff icients for t he re la t ion- sh ips b e t w e e n h e a r t m a s s a n d i n c u b a t i o n age a t i n c u b a t i o n t e m p e r a t u r e s o f 36, 38 a n d 40° C were 0.96, 0.95 a n d 0.99, respect ively . T h e m e a n h e a r t m a s s was g rea te s t a t 40° C a n d leas t in t he e m b r y o s i n c u b a t e d a t 36°C b u t the d i f fe rences wi th c o n t r o l s were n o t s ta t i s t ica l ly s ign i f ican t (Tab le 2).

Lungs. L u n g m a s s a lso i nc reased wi th i n c u b a t i o n age (Fig. 3). T h e co r r e l a t i on coeff icients for the r e l a t i o n s h i p s were 0.95, 0.95 a n d 0.91 for i n c u b a t i o n t e m p e r a t u r e o f 36, 38 a n d 40°C, respect ively. In

pipped eggs, there was a marked increase in lung mass at incubation temperatures of 38 and 40°C, but not at 36°C. When the mean values at the three temperatures were compared, only the difference between incubation temperatures of 36 and 38°C was statistically significant (Table 2).

Brain. The increase in brain mass with incubation age was best represented by second-order regression lines with correlation coefficients of 0.97, 0.96 and 0.98, in eggs incubated at 36, 38 and 40°C, respect- ively. The mean brain mass was highest in the eggs at 40°C and least in the 36°C group. The difference between the eggs incubated at 36°C and the control embryos was statistically significant (Table 2). The

Table 2. Mean organ mass (±SD) in embryos incubated at 36, 38 or 40°C

Incubation temperature (°C) Organ mass (g) 36 38 40

Heart 0.066 + 0.061 0.097 ± 0.077 0.138 + 0.095 Lungs 0.067* + 0.040 0.142 + 0.109 0.165 + 0.104 Brain 0.226* + 0.148 0.397 ± 0.316 0.554 + 0.324 Eyeballs 0.448* ± 0.247 0.593 + 0.305 0.706 + 0.267 leg muscles 0.252* ± 0.259 0.843 + 0.729 1.211 + 0.948 Pectoral muscles 0.097* _+ 0.077 0.245 + 0.279 0.279 + 0.152 Stomach 0.111 * ± 0.139 0.637 + 0.729 1.000 + 0.969 Intestine 0.073* ± 0.069 0.415 ± 0.372 0.509 ± 0.528 Liver 0.095* ± 0.087 0.248 ± 0.219 0.368 ± 0.333

*Significantly different from controls (38°C).

TB 17/6--E

342

0.6

0.5

0.4

o t~

0.3

"d ", 0.2 o *d Q)

m 0.1

0.0 5

3.5

o [] Do

o v v o

o

o 8

[] i 10 15 20

Q. ZHANG and G. C. WmTTOW

25

,3.0 O

2.5

N 2.0

~ 1.5

,_~ 1.0 ~ t 3

0.5

0.0 ~ . . . . . 10 15 20 25

Duration of incubation (days)

Fig. 5. Embryonic pectoral and leg muscle mass in eggs incubated at different temperatures. Notations as in Fig. 2.

v

o

o r / ]

3.5

3.0

2.5

2.0

1.5

1.0

0.5

0.0

2.0

i I i

D

10 15 20 i i i

difference in brain mass between the control eggs and those incubated at 36°C increased as incubation age increased (Fig. 4) but brain mass in the control eggs and those kept at 40°C increased in parallel, with age.

Eyeballs. The mass of the eyeballs increased rapidly very early in incubation and more slowly thereafter (Fig. 4). This was true for the control eggs and those incubated at 40°C, but not for the 36°C group. In the latter, the brain growth could be represented by a linear regression with a correlation coefficient of 0.97. This disparity in the nature of the growth curves at different temperatures resulted in significant differ- ences between eggs incubated at 36°C and control eggs between 11 and 21 days but, overall, the differ- ences were not significant (Table 2). There were no significant differences between eggs incubated at 38 and 40°C.

Pectoral and leg muscles. The pectoral and leg muscles differed in the nature of their growth. The leg muscles grew exponentially at all incubation tempera- tures but this was true for the pectoral muscles only in eggs incubated at 36°C (Fig. 5). At this tempera- ture (36°C), pectoral muscle was too small to dissect for the first 13 days of incubation. At incubation temperatures of 38 and 40°C pectoral muscle growth rates diminished with age. Pectoral muscle mass was clearly less at 36°C during the final six days of incubation than in control eggs and this was also the case for the leg muscles (Fig. 5; Table 2). Overall, the

25

1.5

~ 1.0

0.5 V

o.o -~_'~- - - ~ ? 10 15 20 25

1.2 ~ , B

1.0 []

~ 0.8

~ o.6

~ 0 .4

0 .2

0 .0 . . . . 10 15 20 25

Duration of incubation (days)

Fig. 6. Embryonic stomach, intestine and liver mass in cggs incubated at different temperatures. Notations as in Fig. 2.

differences between the leg muscles and pectoral muscles at 38 and 40°C were not significant (Table 2).

Stomach, intestine and liver. The growth of the stomach, intestine and liver was exponential at all three temperatures (Fig. 6). The regression co- efficients were all above 0.91. Growth rates were highest at 40°C and lowest at 36°C. Overall, the difference between incubation temperatures of 40 and 38°C was not significant (P > 0.05) but the difference between incubation temperatures of 38 and 36°C was

Table 3. Effect of incubation temperature on organ maturity

Dry/wet mass x 100

Organ 36°C 38°C 40°C

Heart I 1.429 12.474 14.928 Lung mass 9.239 11.972 14.424 Pectoral muscles 9.701 12.245 20.538 Leg muscles 10.437 14.472 17.690 Liver 18.476 24.960 30.978 Stomach I 1.532 12.339 16.100 Intestine 9.550 I 1.590 14.283 Eyeballs 4.799 5.245 5.921 Brain 10.044 11.360 13.610

Embryonic organ growth and temperature 343

12 , , , 6 , , )

,ot j t ,

":t t o / n _;.,W, , , /

5 10 15 2 0 2 5 5 10 15 2 0 2 5 2 . 0 , , , , 2 . 5 . , , ) .

, f i t 2 . 0 Q D ~'. 'I o ~ Z o I~

I o % ~ I ~. ' ~ o O - 0

=-'.° I J// YJ 1 1

o o ; ",'o-:; ,'o ,'~ o o , , " 5 10 15 20 ' 2 5

10 , ) j l , 3 . 5 / l i ~ _ _

t , , '°

~ 15 v ~ w 4 ~ •

~ 1.o

'~ 2 ~ 0 . 5

0 O 0 5 10 15 2 0 2 5 5 10 15 2 0 25

D u r a t i o n o f i n c u b a t i o n ( d a y s )

Fig. 7. Embryonic dimensions in eggs incubated at different temperatures. Notations as in Fig. 2.

statistically significant (Table 2) and the difference between growth rates at 38 and 36°C increased as incubation age increased.

Tissue maturity In general, the maturity of the tissues, as indicated

by the inverse of the water content, was lower than the control values at 36°C, and higher at 40°C (Table 3). At 36°C, the most affected tissues were the leg muscles and the least, the heart. In the eggs incubated at 40°C, the pectoral muscles had the highest matu- rity compared with controls, while the eyeballs were closest to control values.

Body dimensions The overall length of the embryo and the length

of the leg, wing, neck and toe could be represented by linear regressions of length on incubation age (Fig. 7), at all incubation temperatures. The growth rate of the culmen, on the other hand, was more logarithmic in nature, decreasing as incubation proceeded. Body lengths were highest at an incu- bation temperature of 40°C, and lowest at 36°C. All lengths, except the culmen, were significantly lower at 36°C than at 38°C (P < 0.05) but only

neck length was significantly longer at 40°C than at 38°C.

D I S C U S S I O N

The oxygen consumption of the embryos incubated at 40°C was higher than that of embryos incubated at 38°C which, in turn, was significantly higher than that of eggs incubated at 36°C. However, the ~O 2 was measured at the incubation temperature, i.e. at 40, 38 or 36°C. Therefore it would be expected that "v'O2 would be higher at the higher temperatures as a result of the van't Hoff-Arrhenius effect on chemi- cal reaction rates. In Fig. 8(A) the mean ~/O2 measured at each incubation temperature is plotted (top curve) together with the expected values (lower curve) for incubation temperatures of 38 and 40°C assuming (I) that the Q~0 of the effect of incubation temperature on ~/O2 is 2, and (2) that the increase in ~O2 with increasing incubation temperature is due solely to this QJ0 effect. It is clear that only a small part of the difference in ~'O2 at different incubation temperatures can be attributed to the van't Hoff-Arrhenius effect of temperature on chemical reaction rates.

344 Q. ZHANG and G. C. WHITTOW

500

~' 400 fl_ I-

,~ 300

.~. 200

O I 00

(A)

0 I I I 36 38 40

500

400

300

200

1oo

(B)

0 I I L 36 38 40

Incubot ion tempero tu re (*C)

Fig. 8. Measured oxygen consumption ('v'O2) of embryos incubated at 36, 38 and 40°C (O) together with the theor- etical increase in ~'O2 assuming a Ql0 of 2 (V). (B) "v'O2 at incubation temperatures of 38 and 40°C expressed as a percentage of the value at 36°C before (l-q) and after (O)

correction (~O2/embryo mass) of ¢¢O2 for body mass.

Embryos were larger in eggs incubated at 40°C, and smaller in embryos incubated at 36°C, than in controls (38°C). Embryo size must also have con- tributed to the differences in 902 at different incu- bation temperatures (Zhang and Whittow, 1992). In Fig. 8(B) the values for mean 902 measured at 38 and 40°C (top curve) are presented as a percentage of the value at 36°C. Also shown (lower curve) are the values after correction for the variations in embryo mass. It is clear that the differences in body mass accounted for much of the difference in 902.

Apart from the quantitative effects of variations in incubation temperature on 902, there was a qualitat- ive effect: in embryos incubated at 36°C, there was evidence of a plateau in VO 2, followed by an increase in pipped eggs. This plateau occurred at incubation temperatures of 38 and 40°C, and it has been reported by others (e.g. Rahn et al., 1974). The plateau is believed to be the result of a constraint on the embryonic oxygen consumption imposed by the gas conductance of the shell (see Whittow and Tazawa, 1991). When the oxygen consumption of the embryo equals the maximal rate at which oxygen can diffuse through pores in the shell, the oxygen consumption cannot increase further unless the shell is fractured (Whittow, 1984, 1985). In the present investigation the p!ateau in oxygen consumption occurred when the VO 2 was approx. 600ml/day. In the eggs incubated at 36°C, the 902 was always well below this level. Presumably, the 902 in eggs incubated at 36°C was always lower than the possible maximal rate of diffusion of oxygen into the egg.

The mass and overall length of the embryo were greatest at an incubation temperature of 40°C and

least at the lowest incubation temperature used (36°C). Although egg temperatures were not measured in the present investigation, it is safe to assume that they reflected the incubation temperature (Tazawa et al., 1989). Therefore the most likely explanation for the higher growth rates at the higher temperatures is a van't Hoff-Arrhenius effect--the direct effect of temperature on chemical reaction rates. However, the differences between values at the control incubation temperature of 38°C and at 36°C were much larger, in general, than the differences between incubation temperatures of 38 and 40°C. This is not the expected effect of the van't Hoff-Arrhenius relationship, which is exponential. It is possible that the growth rate at 38°C represents the maximal or near-maximal growth rate possible. Thus, the relatively small effects of increasing incubation temperature from 38 to 40°C may partly reflect the "throttling" effect of the shell conductance on gas exchange and growth. If this was the explanation, growth rates would be expected to differ during the first half of incubation, when the gas conductance exceeds the oxygen consumption of the embryo, and again, after pipping has occurred. Figure 2 shows that neither of these possibilities occurs. Growth rates at 38 and 40°C are parallel throughout incubation.

There were not only differences in overall growth at the different incubation temperatures but also differences in the relative growth of organs (Table 4). Thus, at 36°C, the overall growth of the embryo was only 38.4% of that at 38°C, but the eyeballs were largely "spared" this reduction in growth while the stomach incurred a much greater reduction than did the embryo as a whole. At 40°C, the embryo mass was 152.2% of the value at 38°C. Stomach growth exceeded that of the entire embryo but the eyeballs were spared much of the enhanced growth at high temperature. Thus, the growth of the stomach was

Table 4. Effect of incubation temperature on organ growth

Incubation temperature

Organ mass 36'C 38"C 40°C

Whole embryo mean (g) 4,772 12.416 18.894 % of control* 38.4 100.0 152.2

Heart (g) 0.0658 0.097 0.138 % of control 67.8 100.0 I42.3

Lungs (g) 0.067 0.142 0.165 % of control 67.8 100.0 116.2

Pectoral muscles (g) 0.097 0.245 0.297 % of control 39.6 100.0 121.22

Leg muscles (g) 0.252 0.843 1.221 % of control 29.9 100,0 144.8

Liver (g) 0,0958 0.248 0.368 % of control 38.6 100.0 148.4

Stomach (g) 0. I 11 0.637 1.000 % of control 17.4 100.0 157.0

Intestine (g) 0,0733 0.415 0.509 % of control 17.7 100.0 122.7

Eyeballs (g) 0.448 0.593 0.706 % of control 75.6 100.0 119.1

Brain (g) 0.226 0.397 0.554 % of control 56.9 100.0 139,5

*Using the 38°C group as a control,

Embryonic organ growth and temperature 345

particularly susceptible to changes of incubation tem- perature while the eyeballs were relatively resistant to temperature variations. The organ that followed the whole embryo most closely, in terms of its pro- portional changes at the three incubation tempera- tures, was the liver.

The effects of incubation temperature on organ growth reported in the present investigation are analogous to the effects of hypoxia and hyperoxia on avian embryonic organ growth described by McCutcheon et al. (1982) and Stock et al. (1983, 1987). Thus, in eggs of the domestic fowl, the heart and brain are "spared" the diminution in embryonic growth that occurs during hypoxia (Smith et al., 1969; Williams and Swift, 1988; Spiers and Baummer, 1990; Tazawa et al., 1988). Liver growth, on the other hand, was stunted more than that of the embryo as a whole. In hyperoxic eggs, embryonic growth was enhanced; the acceleration of growth of the heart exceeded that of the embryo as a whole, but the brain was spared this increased growth and liver growth was not affected by hyperoxia. A further analogy can be drawn between the effects of incubation tempera- ture on embryonic organ growth and the effects of exposure to an electromagnetic field (EMF) during incubation (Zhang et al., 1992). Thus, incubation at 40°C or exposure to an EMF of 2 G induced an increase in overall growth of the embryo, while incubation at 36°C or exposure to an EMF of 1 G repressed growth. However, the pattern of changes in organ growth induced by temperature differed from that produced by exposure to an EMF. These com- parisons suggest that induced variations in overall growth of the embryo may be achieved by different patterns of effects on organ growth, depending upon the growth-provoking or growth-repressing agent.

Overall, the maturity of the organs, as evidenced by their percentage water content, changed congruently with their growth. Thus, growth and maturity were repressed by incubation at 36°C and increased at 40°C (Tables 3 and 4). However, the data for individ- ual organs revealed disparities between the effects of temperature on growth and maturity. For example, the growth of the stomach was stunted by incubation at 36°C compared with incubation at 38°C, but the maturity of the stomach was relatively high (Table 3). At 40°C, the growth of the pectoral muscles was moderately increased but the increase in the maturity of pectoral muscle exceeded that of any other tissue.

REFERENCES

Ackerman R. A., Whittow G. C., Paganelli C. V. and Pettit T. N. (1980) Oxygen consumption, gas exchange and growth of embryonic Wedge-tailed Shearwaters (Pu~inus pacificus chlororhynchus ). Physiol. Zoiil. 53, 210-221.

Barott H. G. (1937) Effect of temperature, humidity and other factors on hatch of hens' eggs and on energy metabolism of chick embryos. U.S. Department of Agri- culture Technical Bulletin, No. 553, pp. 1-45.

Dunnett C. W. (1955) A multiple comparison procedure for comparing several treatments with a control. J. Am. statist. Ass. 50, 1096-1121.

Freeman B. M. (1984) Physiology and Biochemistry of the Domestic Fowl, pp. 164-167. Academic Press, New York.

Freeman B. M. and Vince M. A. (1974) Development of the Avian Embryo, pp. 1-123. Chapman & Hall, London.

Kuroda O., Matsunaga C., Whittow G. C. and Tazawa H. (1990) Comparative metabolic responses to prolonged cooling in precocial duck (Arias domestica) and altricial pigeon (Columba domestica) embryos. Comp. Biochem. Physiol. 95A, 407--410.

McCutcheon I. E., Metcalfe J., Metzenberg A. B. and Ettinger T. (1982) Organ growth in hyperoxic and hy- poxic chick embryos. Respir. Physiol. 50, 153-163.

Rahn H., Paganelli C. V. and Ar A. (1974) The avian egg: Air cell gas tension, metabolism and incubation time. Respir. Physiol. 22, 297-309.

Romanoff A. L. (1960) The Avian Embryo; Structural and Functional Development, pp. 560-741. Macmillan, New York.

Smith A. H., Burton R. R. and Besch E. L. (1969) Develop- ment of the chick embryo at high altitude. Fedn Proc. Fedn Am. Socs exp. Biol. 28, 1092-1098.

Sotherland P. R. and Rahn H. (1987) On the composition of bird eggs. Condor 89, 48-65.

Spiers D. E. and Baummer S. C. (1990) Embryonic develop- ment of Japanese quail (Coturnix coturnix japonica) as influenced by periodic cold exposure. Physiol. Zoiff. 63, 516-535.

Stock M. K. and Metcalfe J. (1987) Modulation of growth and metabolism of the chick embryo by a brief (72-hr) change in oxygen availability. J. exp. Zool., Suppl. 351-356.

Stock M. K., Francisco D. L. and Metcalfe J. (1983) Organ growth in chick embryos incubated in 40% or 70% oxygen. Respir. Physiol. 52, 1-11.

Tazawa H., Wakayama H., Turner J. S. and Paganelli C. V. (1988) Metabolic compensation for gradual cooling in developing chick embryos. Comp. Biochem. Physiol. 89A, 125-129.

Tazawa H., Whittow G. C., Turner J. S. and Paganelli C. V. (1989) Metabolic responses to gradual cooling in chicken eggs treated with thiourea and oxygen. Comp. Biochem. Physiol. 92.4,, 619-622.

Williams J. B. and Swift K. (1988) Oxygen consumption and growth of northern bobwhite embryos under normoxic and hyperoxic conditions. Condor 90, 187-192.

Whittow G. C. (1984) Physiological ecology of incubation in tropical seabirds. In Tropical Seabird Biology (Edited by Schreiber R. W.), Studies in Avian Biology No. 8, pp. 47-72. Cooper Ornithological Society.

Whittow G. C. (1985) Partition of water loss from the eggs of the Sooty Tern between the pre-pipping and pipped periods. Wilson Bull. 97, 240-241.

Whittow G. C. and Tazawa H. (1991) The early develop- ment of temperature regulation in birds. Physiol. Zodl. 64, 1371-1390.

Zhang Q. and Whittow G. C. (1992) Embryonic oxygen consumption and organ growth in the Wedge-tailed Shearwater. Growth Dev. Aging. In press.

Zhang Q., Tabrah F. L. and Whittow G. C. (1992) Effects of an electromagnetic fields on the embryonic growth and oxygen consumption. J. Bioelect. In press.