Effect of chronic hypoxia during embryonic development on physiological functioning and on hatching and post-hatching parameters related to ascites syndrome in broiler chickens

Abstract

The present study was designed to investigate the effect of different atmospheric pressure on the endogenous functions of broiler chickens during embryonic, hatching and growing periods related to ascites. Eggs from a commercial broiler line were incubated in two similar commercial incubators at high and low altitudes. The effect on embryonic development and physiological functions including hatching parameters, incidence of ascites and growth performance were examined. Embryos incubated at high altitude had higher plasma tri-iodothyronine, thyroxine, corticosteroid and lactic acid levels, and hatched earlier than those incubated at low altitude. Embryonic mortality was higher at high altitude. Chickens that had been incubated at high altitude showed less right ventricular hypertrophy and ascites mortality than those incubated at low altitude. It was concluded that different atmospheric pressure during incubation interacts with the endocrine functions of the embryo and hence affects hatching parameters, thereby influencing ascites susceptibility.

Introduction

Differences in the oxygen requirements between fast and slow growing lines of chickens are believed to be causally linked with the more pronounced occurrence of right ventricle hypertrophy and ascites in the fast growing lines (Peacock et al., 1990). Such occurrences among fast growing lines are even more apparent under conditions that impose an additional metabolic load on the birds, such as low ambient temperature and high altitude (Julian, 1993, 2000; Hassanzadeh et al., 2001, 2003). This may not only be related to an increased oxygen demand, but also to the supply of oxygen that could be involved or altered in these lines selected for rapid growth and efficient food conversion (Decuypere et al., 2000).

The syndrome of ascites is multifactorial and mainly caused by exogenous and/or endogenous factors (Decuypere et al., 2000). The peak of ascites incidence occurs during weeks 5 to 6 of the growing period, but it is thought that the aetiology of the syndrome is initiated much earlier, even during the embryonic stage (Coleman & Coleman, 1991). As the chick embryo consumes 60% more oxygen between the start of pulmonary breathing and hatching compared with the earlier stages (Visschedijk, 1968), it is possible that a shortage of oxygen occurs during this stage. Oxygen and carbon dioxide exchanges and endocrine functions especially tri-iodothyronine (T₃), thyroxine (T₄) and corticosterone are of fundamental importance for embryonic development during incubation and may affect survival of the embryo (Decuypere et al., 1979; Tullett, 1990). Thyroid hormones are involved in the complex process of pipping and hatching in chick embryos (Decuypere et al., 1991) and are important for regulating metabolic rate during the post-hatch period (Decuypere et al., 2000). This becomes even more apparent under adverse environmental conditions; for example, low ambient temperature (Scheele et al., 1992) and high altitude (Hassanzadeh et al., 1999). The findings of Chineme et al. (1995) indicated that the length and/or severity of prenatal hypoxia may influence postnatal characteristics related to ascites. Eggs incubated in an environment with a relatively high concentration of carbon dioxide hatched earlier than in an environment with normal amounts (Buys et al., 1998; Hassanzadeh et al., 2002). The chickens incubated in the environment with increased concentration of carbon dioxide showed a lower incidence of ascites during the growing period because high concentrations of carbon dioxide in the incubation environment might decrease the length of time the embryo experiences hypoxia (Buys et al., 1998; Hassanzadeh et al., 2002).

The partial pressure of oxygen becomes lower with increasing altitude. At sea level, oxygen makes up 20.9% of the atmosphere and the equivalent percentage of oxygen drops approximately 1% for every 500 m rise in altitude (Julian, 2000). The purpose of the present study was to test the hypothesis that there is a metabolic adaptation to environmental hypoxia in broiler chickens (Buys et al., 1998; Hassanzadeh et al., 2002).

Materials and methods

One thousand five hundred eggs from a commercial broiler line (Arbor Acres) were incubated under standard conditions at 37.8°C and a relative humidity of 55% in two similar commercial incubators, one of which was located at high altitude, 2000 m above sea level (in central Iran) and the other at low altitude, at sea level (in northern Iran). Individual egg weights were recorded before incubation and subsequently they were equally and randomly divided over the two low and high altitude situated incubators (750 eggs per incubator). At the end of incubation, the eggs were inspected. Early hatching at 482 h of incubation, final hatching at 508 h of incubation, embryonic mortality and non-fertile eggs were recorded. Blood samples were collected in heparinized tubes from 30 embryos per incubator by cardiac puncture at the non-external pipping stage (460 h of incubation) and in newly hatched chicks for determination of plasma thyroid (T₃, T₄), corticosterone and lactic acid levels as described earlier (Decuypere et al., 1983, 1994; Meeuwis et al., 1989; Hassanzadeh et al., 1997). Plasma lactic acid was measured using Sigma kits 826 (Sigma, St Louis, MO, USA). The assay was based on the spectophotometric measurement (λ=340 nm) of NADH produced by lactate dehydrogenase. The relative embryo weight was calculated as the ratio of embryo weight to egg weight at 460 h of incubation (Dewil et al., 1996; Hassanzadeh et al., 2002). All hatched chicks were weighted when incubation was stopped at 508 h.

At the end of incubation, a total of 240 newly-hatched chicks from each incubator were randomly selected for the follow-up experiment. One-half of the newly-hatched chicks (508 h of incubation) from each incubator were randomly divided and housed at a high-altitude farm (2000 m above sea level in central Iran), while the other half of the chicks (120 chicks) were placed in a low-altitude farm (around sea level in northern Iran). At each altitude, they were again divided over three floor pens (40 chicks per pen) and were reared under a nearly continuous lighting programme (23 h light and 1 h dark per 24 h) until 6 weeks of age. All birds had ad libitum access to commercial broiler crumble food containing 3100 kcal/kg metabolisable energy and 210 g/kg crude protein and drinking water. The temperature was regulated as previously described (Hassanzadeh et al., 2002). Briefly, it was set initially at 33°C and gradually reduced by 1°C every 2 days until 22°C was reached. During the period of 14 to 28 days the electrical heating system was turned off during the night while the minimum environmental temperature did not descend below 15°C.

Body weights and feed intake were recorded every 2 weeks for each pen. From 10 randomly selected birds per group, venous blood samples were taken and collected in heparinized tubes on ice for the separation of plasma and for determination of plasma thyroid hormones, corticosterone and lactic acid concentrations. Mortality was recorded daily and examined for lesions of heart failure and ascites. At the end of the experiment 50 chickens randomly selected from each group were slaughtered. The heart was removed and the atria, major vessels and fat were trimmed off. The right ventricle/total ventricle (RV/TV) ratio was determined and was classified as reported earlier (Julian, 1987). A normal broiler should have a ratio of RV/TV <0.25. Birds having a RV/TV ratio within 0.25 to 0.29 were classified as moderate right ventricular hypertrophy (RVH) and those birds having a ratio over 0.29 were classified as marked RVH. Statistical analysis was performed using the “General linear model procedure” (SAS Institute Inc., 1986). If a significant overall effect (P<0.05) was found, treatment means were compared by using the Scheffé test.

Results

Hatching and embryonic parameters

When eggs were incubated in the high-altitude incubator, earlier hatching at 482 h of incubation was markedly higher (36%) compared with eggs incubated in the low-altitude (2.6%) incubation (Table 1). At the end of incubation, final hatchability was numerically lower at high altitude (81.9%) compared with low altitude (86.5%). In non-hatched eggs, embryonic mortality was numerically higher in the high-altitude incubator (7.7%) compared with the low-altitude incubator (3.6%). The number of non-fertile eggs was similar in both incubators. The relative weights of embryos from the high-altitude incubator tested to be higher, but the difference was not significant (Table 1). In contrast, mean body weights of the newly-hatched chicks at high altitude were significantly (P<0.001) lower compared with chicks hatched at low altitude.

Table 1. Hatching parameters in commercial broiler line eggs that were incubated at high and low altitudes

Parameter	High-altitude hatchery	Low-altitude hatchery
Earlier hatching at 482 h of incubation	259/720^a (36%)	19/720 (2.6%)
Final hatching	590/720 (81.9%)	623/720 (86.5%)
Non-hatched eggs
Total	130/720 (17.9%)	97/720 (13.4%)
Embryonic mortality	56/720 (7.7%)	26/720 (3.6%)
Non-fertile	74/720 (10.2%)	71/720 (9.8%)
Relative embryo weight×100	75.8±0.01 (n=30)	75.3±0.01 (n=30)
Newly-hatched chick weight (g)	48±0.2^B (n=30)	50±0.4^A (n=30)
Within rows, means with no common uppercase superscripts are significantly different (P<0.05).
^a 720 means that 30 of the 750 embryos were sacrificed for blood sample collection.

The results of plasma T₃ and T₄ concentrations, the T₃/T₄ ratio, and corticosterone and lactic acid levels of embryos at the non-external pipping stage and in newly-hatched chicks are presented in Table 2. High-altitude embryos showed significantly higher plasma T₃ (P<0.05) and T₄ (P<0.0001) levels, T₃/T₄ ratios (P<0.05) and higher corticosterone concentrations (P<0.05) compared with low-altitude embryos. However, high-altitude newly-hatched chicks showed lower plasma T₃ (P<0.01) concentrations and lower T₃/T₄ (P<0.01) ratios compared with low-altitude chicks, but no altitude effect on mean plasma T₄ and corticosterone levels was seen at this stage. Both embryos (P<0.001) and newly-hatched chicks (P<0.005) incubated at high altitude showed significantly higher plasma lactic acid concentrations than the low-altitude embryos and newly-hatched chicks.

Table 2. Plasma T₃, T₄ concentrations and T₃/T₄ ratio, lactic acid and corticosterone levels in embryos at 460 h of incubation (non external pipping stage) and in newly hatched chicks of commercial broiler eggs incubated at high and low altitudes. Values are means±SEM. (n=30)

Parameter	High-altitude hatchery	Low-altitude hatchery	P value
Plasma T3 levels (ng/ml)
Embryo	0.99±0.20	0.32±0.11	0.05
Newly-hatched chicks	0.26±0.03	0.47±0.07	0.01
Plasma T4 levels (ng/ml)
Embryo	8.87±0.89	4.54±0.69	0.0001
Newly-hatched chicks	3.04±0.18	3.38±0.21	Not significant
Plasma T3/T4 ratio
Embryo	0.157±0.04	0.062±0.01	0.05
Newly-hatched chicks	0.076±0.01	0.144±0.02	0.01
Plasma corticosterone (ng/ml)
Embryo	27.4±0.89	19.8±3.3	0.05
Newly-hatched chicks	48.7±3.3	48.8±2.7	Not significant
Plasma lactic acid (mg/dl)
Embryo	6.02±0.77	3.6±0.32	0.001
Newly-hatched chicks	30.5±4.2	18.6±2.3	0.005

Ascites mortality and RV/TV ratio

The number of broiler chickens that developed RVH and ascites in the different age and treatment groups, and the RV/TV ratios of surviving chickens that were randomly selected and slaughtered at 6 weeks of age are presented in Table 3. During the 6 weeks of experimental period, 57 (11.9%) of the 480 birds died due to RVH and ascites. The first incidence of ascites mortality occurred at 2 weeks of age in high-altitude broilers and at 5 weeks of age in low-altitude broilers. Ascites mortality was markedly higher in high-altitude conditions (50 birds) compared with low-altitude conditions (seven birds) (20.8% versus 2.9%). Within high-altitude-reared birds, a slightly higher incidence of ascites was observed in broiler chickens that hatched at low altitude compared with high-altitude-hatched chickens (27 birds versus 23 birds). Furthermore, the number of surviving birds that were showing a RV/TV ratio over 0.25 was obviously higher in low-altitude-hatched birds (13 birds ≥0.25 and 19 birds ≥0.29) compared with the high-altitude-hatched birds (5 birds ≥0.25 and 14 birds ≥0.29). However, these were not different at low altitude for both groups incubated at low or high altitude.

Table 3. Ascites mortality and RV/TV ratio in broiler chickens slaughtered at 6 weeks that were hatched and reared in two different altitude schedules

	Ascites mortality						RV/TV
Altitude of incubator–farm^a	Week 2	Week 3	Week 4	Week 5	Week 6	Total	≥25 to >29	≥9
Hh–Hg	−	3	4	8	8	23	5/50^b	14/50
Lh–Hg	2	3	4	9	9	27	13/50	19/50
Hh–Lg	−	−	−	2	1	3	2/50	5/50
Lh−Lg	−	−	−	2	2	4	3/50	4/50
^a Hh–Hg, high-altitude hatchery and high-altitude growing; Lh–Hg, low-altitude hatchery and high-altitude growing; Hh–Lg, high-altitude hatchery and low-altitude growing; Lh–Lg, low-altitude hatchery and low-altitude growing.
^b Number of RVH per 50 chickens.

Post-hatch physiological parameters

Mean plasma thyroid hormone concentrations, plasma corticosterone, lactic acid levels and the results of the statistical analyses are presented in Table 4. Plasma T₃ levels were not significantly affected by the two different altitudes, neither at hatchery nor during the growing period, except at day 28 when high-altitude-hatched chickens showed significantly (P<0.005) lower plasma T₃ concentrations compared with the low-altitude-hatched chickens. A significant (P<0.05) interaction between altitude effects during the hatchery×growing period was observed at day 42 of age.

Table 4. Plasma T₃, T₄, corticosterone and lactic acid levels of commercial broiler chickens that were hatched and reared in two different altitude schedules

	Altitude schedule^a				P value^b
	Hh–Hg	Lh–Hg	Hh–Lg	Lh–Lg	Hatchery	Growing period	Hatchery×growing period
Plasma T3 levels (ng/ml)
Day 7	2.01±0.17	1.83±0.11	1.94±0.14	1.70±0.13	NS	NS	NS
Day 14	1.68±0.13	2.03±0.19	2.09±0.26	2.11±0.15	NS	NS	NS
Day 28	1.45±0.13^B	2.13±0.20^A	1.64±0.12^B	1.94±0.17^AB	0.005	NS	NS
Day 42	0.90±0.12^B	1.04±0.13^A	1.46±0.17^A	0.91±0.06^B	NS	NS	0.05
Plasma T4 levels (ng/ml)
Day 7	6.93±0.9^A	5.62±0.9^A	3.98±0.8^B	3.61±0.5^B	NS	0.001	NS
Day 14	4.36±0.6	5.14±1.1	5.15±0.6	5.90±0.9	NS	NS	NS
Day 28	5.76±0.9^B	5.77±0.7^B	10.04±0.7^A	6.80±0.7^B	0.05	0.001	0.05
Day 42	6.51±0.7^B	7.44±0.9^B	10.25±0.6^A	11.01±0.8^A	NS	0.001	NS
Plasma corticosterone (ng/ml)
Day 7	47.5±3.3	47.8±3.8	54.5±6.3	42.5±8.9	NS	NS	NS
Day 14	39.3±6.3	44.0±4.2	33.5±4.5	41.3±6.5	NS	NS	NS
Day 28	24.8±5.5^AB	18.3±3.1^B	11.5±1.8^B	36.3±7.2^A	NS	NS	0.05
Day 42	23.8±5.5	12.8±1.4	13.1±1.9	12.1±2.8	NS	NS	NS
Plasma lactic acid (mg/dl)
Day 7	14.1±1.6	16.4±0.98	15.4±1.5	17.9±2.6	NS	NS	NS
Day 14	12.1±1.7	19.9±2.1	12.3±1.0	14.6±1.8	NS	NS	NS
Day 28	39.8±5.5^A	35.7±3.3^A	17.8±1.6^B	17.5±0.9^B	NS	0.001	NS
Day 42	38.8±4.7^A	27.7±1.4^A	22.1±2.2^B	22.3±2.9^B	NS	0.001	NS
Data presented as mean±standard error of the mean (n=10).
^a Hh–Hg, high-altitude hatchery and high-altitude growing; Lh–Hg, low-altitude hatchery and high-altitude growing; Hh–Lg, high-altitude hatchery and low-altitude growing; Lh–Lg, low-altitude hatchery and low-altitude growing.
^b NS, not significant. ^A, ^B within rows, values having a common uppercase letter are not significantly different (P<0.05).

Broiler chickens reared at high altitude showed significantly higher plasma T₄ levels compared with those chickens reared in low altitude at day 7 of age. At day 28, only high-altitude-hatched chicks that were reared at low altitude showed significantly higher values of plasma T₄. At day 42, all high-altitude-grown birds showed significantly (P<0.001) lower plasma T₄ concentrations compared with the low-altitude-grown birds.

Plasma corticosterone levels were significantly (P<0.0001) decreased with age in all groups. At day 28, chickens that were hatched and reared at high altitude or at low altitude showed the highest corticosterone levels, while a transfer from high to low altitude or vice versa resulted in lower levels of corticosterone and a significant interaction between the altitude effects of the hatchery×growing period. During the first 2 weeks of the growing period plasma lactic acid levels were not significantly affected by the altitudes, neither in the hatchery nor during the growing period. But from day 28 onwards, broiler chickens that were reared at high altitude condition had significantly (P<0.001) higher plasma lactic acid compared with those chickens reared at low altitude.

Growth performance

At 14 days of age, in both high-altitude and low-altitude farms, broiler chickens that hatched at low altitude showed significantly (P<0.001) higher mean body weight compared with those chickens that had been hatched at high altitude (Table 5). Furthermore, those chickens that were reared at low altitude showed heavier mean body weight (P<0.001) compared with those chickens reared at high altitude. At 28 and 42 days of age, those birds that were grown at low-altitude conditions showed significantly higher (P<0.01) mean body weight compared with high-altitude-grown birds. Feed intake showed the same pattern of differences as for growth, resulting in no significant differences in feed conversion ratio whatever the altitude of incubation or rearing period.

Table 5. Body weight, feed intake and feed conversion ratio in commercial broiler chickens that were hatched and reared in two different altitude schedules

	Altitude schedule^a				P value^b
	Hh–Hg	Lh–Hg	Hh–Lg	Lh–Lg	Hatchery	Growing period	Hatchery×growing period
Body weight (g/chicken)
Day 14	332±10^C	368±5^B	388±3^B	413±24^A	0.001	0.001	NS
Day 28	1032±31^B	1036±63^B	1229±3^A	1226±7^A	NS	0.01	NS
Day 42	1821±33^B	1879±29^B	1936±10^A	1959±6^A	NS	0.01	NS
Feed intake (g/chicken)
Day 1–14	424±9^C	469±12^B	518±28^AB	561±7^A	0.01	0.005	NS
Day 14–28	1183±43^B	1113±60^B	1309±21^A	1330±12^A	NS	0.005	NS
Day 28–42	1702±11	1834±90	1561±46	1669±130	NS	NS	NS
Day 1–42	3309±31	3443±24	3389±62	3563±141	NS	NS	NS
Feed conversion ratio (chicken)
Day 1–14	1.52±0.01	1.56±0.01	1.53±0.07	1.56±0.04	NS	NS	NS
Day 14–28	1.59±0.10	1.68±0.07	1.63±0.03	1.70±0.05	NS	NS	NS
Day 28–42	2.03±0.21	2.18±0.03	2.20±0.03	2.21±0.08	NS	NS	NS
Day 1–42	1.87±0.02	1.88±0.02	1.80±0.02	1.87±0.06	NS	NS	NS
Data presented as mean±standard error of the mean (n=3 pens per schedule).
^a Hh–Hg, high-altitude hatchery and high-altitude growing; Lh–Hg, low-altitude hatchery and high-altitude growing; Hh–Lg, high-altitude hatchery and low-altitude growing; Lh–Lg, low-altitude hatchery and low-altitude growing.
^b NS, not significant. ^A, ^B, ^C within rows, values having a common uppercase letter are not significantly different (P<0.05).

Discussion

The present work demonstrates that differences between the eggs incubated in a hypoxic environment at high altitude or in a normal atmospheric environment at sea level differed not only in the percentage of early (at 482 h) and final (at 508 h) hatchability of incubation, but also in plasma T₃, T₄ levels, T₃/T₄ ratio, corticosterone and lactic acid concentrations in the non-external pipping embryos. Thyroid hormones are known to be involved in the complex processes of transition from allantoic to pulmonary respiration and to play part in the length of the incubation process (Decuypere et al., 1991; Dewil et al., 1996). This was confirmed by the results of Buys et al. (1998) showing a concomitant higher activity of thyroid hormones, earlier pipping and hatching of high CO₂-incubated embryos compared with the normal CO₂-incubated embryos. On the other hand, Decuypere et al. (1983) and Meeuwis et al. (1989) demonstrated that corticosterone is required for peripheral conversion of T₄ to T₃ during the prenatal life. Thus, the higher corticosterone levels in high-altitude-incubated eggs might have served to boost the shift in T₃/T₄ ratio. Considered together, high-altitude incubation seems to favour a higher T₃/T₄ ratio. Both acting together may favour early pipping and hatching as confirmed by Tona et al. (2003). It cannot be excluded here that the stimulated earlier hatching also is reflected in an earlier increase in T₃ and T₄ levels of these embryos at high-altitude incubation, as blood sampling was performed at a similar chronological age (420 h) but probably not at a similar physiological stage. The significantly higher plasma lactic acid levels in embryos incubated at high altitude compared with embryos incubated at low altitude demonstrate anaerobic metabolism as a consequence of the hypoxic conditions at high altitude (Hassanzadeh et al., 1997).

Rouwet et al. (2002) demonstrated that chronic hypoxia during embryonic development induces structural and functional cardiovascular abnormalities (e.g. left ventricular dysfunction) in the near-term chick embryos. These abnormalities may be responsible for the increased mortality of embryo's incubated under high altitude. However, as not all embryos died, this suggests interindividual variation in adaptability to hypoxic condition (Tazawa et al., 1992). These adaptive mechanisms include an increased blood oxygen affinity and/or a decreased oxygen consumption (Hassanzadeh et al., 2002) resulting in subsequent higher embryonic mortality in susceptible embryos at high altitude. The higher embryonic mortality in the high-altitude-incubated eggs is responsible for the lower final hatchability at this altitude.

In newly-hatched chicks plasma lactic acid concentrations were still higher at high altitude compared with low-altitude-hatched chicks, and this is again indicating hypoxic conditions at the high altitude. Plasma T₃ levels and the T₃/T₄ ratio remained, but now in the opposite direction. The significantly higher T₃ levels and T_3/T₄ ratio in low-altitude chicks compared with high-altitude-hatched chicks suggest an enhanced peripheral conversion of T₄ to T₃, and this could be related to prolongation of hatching time of chicks incubated at low altitude. Since no significant difference were observed on relative embryo weight, the longer stay of chicks in the high-altitude hatchery due to their earlier hatching compared with those in the low-altitude hatchery could be most probably responsible for the lower body weight of newly-hatched chicks, as a result of increased water losses.

The observed clinical signs and the occurrence of right ventricular failure of ascitic birds correspond with those reported at high altitude and low environmental temperature (Sillau et al., 1980; Hassanzadeh et al., 2003 , 2004). The hypoxia at high altitude also increased plasma lactic acid levels during the last stage of the growing period.

Structural and endocrine changes often linked with ascites susceptibility may be influenced in early stages of development, even during embryogenesis. It is hypothesized that developmental changes induced by environmental or incubation conditions may play a role in the genotype and environment interaction in ascites susceptibility (Decuypere, 2002). Eggs incubated in an environment with a high concentration of CO₂ hatched earlier than those in an environment with normal CO₂ levels (Buys et al., 1998; Hassanzadeh et al., 2002). Moreover, the chickens incubated in the environment with increased concentrations of carbon dioxide showed a lower incidence of ascites during the growing period (Buys et al., 1997 , 1998). In the present study there was a lower incidence of ascites as well as a reduced RV/TV ratio of chickens slaughtered at 6 weeks that were hatched and grown at high altitude compared with the low-altitude-incubated eggs. This could be related to the decrease in the time the embryo experiences hypoxia, which is present in the air chamber of the embryo during the final stages of incubation, as suggested earlier by Buys et al. (1998) and Decuypere (2002). Furthermore, incubation at high altitude led to initial growth depression in high-altitude and low-altitude birds, resulting in a significantly lower body weight up to 14 days. However, no significant incubation effect on body weight was found at later ages in both altitude groups. It is generally accepted that a slowing down of the initial high growth during the first weeks of broiler raising, followed by catch-up growth, may have a beneficial effect on ascites incidence (Buys et al., 1998; Hassanzadeh et al., 2003 , 2004), which is in accordance with our data. Indeed, the lower growth rate and hence lower body weights attained during the first weeks of chicks hatched under hypoxic incubation conditions were accompanied by a lower ascites incidence at high altitude, as well as a reduced RVH and RV/TV ratio of surviving chickens at 6 weeks of age. Differences in growth curves between the high-altitude and low-altitude-grown birds with the concomitant differences on plasma lactic acid levels confirmed earlier findings of Hassanzadeh et al. (1999), demonstrating that birds from high altitude are seriously suffering from hypoxia and RVH that could consequently lead to decrease final body weight at the slaughtered age. However, Olkowski et al. (1999) and Olkowski and Classen (1998) have published results indicating that RVH and right ventricular failure are secondary to left ventricular failure. This may be brought into relation with changed thyroid function and the lower thyroid hormone levels in postnatal birds when incubated at high altitude because subclinical hypothyroidism causes left ventricle dysfunction, at least in mammals (Fazio et al., 2004).

Post-hatch data on T₃ clearly reflect the age-related decrease in T₃ levels in all groups (Kühn et al., 1982). Thyroxine clearly seems to be influenced by altitude during rearing of chickens with initial higher values (at 7 days) at high altitude compared with low altitude. But at 28 and 42 days, the reverse was observed with a lower T₄ at high altitude compared with low altitude. There was no difference between altitude groups at 14 days of age. Age-related increases were observed at low altitude as reported earlier (Kühn et al., 1982). Post-hatch results on corticosterone indicate an age-related decrease, confirming earlier findings of Decuypere et al. (1989).

In conclusion, our results showed that exposure of the embryos to chronic hypoxia at high altitude leads to changes of the physiological functions of embryos. This demonstrates again that, although the peak of ascites incidence and RVH occurs at the end of the growing period, there are strong indications that the aetiology of the syndrome may be traced as far back as the embryonic stage. The connection between incubation conditions and the pathological processes involved in the development of ascites needs further investigation.

Acknowledgments

This research was funded by the research committee of Tehran University, Faculty of Veterinary Medicine and the Laboratory for Physiology and Immunology of Domestic Animals, K.U. Leuven, Belgium. V. Bruggeman is a postdoctoral fellow of the F.W.O.-Flanders, Belgium. G. Nackaerts is acknowledged for technical assistance. Dr Chark Kar, Dr F. Fahimi and Parnia Far are acknowledged for providing incubators and farm facilities. This work was funded by the F.W.O.-Vlaanderen, project number G0286.04.