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Poult Sci 2007. 86:1384-1389
© 2007 Poultry Science Association
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PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION

Blood Characteristics for High Altitude Adaptation in Tibetan Chickens1

H. Zhang*, C. X. Wu*,2, Y. Chamba{dagger} and Y. Ling*

* College of Animal Science and Technology, China Agricultural University, Bejing, China 100094; and {dagger} College of Agriculture and Animal Husbandry, Tibet University, Linzhi, Tibet, China 860000

2 Corresponding author: chxwu{at}public.bta.net.cn


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tibetan chickens, a unique chicken breed native to high altitude, have good adaptation to hypoxia. The experiment was conducted to determine the adaptive blood characteristics in Tibetan chickens. Fertile eggs from Tibetan and Dwarf Recessive White chickens were incubated, and the chicks were reared until 10 wk of age at low altitude (100 m) and high altitude (2,900 m). At 1 d and 2, 6, and 10 wk of age, the hematological characteristics, blood gas value, and blood volume were measured. Tibetan chickens had more red blood cells (RBC), smaller mean cell volume, lower pH and partial pressure of oxygen, and higher partial pressure of carbon dioxide at high altitude and had lower blood volume, erythrocyte volume, and plasma volume at low and high altitude than Dwarf Recessive White chickens. Tibetan chickens reared at high altitude retained a high level of RBC and a stable level of hematocrit from younger to older, but Dwarf Recessive White chickens reared at high altitude presented an increase in RBC and hematocrit values. It was concluded the adaptation was achieved in Tibetan chickens by increase in RBC and blood oxygen affinity, decrease in mean cell volume, and reducing susceptivity to hypocapnia.

Key Words: Tibetan chicken • high altitude adaptation • hematological characteristic • blood gas • blood volume


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Tibetan chicken (Gallus gallus), a montane chicken breed, has a very wide distribution at altitudes of 2,200 to 4,100 m in the Qinghai-Tibet Plateau. Although the birds are exposed to hypobaric hypoxia at these altitudes, this breed, with a history of domestication more than 1,000 yr at high altitude, breeds successfully and has good resistance to chronic mountain sickness (Zhang et al., 2005, 2006). Tibetan chickens have good adaptations to high altitude and therefore are good subjects for investigating the mechanisms of adaptation.

Monge and Leon-Velarde (1991) pointed out, in an excellent review about physiological adaptation to high altitude in mammals and birds, that a high O2-hemoglobin (Hb) affinity, a moderate or absent polycythemic response, a low venous partial pressure of oxygen (pO2), a thin-walled pulmonary vascular tree, and the absence of chronic mountain sickness are characteristics of a genotypically adapted high-altitude mammal or bird, and these conditions are maintained at sea level and are transmitted to the descendants. Bar-headed geese (Anser indicus), a species that breeds on the Tibetan Plateau and migrates from near sea-level conditions to elevations as high as 9,200 m, had a high oxygen affinity and invariable values of red blood cells (RBC), hematocrit (Hct), Hb, and mean cell volume (MCV) in conditions of sea level or high altitude (Black and Tenney, 1980). Ye et al. (1994) reported that highland native animals had higher RBC numbers and smaller MCV, which are advantageous for oxygen transport. Blood gases are sensitive to the hypoxic environment, with reductions in the arterial pO2 matching the reduction in atmospheric pO2 in Peking ducks and Bar-headed geese (Faraci et al., 1985). Increases in blood volume (BV) also are important in increasing hypoxic tolerance (Birchard and Tenney, 1990), but potential changes in BV in chickens at different altitudes have not been reported previously.

Several facets of high altitude adaptation have been evaluated in lowland chickens (Leon-Velarde and Monge, 2004), but there are few evaluations of chickens that are native to high altitude. The question addressed in the present study concerns how hematological characteristics, blood gases, and BV compare between high altitude native chickens and lowland chickens, both at low and high altitudes, and how the Tibetan chickens adapt to high altitude hypoxia in blood characteristics.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Tibetan chickens (Gallus gallus) from Linzhi, Tibet (2,900 m altitude) were transported to Beijing (100 m altitude) in 2002, where they were bred for 2 generations. One thousand fertile eggs were collected from the second-generation Tibetan chickens (TC) and Dwarf Recessive White chickens (DRWC), a breed that normally is reared at sea level. Half of the eggs were transported to Linzhi by air. Eggs were incubated, and chicks were reared, at Linzhi and Beijing. The managements and feeding regimen were the same for all the birds.

Blood samples (about 0.5 mL) were obtained by direct cardiac puncture into heparinized syringes from chicks at hatch and at 2, 6, and 10 wk of age. Less than 2 min was required to complete the entire sampling procedure. In general, most chicks became quiet and gentle in the palm and shut their eyes while the blood was sampled. The bright red arterialized blood from the left ventricle was obviously different from the dark red mixed venous blood from the right ventricle. Because both bloods were clearly distinguishable by color, an eventual mixing of bloods could be readily recognized, and mixed samples were not used for blood gases. The number of samples for each group was not less than 10 at each sampling age.

Red blood cell counts (million/mL) were measured using a hemocytometer by the Hagem dilution method. Hematocrit (%) was measured by centrifuging 5 min at 13,600 x g. Hemoglobin (g/100 mL) concentration was measured spectrophotometrically using E540 = 11.0 mm–1·cm–1 on a heme basis. Mean cell volume (µm3) was calculated from RBC and Hct measurements (Dacie and Lewis, 1975). Blood volume was measured using a modification of the Evans Blue dye dilution technique (El-Sayed et al., 1995) in 10-wk-old chicks only. Total BV was calculated from Hct and PV (mL/kg) measurements. Erythrocyte volume (mL/kg) was calculated as the difference between total BV and plasma volume. Blood gases including pH, partial pressure of CO2 pCO2, mmHg), and pO2 (mmHg) were determined at each sampling time using a calibrated blood gas analyzer (ABL-5, Radiometer, Copenhagen, Denmark) at 41°C.

Statistical analyses were performed using a 3-way AN-OVA, and the least square means were calculated for multiple range test. Analyses of variance were performed to assess the statistical significance of the effects of altitude, breed, age, and the interactions for hematological parameters and blood gases, and the effects of altitude, breed, sex, and interactions for BV. The analysis was carried out with software of SAS 8.02 (SAS Inst. Inc., Cary, NC).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hematological Characteristics
A summary of effects of altitude, breed, age, and the interactions on hematological parameters is presented in Table 1Go. The age of 1 d and 2, 6, and 10 wk were divided into 2 periods, younger (1 d and 2 wk) and older (6 and 10 wk).


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  		Table 1. Summary of hematological characteristics and the effects of altitude, breed, and age; means value ± SE and t-value; or F- and P-values for statistical analyses1
 
Effects of altitude, breed, age, and the interactions of altitude x breed, and altitude x breed x age on RBC and Hct were significant (P < 0.001; Table 1Go). The mean of RBC value for birds reared at high altitude (2.86 million/mL) was significantly higher than for birds reared at low altitude (1.77 million/mL). Similar degrees of significance in RBC were seen in favor of TC (2.47) compared with DRWC (2.16) and older (2.55) compared with younger birds (2.08). The TC had significantly more RBC (3.17) than the DRWC (2.54) when they both were reared at high altitude, but they had similar RBC value at low altitude. The RBC increased significantly from younger to older in TC and DRWC at low and high altitudes, but the difference was the biggest in DRWC reared at high altitude.

The Hct values were influenced also by altitude, breed, age, and the interactions of altitude x breed and altitude x breed x age were significant. The mean Hct value of the birds reared at high altitude (36.49%) was increased compared with the birds reared at low altitude (29.73). Similar levels of significance were recorded between TC (32.12) and DRWC (34.09), and younger (31.21) and older (35.01). The TC had a significantly lower Hct value (35.14) than the DRWC (37.84) when they both were reared at high altitude, but did not at low altitude. At high altitude the Hct value in TC increased insignificantly from younger to older (34.46 to 35.82), but that value in DRWC increased significantly (35.44 to 40.34).

There was a significant increase in Hb value in the birds reared at high altitude (10.45 g/100 mL) compared with those birds reared at low altitude (9.49). Similar change was also recorded in older birds (10.54) compared with younger ones (9.40). The effects of breed and interactions were not significant (P > 0.05).

Table 1Go shows that the mean MCV value for the high altitude birds (131.7 µm3) was significantly lower than for the low altitude birds (171.1). The TC had significantly smaller red cells (139.7) than the DRWC (162.3); especially at high altitude the TC had much lower MCV value (111.5) than the DRWC at high altitude (152.1). When they both were reared at low altitude there was not a significant difference in MCV value between the 2 breeds. There was a tendency for TC and DRWC to be smaller in older (143.2) than in younger birds (158.7). The interaction of altitude x breed x age on MCV is significant (P < 0.001). A significant decrease in MCV value from younger to older showed in the TC reared at lowland and in the DRWC reared at highland.

Blood Gases
As shown in Table 2Go, the venous pH values were affected significantly by altitude, breed, and the interaction between them. The mean pH value of birds at high altitude (7.41) significantly increased compared with that of birds at low altitude (7.36; P < 0.001). The TC had significantly lower pH value (7.40) than the DRWC (7.42) when they were reared at high altitude (P < 0.01), but the difference between the 2 breeds was not significant when they were reared at low altitude.


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  		Table 2. Summary of blood gases (mmHg) and the effects of altitude, breed, and age; means value ± SE and t-value; or F- and P-values for statistical analyses1
 
The mean venous pCO2 value of the high altitude birds (37.3 mmHg) was significantly reduced compared with the low altitude birds (41.0; P < 0.001). Similar level of significance was recorded between TC (40.5) and DRWC (37.7; P < 0.001). A significant difference in pO2 value between the 2 breeds was present only when they were reared at high altitude (P < 0.001).

Table 2Go shows that there were significant effects of altitude and breed on venous pO2 value. Birds reared at high altitude had a significantly lower venous pO2 value (50.3 mmHg) than birds reared at low altitude (52.5; P < 0.05). There was also a tendency to be lower in TC (49.1) than in DRWC (53.6; P < 0.001); there was no further significant difference in venous pO2 with age.

The mean arterial pO2 value for birds reared at high altitude (87.8 mmHg) was significantly lower than for birds reared at low altitude (97.8; P < 0.001). The TC had lower arterial pO2 (90.5) than the DRWC (95.0) and older birds (89.9) lower than younger birds (95.7). The interactive effects of altitude, breed, and age on pO2 were not significant (P > 0.05).

Blood Volume
A summary of the effect of altitude, breed, sex, and the interaction of each on birds’ BV is presented in Table 3Go. There were significant reductions in BV and PV values in the birds at high altitude (93.0 and 55.9 mL/kg) compared with those birds at low altitude (101.7 and 68.8). However, the mean EV value for the birds at high altitude (37.2) was significantly higher than for the birds at low altitude (32.9; P < 0.05). The TC had lower values of BV (80.5), PV (54.6), and EV (25.9) than the DRWC (114.2, 70.1, and 44.1, respectively), and males had higher values of BV (107/4), PV (68.8), and EV (38.6) than females (87.3, 55.9, and 31.4, respectively).


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  		Table 3. Summary of blood volume (BV; mL/kg) and the effects of altitude, breed, sex; means ± SE and t-value, or F- and P-values for statistical analyses1
 
The interactive effects between altitude and breed were significant for BV and PV, but were not for EV. The BV, EV, and PV values in the TC were relatively stable from low to high altitudes (P > 0.05), but the BV and PV values of the DRWC at high altitude decreased compared with those at low altitude (P < 0.001), only the EV values didn’t change markedly (P > 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During continuous exposure to high altitude, animals develop several physiological responses to make it possible to live in a low O2 environment. Increase of RBC numbers is one of the most important hematological acclimatizations, and it appears after 1 to 2 wk (Monge and Leon-Velarde, 1991). In present study, when the chickens were maintained at high altitude, they all had increases in RBC, Hct, and Hb induced by hypobaric hypoxia. An increase in RBC and Hb increases the oxygen-carrying capacity of blood and acts as a compensatory mechanism to the stimulus of reduced oxygen saturation (Yersin et al., 1992). Tibetan chickens reared at high altitude had higher Hct than chickens reared at low altitude but had lower Hct than lowland native chickens reared at high altitude, which was consistent with high altitude native white-tailed ptarmigan (Carey and Martin, 1997) and a long resident on high altitude, the Tibetan people (Wu et al., 2005). Further, at high altitude an increase in RBC and a decrease in MCV occurred simultaneously in TC, so the total surface of RBC was enlarged, which was advantageous for hemoglobin to bind oxygen and would prevent partly the increase of blood viscosity that resulted from polycythemia. Similar results were reported about high altitude native mammals (Bunn and Poyton, 1996; Wu et al., 2005). So the increase in RBC and the decrease in MCV may be the common hematological mechanism for mammals and birds to adapt to high altitude hypoxia. Tibetan chickens reared at high altitude remained a high level of RBC and a stable level of Hct from younger to older, but DRWC reared at high altitude presented an increase from younger to older in RBC and Hct values. The results indicated that at low O2 environment the higher level of RBC in TC was genetic adaptation but in DRWC was a physiological compensatory response.

Reduced environmental oxygen availability at high altitude stimulates ventilation, which plays an important role in maintaining an adequate oxygen transfer to the blood for mammals and birds (Monge and Leon-Velarde, 1991), but also increases the exhalation of CO2 and results in hypocapnia (Besch et al., 1971). So the high altitude natives blunt hypoxic ventilatory response, serving to counteract acid-base problems arising from hyperventilation (Hochachka et al., 1999). At high altitude, TC exhibited significant lower value in pH and higher value in pCO2 than DRWC. The results indicated TC were less susceptible to hypocapnia than DRWC under conditions of hypoxia. According to Powell (1990), the most important consequence of acclimatization in ducks was hypocapnia and not enhanced O2 delivery. During acute severe hypoxia, the pO2 declined in bar-headed geese of the Himalayas and lowland Pekin ducks (Faraci et al., 1984, 1985). Consistent with the results, we observed that pO2 of TC and DRWC were lower at high altitude than that at low altitude. Furthermore, TC had lower pO2 than DRWC, which might be because TC had higher hemoglobin-oxygen affinity of blood than lowland chickens. The higher affinity may increase oxygen saturation of blood and acts as to compensate for the reduced partial pressure of oxygen and is considered to be characteristic of genotypically adapted high altitude mammals or birds (Monge and Leon-Velarde, 1991). A number of montane species, such as bar-headed and Andean geese (Weber et al., 1993) and Tufted ducks (Lutfullah et al., 2005), had special mutations that altered the amino acid residues in Hb and increased O2 affinity. We found in the other experiment that the Hb amino acid sequences of TC had a functional mutation of Met-32(B13)-Leu in {alpha}D globin chain, which evidently causes the increase in oxygen affinity (Gou et al., 2005).

Chronic exposure to hypoxia elicited a decrement in BV and PV for microswine (Durkot et al., 1996) and human (Robach et al., 2000; Heinicke et al., 2003). We observed a similar result in DRWC. However, in TC the values for BV, PV, and EV did not vary when compared at low and high altitudes. Furthermore, we found that TC had lower BV, PV, and EV than DRWC, but there were some reports that many high altitude native animals had higher BV or EV (Birchard and Tenney, 1990; Claydon et al., 2004). There were higher BV, PV, and EV in males than in females, but the effect of sex was independent of altitude, so the difference in BV, PV, and EV between males and females was not related to hypoxic response. Whether the amount of BV is in some way related to oxygen transport and hypoxic adaptation requires further study.


   FOOTNOTES
 
1 The research was supported by the 973 Project (2006CB102101) of China and the Project of National Fundamental Platform for Scientific Work (2005DKA21100-02). Back

Received for publication January 17, 2007. Accepted for publication March 21, 2007.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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Dacie, J. V., and S. M. Lewis. 1975. Practical Haematology. Churchill Livingston, Edinburgh, UK.

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