Hypoxia and hypercapnia during incubation of chicken eggs : effects on development and subsequent performance E. DECUYPERE1*, O. ONAGBESAN1, L. DE SMIT1, K. TONA1, N. EVERAERT1, A. WITTERS1, M. DEBONNE1, E. VERHOELST2, J. BUYSE1, M. HASSANZADEH3, J. DE BAERDEMAEKER2, L. ARCKENS4 and V. BRUGGEMAN1 1

Faculty of Bioscience Engineering, Department of Biosystems, Division Livestock-NutritionQuality, University of Leuven, 3001 Heverlee, Belgium. 2 Faculty of Bioscience Engineering, Department of Biosystems, Division of Mechatronics, Biostatistics and Sensors (MeBioS), University of Leuven, 3001 Heverlee, Belgium. 3 Faculty of Veterinary Medicine, University of Tehran, Iran. 4 Biology Department, Animal Physiology and Neurobiology Section, Laboratory for Neuroplasticity and Neuroproteomics, University of Leuven, 3000 Leuven, Belgium. ∗ Corresponding author: E. Decuypere, [email protected]

Oxygen (O2) and carbon dioxide (CO2) exchanges are of fundamental importance for embryonic development during incubation. They may not only affect livability of the embryo, but also affect embryonic development, hatchability, pipping and hatching events as well as later development and functioning. The effects of gaseous environment not only depend on the degree of hypoxia and hypercapnia, but even more on the time of development at which the embryo is exposed to them. O2 uptake of the egg increases exponentially as the embryo grows rapidly during the first two weeks of incubation. The surface area of the area vasculosa and the growth rate of the chorioallantois membrane are closely linked to O2 uptake. Therefore hypoxia and/or hypercapnia during the first half of incubation have been hypothesized to stimulate blood vessel development and this is currently under investigation. Recent studies have demonstrated that a gradual increased hypercapnia during the first 10 days of incubation enhanced embryo growth, stimulated early hatching and improved hatchability. Changes in pH as well as differential protein expression in the chick embryo chorioallantois membrane were studied and are presented elsewhere at this conference. At the end of incubation, hypoxia or hypercapnia may also induce physiological changes that are related to the timing and the control of pipping and hatching events as well as to cardiovascular or pulmonary changes, altering the sensitivity for ascites in later life. It was shown that increasing CO2 towards the end of incubation acts as a hatching stimulus; also hypoxic conditions at this period affect the hatching process as well as corticosterone secretion. In ovo administration of glucocorticoids also affects hatching events but this is highly dependent on the developmental stage of the embryo. High altitude incubation equally affects hatching events as well as hormonal levels. These results indicate that late prenatal hypoxia as well as hypercapnia may be beneficial for a lower incidence for ascites during the growing period of broilers. However the degree and timing of these treatments are crucial for their epigenetic or long term effects. Moreover, early or late hypercapnia and/or hypoxia are different phenomena, related to other developmental and physiological processes with different consequences in later life.

Keywords: O2; CO2; incubation; development

Oxygen (O2) and carbon dioxide (CO2) exchanges are of fundamental importance for embryonic development during incubation, together with a number of other physical factors that have to be controlled in the incubator. They may not only affect livability of the embryo, but also affect embryonic development, hatchability, pipping and hatching events as well as later development and functioning (Decuypere et al., 2001; Tona et al., 2005). In this review we will focus on the gaseous environment of the embryo, and these effects not only depend on the degree of hypoxia and hypercapnia, but even more on the time of development at which the embryo is exposed to them. O2 uptake of the egg increases exponentially as the embryo grows rapidly during the first two weeks of incubation (Tazawa, 1980). The surface area of the area vasculosa and the growth rate of the chorioallantoic membrane are closely linked to O2 uptake (Tazawa and Whittow, 2000). Therefore hypoxia and/or hypercapnia during the first half of incubation have been hypothesized to stimulate blood vessel development. Blacker et al. (2004) found that hypoxia accelerated the developmental process of chick embryo. Other studies have demonstrated that a gradual increased hypercapnia during the first 10 days of incubation enhanced embryo growth, stimulated early hatching and even improved hatchability (Gildersleeve and Boeschen, 1983; Hogg, 1997; De Smit et al., 2006). The study by De Smit et al. (2006) investigated the effect of non-ventilation of the incubator during the first 10 days of incubation on CO2 concentration in the incubator as well as on different physiological and performance parameters in embryos and post-hatch chickens. 60 NON-VENTILATED VENTILATED

* 50

pO2/pCO2

40

* 30

*

20

** 10

* *

0 8

10

12

14

16

18

20

22

INCUBATION DURATION (days)

Figure 1: Changes in the ratio of the partial pressure of O2 and CO2 in the air cell of the developing egg. (n = 15) * P < 0.05; ** P < 0.0001

The CO2 concentration in the ventilated (V)-incubators remained below 0.1 % in these first 10 days while in the non-ventilated (NV)-incubators CO2 levels rose to 1-1.5 %. Changes in albumen pH, important for early embryonic developmental processes, as well as differential protein expression in the chick embryo and CAM were studied and are presented elsewhere at this conference (Bruggeman et al., De Smit, et al.). The eggs of the NV-incubation showed higher pCO2 levels in the air cell (figure 1) from embryonic day 10 until 14 compared to the eggs of the V-group, and they showed an accelerated embryonic growth. Eggs incubated under NV conditions hatched earlier and the spread of hatch was narrower (figure 2). This is related with a higher plasma corticosterone and T3 level and higher pCO2 in the air cell at internal pipping in the NV-group. These parameters are related to each other as corticosterone has been implicated in the maturation of thyroid hormone metabolism (Decuypere et al., 1983) and glucocorticoids and thyroid hormones are involved in the preparation for pipping and hatching process (Decuypere et al., 1991).

EXPERIMENT 2

HATCHING PERCENTAGE PER 2 HOURS (%)

HATCHING PERCENTAGE PER 2 HOURS (%)

EXPERIMENT 1

30 NON-VENTILATED VENTILATED

25

20

15

10

5

0 460

470

480

490

500

30 NON-VENTILATED VENTILATED 25

20

15

10

5

0 460

510

470

480

490

500

510

INCUBATION DURATION (h)

INCUBATION DURATION (h)

A

B Experiment 1 NV (n = 120)

Average hatching time (h) % hatch

484.70 ± 0.50

b

89.25% a

Experiment 2

V (n = 120) 494.61 ± 0.64

NV (n = 120) a

85.35% b a

Average chick mass at hatch (g)

54.14 ± 0.71

Relative chick weight at hatch (%)

74.9 ± 0.2 % ab

54.04 ± 0.99

480.56 ± 0.55

V (n = 120) c

92.53%a a

75.7 ± 0.3 % a

495.5 ± 0.56 a 91.60%a

b

50.36 ± 0.33 b

75.0 ± 0.2 % ab

74.5 ± 0.2 % b

50.64 ± 0.34

C Figure 2: Spread of hatch. Average hatching time, %hatch, chick masses and relative chick weights at hatch (C) and comparative percentage of hatched chicks of the non-ventilated (NV) and ventilated (V) incubation group in experiment 1 (A) and experiment 2 (B). a,b,c

Means without common superscript letters within a row are significantly different (P < 0.05)

During the post-hatch period, the chicks of the NV-group had a different relative growth pattern, reaching their maximum relative growth already after 1 week, while this was somewhat postponed in chicks from the V-group. While severe hypoxia prenatally has repercussions on the vascular development of the chick, inducing hypertrophy of the heart (Villamor et al., 2004) and the aorta (Rouwet et al., 2002), nonventilation during the first 10 days resulted in a significantly increased luminal diameter of the aorta while the wall/lumen ratio decreased compared to the V-group (figure 3). All these results point to more or less persistent, epigenetic effects of hypoxia/hypercapnia during critical or sensitive periods of incubation. At the end of incubation, hypoxia or hypercapnia may also induce physiological changes that are related to the timing and control of pipping and hatching events as well as to cardiovascular or pulmonary changes, altering the sensitivity for ascites later in life. Increasing CO2 towards the end of incubation acts as a hatching stimulus but also the hypoxic condition of high altitude incubation equally affects hatching events as well as hormonal levels. This was clearly demonstrated by a study of Hassanzadeh et al. (2004). Embryos incubated at high altitude had higher plasma triiodothyronine, thyroxine, corticosterone levels and hatched earlier than those incubated at low altitude. Moreover, chickens that had been incubated at high altitude show less right ventricular hypertrophy and ascites mortality than those incubated at low altitude and reared at high altitude. Although later prenatal hypoxia as well as hypercapnia may be beneficial for a lower incidence for ascites during the growing period of broilers, early hypercapnia as induced by NV during the first 10 days of incubation may result in an increased sensitivity for ascites-inducing factors (De Smit et al.,

unpublished results). This indicates that the timing of these treatments is crucial for their long term epigenetic effects. Moreover, early or late hypercapnia and/or hypoxia are different phenomena, related to other developmental and physiological processes with different consequences in later life.

NON-VENTILATED

600

VENTILATED

P < 0.05

1,5 1,4

1,2 1,1

400

WALL/LUMEN RATIO

LUMINAL DIAMETER (µm)

P < 0.001

1,3

500

300

200

1 0,9 0,8 0,7 0,6 0,5 0,4 0,3

100

0,2 0,1

0 NON-VENTILATED

VENTILATED

LUMINAL DIAMETER

0 NON-VENTILATED

VENTILATED

WALL/LUMEN RATIO

Figure 3: Comparison of the luminal diameter and the wall/lumen ratio of hematoxyline/eosine stained sections of the ascending aorta of chick embryos of the NV-group and the V-group at IP-stage.

The importance of timing in interfering with or changing physiological processes was nicely illustrated by a recent study of Tona et al. (2006) administering a synthetic glucocorticoid (dexamethasone) at 2 stages of development (d16 or d18) in embryos that had been incubated under NV or V conditions for the first 10 days. Dexamethasone injected at day 18 increased T3 levels at IP and advanced hatching as well as shortening the hatching process. Injecting at day 16 however did not result in increased T3 levels at IP while the effects on hatching and hatching process were reversed. Moreover hatchability was significantly decreased when dexamethasone was injected at day 16 (and even more in the V than in the NV group). Since T3 and glucocorticoids have been implicated in the hatching process, the untimely elevation of glucocorticoid at day 16 may be detrimental to the embryo when it was not capable of initiating physiological response to its endocrine stimuli. Moreover, injections of dexamethasone at day 16 caused a rebound effect on the functioning of the hypothalamopituitary-adrenal (HPA) axis in early postnatal life which was not observed in chickens injected at day 18 of their incubation. This disturbance in HPA axis establishment may cause an increased functioning and has been documented and reviewed earlier (Decuypere and Michels, 1992). This points to the importance of critical phases in development and carry-over effects in later life when interfering with these critical periods.

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