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Poult Sci 2007. 86:904-908
© 2007 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Blood Gas, Hemoglobin, and Growth of Tibetan Chicken Embryos Incubated at High Altitude

Z. H. Wei*,{dagger}, H. Zhang*, C. L. Jia*,{dagger}, Y. Ling*, X. Gou*, X. M. Deng* and C. X. Wu*,1

* College of Animal Science and Technology, China Agricultural University, Beijing, 100094, China; and {dagger} College of Animal Science and Technology, Northwest A&F University, Shaanxi, 712100, China

1 Corresponding author: lingzi{at}cau.edu.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metabolism and hatchability are impaired when chicken eggs laid at sea level are incubated at high altitude. The Tibetan chicken is an excellent local poultry breed that inhabits altitudes of 2,900 m and has a hatchability of approximately 75% at that altitude. To understand how Tibetan chicken embryos develop successfully at high altitude, we compared blood gas, pH, hemoglobin concentrations and embryo mass for Tibetan chicken embryos (T) and for embryos from a dwarf breed (D) that normally is reared at sea level. The 2 breeds (T and D) and 2 incubation altitudes (2,900 m = high, H; and 100 m = low, L) were compared at 9, 12, 15, and 18 d of incubation. Embryo weights were lower for the high altitude groups (TH, DH) than for the low altitude groups at all stages of incubation. The embryo mass of TH appeared to increase more quickly than that of DH. Compared with DH, TH embryos had lower arterialized oxygen partial pressure on d 18, higher venous carbon dioxide partial pressure from d 12 to 18, and higher hemoglobin concentration and lower venous blood pH values on d 12 and 15. These findings indicate that the ability of the Tibetan chicken embryos to adapt to the high altitude may be due to the increase in hemoglobin concentration, which augments the blood oxygen-carrying capacity. In addition, the higher venous carbon dioxide partial pressure and lower venous blood pH promote unloading of oxygen from hemoglobin.

Key Words: Tibetan chicken embryo • blood gas • hemoglobin • growth • high altitude


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Birds are the most successful vertebrate class in terms of survival and reproduction at high altitude (Black and Snyder, 1980). However, there is little research published on native high altitude domestic poultry. The Tibetan chicken is an excellent local poultry breed that inhabits altitudes of 2,900 m and has a hatchability of approximately 75% at that altitude. When sea-level-dwelling chicken eggs are moved to high altitude, the hatchability is only 37%, compared with a normal hatchability of about 90% at sea level according to our previous work (Zehui and Changxin, 2005). Some investigators have shown that this decrease in hatchability is mainly due to the decrease in the partial pressure of oxygen with altitude (Stephen and Ploog, 1967). Because the lungs of the chicken embryo are not functional until the end of incubation, oxygen moves into eggs mainly by diffusion through microscopic pores in the shell, then through the shell membranes into chorioallantoic capillaries, and ultimately into the tissues by blood transport. Carbon dioxide moves in the opposite direction (Wangensteen and Rahn, 1970–1971; Carey et al., 1993). Adaptations to potentiate oxygen transport into embryos under hypoxia may occur at any one or all of these steps (Snyder et al., 1982b). Recently, we studied eggshell conductance and found that the eggshell conductance of Tibetan chickens was significantly lower than that of the sea-level chicken, which would not appear to be beneficial for oxygen transport. Therefore, we compared blood gas, blood pH, hemoglobin concentration, and mass of Tibetan chicken embryos with those of the sea-level chicken at 2,900 m and 100 m to understand how Tibetan chicken embryos develop successfully at high altitude.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Eggs and Incubation
Eggs from a flock of Tibetan hens (T) were gathered and incubated at the poultry farm in the College of Agriculture and Animal Husbandry of Tibet University at Tibet (2,900 m = high, H), which were designated as the Tibetan chicken eggs incubated at high altitude (TH). One-day-old chicks produced by this flock were transported to a farm at China Agriculture University in Beijing (100 m = low, L). Eggs laid by these chickens after maturity were collected and incubated in Beijing, which were designated as the Tibetan chicken eggs incubated at sea level (TL). Eggs from a single flock of Dwarf chicken hens (D) were randomly divided into 2 groups. One group (DL) was set in the incubator together with TL eggs at 100 m, whereas the second group (DH) was transported to Tibet and set in the incubator together with TH eggs at 2,900 m. Eggs were incubated at 37.5° C, relative humidity of about 55%, and turned every 4 h.

Blood Sampling and Blood Gas Analysis
About 0.5 mL of arterialized blood (from an allantoic vein) or venous blood (from an allantoic artery) was collected into a heparinized 1.0-mL syringe via a 23-gauge needle from eggs of 12, 15, and 18 d during incubation; however, on d 9 only 120 µL of blood was collected. The time from taking eggs out of the incubator to blood withdrawal was less than 5 min. After sampling, 120 µL of the blood sample was immediately analyzed with a Radiometer ABL5 blood-gas analyzer (Radiometer, Co-penhagen, Denmark) for determination of PO2, PCO2 , and pH; the remaining blood was used for the measurement of hemoglobin concentration. Because the temperature inside the eggs has been found to be higher than the incubator temperature during the second half of incubation (Tazawa et al., 1983), the temperature for analysis was set at 39° C.

Hemoglobin
Hemolyzed blood was centrifuged at 12,000 x g. The supernatant was used for the measurement of hemoglobin concentration by a cyanmethemoglobin method (Tazawa et al., 1988).

Body Mass
Embryos were taken out of eggs and decapitated. Then, embryos were freed from yolk and embryonic membranes, blotted, and weighed.

Statistics
The results were presented throughout the text and figures as LMS ± SE. All data were analyzed by GLM procedure of SAS 8.0 (SAS Institute Inc., Cary, NC). The model was as follows: Yij = µ+ Ai + Bj + {varepsilon}ij, where Yij is venous or arterialized CO2 or O2 partial pressure, or venous or arterialized blood pH, or hemoglobin concentration. In this model, µ is the overall mean, Ai is altitude (i = 1, 2), Bj is breeding (j = 1, 2), and {varepsilon}ij is the residual error term. In all analyses, nonsignificant interactions were deleted from the model. Differences between embryonic mass were examined using GLM, and initial egg mass was used as a covariate for embryo mass. The significance of differences between means was determined with the PDIFF option of the LSMEANS statement of SAS 8.0 software. Differences between means were considered significant at the P < 0.05 level.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Blood Gases
Venous and arterialized PCO2 of chicken embryos gradually increase during incubation at sea level or high altitude, whereas PO2 decreases. At sea level, venous and arterialized PCO 2 or PO2 in TL and DL embryos did not differ significantly throughout the incubation (Figure 1Go). At high altitude, venous CO2 partial pressure (PvCO2) of TH embryos was significantly higher than that of DH embryos from d 12 to 18, but venous O2 partial pressure (PvO2) did not differ significantly between TH and DH embryos. Arterialized CO2 partial pressure (PaCO2) of TH embryos was significantly higher than that of DH embryos only on d 15, whereas arterialized O2 partial pressure (PaO2) of TH embryos was significantly lower than that of DH embryos only on d 18.


Figure 1
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  		Figure 1. Arterialized (PaO2 and PaCO2) and venous (PvO2 and PvCO2) blood gases of Tibetan and Dwarf chicken embryos incubated at 2,900 m and sea level. a–cWithin each incubation period, values sharing no common letter differ (P < 0.05). DH = Dwarf chicken eggs incubated at high altitude; DL = Dwarf chicken eggs incubated at sea level; TH = Tibetan chicken eggs incubated at high altitude; and TL = Tibetan chicken eggs incubated at sea level.

 
The PvCO2 and PaCO2 of TH embryos were significantly lower than those of TL embryos with the exception of d 9; this pattern also occurred in the Dwarf chicken embryos. The PvO2 of TH embryos were significantly lower than that of TL embryos on d 9, which was reversed on d 18. The PaO2 of TH embryos were significantly lower than that of TL embryos on d 9, 12 and 15; however, on d 18, this was reversed. The PvO2 and PaO2 of DH embryos were significantly lower than that of DL embryos on d 9 and 12, but on d 18 this was also reversed.

Blood pH
At sea level, arterialized blood pH values of TL embryos were significantly higher than those of DL embryos on d 18, but on other determined days there were no significant differences (Figure 2Go). At high altitude, arterialized blood pH values of TH embryos were significantly lower than that of DH only on d 15. Venous blood pH values of TH embryos were significantly lower than that of DH embryos except on d 9, when there was no significant difference.


Figure 2
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  		Figure 2. The arterialized and venous blood pH values of Tibetan and Dwarf chicken embryos during development. a–cWithin each incubation period, values sharing no common letter differ (P < 0.05). DH = Dwarf chicken eggs incubated at high altitude; DL = Dwarf chicken eggs incubated at sea level; TH = Tibetan chicken eggs incubated at high altitude; and TL = Tibetan chicken eggs incubated at sea level.

 
Comparing the sea level results with the high altitude results, the arterialized blood pH value of DH embryos was significantly higher than that of DL embryos (except on d 9 when there was no significant difference). Venous blood pH values of DH embryos were significantly higher than those of DL embryos on d 12 and 15. Arterialized and venous blood pH values of TH embryos were significantly higher than those of TL embryos only on d 12 and 15, respectively.

Embryo Mass
During the incubation period, the embryo mass of both high altitude groups was significantly smaller than that of the sea-level groups (Figure 3Go). On d 9 and 12, the embryo mass of DL embryos was significant smaller than that of TL embryos, and this was reversed on d 15 and 18. At high altitude, the embryo mass of DH embryos was significantly smaller than that of TH on d 18.


Figure 3
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  		Figure 3. The embryo mass of Tibetan and Dwarf chicken embryos incubated at high altitude and sea level. a–cWithin each incubation period, values sharing no common letter differ (P < 0.05). DH = Dwarf chicken eggs incubated at high altitude; DL = Dwarf chicken eggs incubated at sea level; TH = Tibetan chicken eggs incubated at high altitude; and TL = Tibetan chicken eggs incubated at sea level.

 
Hemoglobin
At sea level, hemoglobin concentration (Table 1Go) of TL embryos was significantly higher than that of DL embryos only on d 12, and at high altitude hemoglobin concentration of TH embryos was significantly higher than that of DH embryos on d 12 and 15. In the Dwarf chicken group, hemoglobin concentration of DH embryos was significantly higher than that of DL embryos only on d 18. In the Tibetan chicken group, hemoglobin concentration of TH embryos was significantly higher than that of TL embryos on d 15 and 18.


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  		Table 1. The hemoglobin concentration (g/100 mL) of Tibetan chicken and Dwarf chicken embryos incubated at 2,900 and 100 m1
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A number of studies have shown that chronic hypoxia decreases the growth of developing chicken embryos (Beattie and Smith, 1975; McCutcheon et al., 1982; Dzialowski et al., 2002). Similarly, in our study, the embryo mass of high-altitude groups was significantly smaller than that of the sea-level groups regardless of breed. However, Snyder et al. (1982a) have reported that embryos of bar-headed goose (a species native to high altitude) are able to maintain normal rates of growth and metabolism when incubated under conditions of hypoxia corresponding to an altitude of 3,700 m. Interestingly, in our study, we found the embryo mass of TH increased faster than that of DH, which can survive until 18 d, although at high altitude the rate of Tibetan chicken embryo growth was impaired. Handrich and Girard (1985) have reported that embryo mass and oxygen consumption were strongly correlated. Our study shows that Pv CO2 of TH embryos was higher than that of DH embryos from d 12 to 18, which indicates TH has higher oxygen consumption than DH. The initial step in gas transport for the avian embryo is diffusion across the eggshell. However, according to the previous study, the eggshell conductance of the Tibetan chicken was lower than the Dwarf chicken eggshell, which appears not to be beneficial to oxygen transport. (Zehui and Changxin, 2005). Thus, TH embryos must have other physiological adaptations at the blood and tissue level. By the determination of PaO2, we also found that PaO2 of TH embryos was significantly lower than that of DH embryos on d 18. Therefore, it is possible that TH embryos can maintain relatively normal growth by increasing the capacity for oxygen transport by blood under conditions of hypoxia.

In avian embryos, the greatest part of the resistance of the inner barrier to oxygen transport has been attributed to the oxygen binding in the blood (Wagner-Amos and Seymour, 2003; Leon-Verlarde and Monge, 2004). An increase in hemoglobin concentration augments the blood oxygen-carrying capacity (Snyder et al., 1982b). We have found that the hemoglobin concentration of TH was significantly higher than that of DH on d 12 and 15 at high altitude. In general, the relative increase in hemoglobin concentration is greater in natives of high altitude than in sojourners (Monge and Leon-Velarde, 1991). This is consistent with observations of mammals living at altitude. On d 18, the significance between TH and DH was impaired, which may be because only individuals having a higher hemoglobin concentration could survive at this time. The DH embryos and TH embryos had significantly higher hemoglobin concentrations compared with DL and TL embryos on d 18. Similarly, Carey et al. (1993) has also reported that the hemoglobin concentration of lowland and mountain coot (Fulica americana peruviana) embryos increased significantly with the developmental increase in body mass, and high altitude coot embryos measured at 4,200 m have significantly higher hemoglobin concentrations than sea level coot embryos. However, an obvious increase in hemoglobin concentration of TH occurred relatively earlier than that of DH. Dzialowski et al. (2002) also discovered that hemoglobin levels increased in response to hypoxia during late stages of development in chicken embryos.

At sea level, there were no significant differences between TL and DL on venous blood pH values. However, venous blood pH values of DH were significantly higher than those of DL on d 12 and 15, which could cause a leftward shift in the blood O2 dissociation curve. As venous blood pH values of TH were significantly lower than that of DH during the middle and late stage, so the blood O2 dissociation curve of TH lies to the right of DH. This suggests that the lower pH values of venous blood of Tibetan chicken embryos when compared with Dwarf chicken embryos promotes O2 unloading from hemoglobin at high altitude.


   ACKNOWLEDGMENTS
 
This work was supported by the Key grant Project of Chinese Ministry of Education (No. 10404). The authors express their sincere appreciation to the College of Agriculture and Animal Husbandry of Tibet University for providing the high altitude experimental base.

Received for publication August 18, 2006. Accepted for publication November 22, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Beattie, J., and A. H. Smith. 1975. Metabolic adaptation of the chick embryo to chronic hypoxia. Am. J. Physiol. 228:1346–1350.[Abstract/Free Full Text]

Black, C. P., and G. K. Snyder. 1980. Oxygen transport in the avian egg at high altitude. Am. Zool. 20:461–468.[ISI]

Carey, C., O. Dunin-Borkowski, F. Leon-Velarde, D. Espinoza, and C. Monge. 1993. Blood gas, pH and hematology of montane and lowland coot embryos. Respir. Physiol. 93:151–163.[ISI][Medline]

Dzialowski, E. M., D. Plettenberg, A. N. Elmonoufy, and W. W. Burggren. 2002. Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos. Comp. Biochem. Physiol. 131(A):713–724.[Medline]

Handrich, Y., and H. Girard. 1985. Gas diffusive conductance of sea-level hen eggs incubation at 2900 m altitude. Respir. Physiol. 60:237–252.[ISI][Medline]

Leon-Velarde, F., and C. Monge. 2004. Avian embryos in hypoxic environments. Respir. Physiol. Neurobiol. 141:331–343.[ISI][Medline]

McCutcheon, I. E., J. Metcalfe, A. B. Metzenberg, and T. Ettinger. 1982. Organ growth in hyperoxic and hypoxic chick embryos. Respir. Physiol. 50:153–163.[ISI][Medline]

Monge, C., and F. Leon-Velarde. 1991. Physiological adaptation to high altitude: Oxygen transport in mammals and birds. Physiol. Rev. 71:1135–1172.[Free Full Text]

Snyder, G. K., C. P. Black, and G. F. Birchard. 1982a. Development and metabolism during hypoxia in embryos of high altitude Anser indicus, versus sea level, Branta canadensis geese. Physiol. Zool. 55:113–123.

Snyder, G. K., C. P. Black, G. F. Birchard, and R. Lucich. 1982b. Respiratory properties of blood from embryos of highland vs. lowland geese. J. Appl. Physiol. 53:1432–1438.[Abstract/Free Full Text]

Stephen, B. F., and H. P. Ploog. 1967. Incubation of chicken eggs at high altitudes, 10500 ft. (3200 m). World’s Poult. Sci. J. 23:346–354.[ISI][Medline]

Tazawa, H., S. Nakazawa, A. Okuda, and G. C. Whittow. 1988. Short-term effects of altered shell conductance on oxygen uptake and hematologyical variables of late chicken embryos. Respir. Physiol. 74:199–210.[ISI][Medline]

Tazawa, H., A. H. J. Visschedijk, J. Wittmann, and J. Piiper. 1983. Gas exchange, blood gases and acid-base status in the chick before, during and after hatching. Respir. Physiol. 53:173–185.[ISI][Medline]

Wagner-Amos, K., and R. S. Seymour. 2003. Effect of local shell conductance on the vascularisation of the chicken chorioallantoic membrane. Respir. Physiol. Neurobiol. 134:155–167.[ISI][Medline]

Wangensteen, O. D., and H. Rahn. 1970–1971. Respiration gas exchange by the embryos. Respir. Physiol. 11:31–45.

Zehui, W., and W. Changxin. 2005. A relation of eggshell conductance of Tibetan chicken to its water loss. J. China Agri. Uni. 10:41–44.




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