Bactrian camels (Camelus bactrianus) has physio-biochemical adaptation to high altitude

The high altitude region of India is characterized by harsh climatic conditions that may have an adverse impact on growth and metabolic functions of camels. Thus, the main aim of this study was to evaluate different adaptation parameters related to morphological, physio-biochemical and haematological indices in low lander Dromedarian and high lander native Bactrian camels at high altitude. To the best of our knowledge, no studies have been conducted so far to evaluate these different adaptation parameters in both breeds of camels to understand their adaptive mechanism in high altitude. Therefore, the present study was conducted to evaluate these adaptation parameters in Dromedarian and Bactrian camels. All morphological parameters were within the normal ranges in both the breeds. However, girth of hump, and skin thickness of shank and abdomen were towards the higher side of normal range in Dromedarian camels The heart rate was signicantly high and rectal temperature was low in Dromedarian camels than native Bactrian camels (P < 0.05). Interestingly, the erythrocytes sedimentation rate, lymphocytes and platelets counts were signicantly high and above the reference range in Bactrian as compared to Dromedarian(p < 0.05), whereas MCV, leukocytes and neutrophils were towards higher side of normal range in Dromedary. Similarly, aspartate aminotransferase (AST), alanine aminotransferase (ALT) were also signicantly high, whereas glucose and triglycerides levels were low in native Bactrian as compared to Dromedarian(p < 0.05). These ndings suggested that there is species difference in adaptation parameters in response to high altitude. Further, native high lander Bactrian camel having better metabolic adaptation and non-glucose energy substrates dependent metabolism. These parameters could be useful for evaluating their health conditions and load carrying performance for further selection of elite animals as pack animals at high altitude.


Introduction
Camel is an even-toed ungulate under the genus Camelus that have characteristic fatty deposits known as "humps" at its back. The camels have been domesticated as livestock for milk, meat, bre, transportation and racing. As working animals, they are uniquely suited for patrolling and load carrying in desert and hilly areas. There are three species of camel mainly single hump camel (Camelus dromedarius), double hump camel (C. bactrianus) and wild Bactrian camel (C. ferus). Single hump camels are generally known as Dromedarian or Arabian camel. Dromedarian are usually found in the Horn of Africa, Middle East and South Asia, while double humped camels are generally known as Bactrian and found in Central Asia (Wu et al., 2014). The wild Bactrian camel are now critically endangered. They can adapt easily in hot desert and harsh conditions due to their ability to remain without drinking water for extremely long periods and having uctuating body temperature.
In India, Bactrian are generally found in the cold desert region of Nubra Valley, Partapur where temperature ranges from + 30°C to -40°C, while Dromedarian are found in the Rajasthan and Gujarat state (Rosati et al., 2005). The total population of Bactrian and Dromedarian camels in India is around 210 and 2,45,000, respectively. The camels can work comfortably in the extremes of altitude and temperature. The camels are known for their distinctive ability to travel on hilly rough areas, and can carry weight up to 200 to 250 kg. On the other hand, pack animals such as mules and ponies can carry only limited quantities of 60 to 70 Kg load (Vivek et al., 2018). However, an in-depth study is further needed for understanding their physiology, and adaptation mechanism for their use as pack animals at high altitude.
The trans Himalayan high altitude region of India is characterized by low atmospheric pressure, extreme temperature variation of -35°C to 40°C, low humidity, high UV-radiation and low partial pressure of oxygen. All these adverse climatic conditions leads to oxidative stress, impaired immune system, poor growth and reproductive health in livestock at high altitude (Kumar et al., 2019). The most important stress factor at high altitude is hypobaric hypoxia which is considered as a severe physiological stress factor. It could affect physiological, serum biochemical and haematological parameters which are important indicator of the animals' health status. However, camels have some unique adaptability to these stress conditions as serum biochemical adaptation parameters in cold desert high altitude region. The analysis of blood components can also help the clinician for easy evaluation of the health status of animals. So far no studies have been done on the analysis of these physio-biochemical parameters to evaluate their feasibility for load carrying and patrolling in high altitude region, which can be helpful in selection of elite breeds from different regions. Therefore, the main aim of this study was to evaluate the effect of high altitude on physiological and biochemical parameters in low lander Dromedarian and native high lander Bactrian camel. Findings of this study could be helpful in diagnosis of disease and in examination of the endurance level of camels for logistic use in Ladakh region. This study will also be helpful in establishing a base line of reference values for physiological, biochemical and haematological parameters in normal and healthy camels at high altitude to highlight the effect of altitude.

Material And Methods
Compliance with ethical standards Animals and sampling procedure This study was conducted at Defence Institute of High Altitude Research (DIHAR), Leh-Ladakh which is located at 3500 meter mean sea level (msl). Total four low lander Dromedarian and four native high lander Bactrian camels were selected for sampling. The Dromedarian camels were introduced for research in high altitude region from Rajasthan, and they were slowly moved in stages from Bikaner to Kashmir to Ladakh. All the camels were normal and healthy during the sampling period. Total 10 ml of blood sample was collected from the jugular vein in morning for separation of serum and analysis of different parameters.

Determination of morphological and physiological parameters
All morphological parameters were recorded as described by Yosef

Determination of serum oxidative stress parameters:
The total antioxidant status in serum sample was estimated by Ferric Reducing Antioxidant Power (FRAP) assay as per Benzie and Strain (1996) method. However, free radical scavenging activity was measured by 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging assay as described by Blois (1958) and Abe et al. (2000). The radical scavenging activity was calculated as per following equation:

See equation 1 in the supplementary les.
where A 0 is the absorbance of the control (water instead of sample), and A 1 is the absorbance of the sample.

Statistical analysis
Results were expressed as means ± standard error (SE) and analyzed statistically by independent ttest using SPSS statistical software (Version 24, IBM Corporation, USA). P-values less than 0.05 were considered statistically signi cant.

Results And Discussion
Morphological parameters Among all the morphological parameters, body height, body length and neck length were signi cantly higher, whereas girth of shank was lower in low lander Dromedarian as compared to native high lander Bactrian camels (p<0.05; Table 1). This variation in morphological parameters could be due their breed characteristics, as there body size is different in all the habitats. However, no signi cant variation was recorded in height of hump, distance b/w the eyes, girth of abdomen, and face length (p>0.05). Interestingly, body height, body length, height of hump were within the normal range of Dromedary, but lies towards higher side. Moreover, hump girth, and skin thickness of abdomen and shank were signi cantly higher in low lander Dromedarian as compared to native Bactrian (p<0.05). These are important morphological adaptation parameters which varies in cold and hypobaric hypoxia environment (Faye et al., 2001). The skin thickness and hump size are dependent on subcutaneous deposition of adipose tissue, which are primary source of non-glucose energy substrates in animals adapted to cold environment at high altitude (Faye et al., 2001;Kamili et al., 2006). Hence, these nding are indicative of morphological adaptation process of Dromedarian to cold oxidative stress conditions. However, high lander Bactrian camels have adapted very well in these conditions as indicated by lower girth of hump and skin thickness as compared to low lander camels. In the present study, these low lander Dromedarian camels were introduced to high altitude two years back. Hence, these Dromedarian camels were under the process of adaptation, and exhibited higher girth of hump, and skin thickness at shank and abdomen region. reports in literature on morphological parameters study of Bactrian camels. This is the rst report on comparative study of all morphological parameters indicative of adaptation in low lander and native high lander camels to high altitude region. These morphological parameters and their reference ranges can also be helpful in the evaluation of overall growth of animal for further selection of elite breeds for logistic purpose e.g. endurance exercise, load carrying and patrolling.

Physiological parameters
Among all the physiological parameters, heart rate was signi cantly high, whereas the rectal temperature was low in Dromedarian camels than the Bactrian camels (p<0.05; Table 1). The heart rate and rectal temperature were within the normal reference range in native Bactrian camels. However, low lander Dromedarian camels showed very high heart rate which lies beyond the reference range of camel. This parameter at higher level is an indicative of adaptation changes in Dromedarian camels. High altitude stress conditions induces hormonal and neuronal changes in body by increasing the sympathetic nervous system activity (Simmonyi, 2014). This leads to a signi cant increase in blood pressure and heart rate in Dromedarian camels at high altitude. Interestingly, the rectal temperature was lower than the normal reference range in Dromedarian camels, which is indicative of poor energy metabolism. The body homeostasis is an important indicator of physiological status of animal adaptation to particular environment (Mohammed et al., 2007). However, both these parameters were within the normal range in Bactrian camel. These ndings revealed that Bactrian camels are more adapted to these extreme conditions than the low lander Dromedarian camels even after two years of rearing of low lander at high altitude. The body temperature and heart rate are dependent on body metabolism and animal response to stress. Therefore, heart rate and rectal temperature of Dromedarian camel indicates these animals are having poor metabolism and adaptation response to stress prevalent at high altitude.

Hematological parameters
Among all hematological parameters, platelets, ESR and lymphocytes levels were signi cantly high in native high lander Bactrian camels (p<0.05; Table 2). However, total leukocytes and neutrophils count were higher in low lander Dromedarian as compared to Bactrian camels (p<0.05). There was no signi cant variation in hemoglobin (Hb), PCV, erythrocytes, monocytes, eosinophil, basophils, MCV, MCH and MCHC levels between the two breeds of camels (P>0.05). The erythrocyte size of Dromedarian camel was signi cantly higher than the Bactrian camel (p<0.05; Table 2 and Fig 1, 2). Blood Hb concentration and erythrocytes count were almost similar and within the range in both the breeds.
Despite similar erythrocyte count and Hb levels, MCV (mean corpuscular volume) values in Dromedarian camels were greater than the Bactrian and normal reference range reported in literature ( Table   2). This may be due to large size of immature erythrocytes which are also known as reticulocytes (Fig 2). The reticulocytes are capable of more cellular oxygen transport due to their high a nity to oxygen. The low lander Dromedarian camel rearing at high altitude need more e cient cellular oxygen transport system to adapt under hypobaric-hypoxia conditions of high altitude (Adili et al., 2013). The microscopic size measurements also revealed larger size of erythrocytes in Dromedarian camel (Fig. 2). The large size of erythrocytes are indicative that low lander camels are under the process of haematological adaptation to hypobaric hypoxia prevalent at high altitude (Banerjee et al., 1962).
The total leukocytes count was signi cantly high in low lander Dromedarian as compared to native Bactrian camels. Increase in leukocytes count could be due to compromised immune system under stressful conditions (Su et al., 2018). More leukocytes are required to cope up with these severe environmental stress condition in order to survive. Therefore, this increase in leukocytes count could be due to stress factors prevalent at high altitude hypoxia conditions, which triggers immune response for homeostasis maintenance (Ouajd and Kamel, 2009). However, low leukocytes count in Bactrian camel along with higher lymphocytes counts are indicative of better adaptation response to high altitude.
The morphological changes in neutrophils in the form of band shaped and segmented neutrophils were observed in both the breeds of camels ( Fig. 1 and 2). However, these morphological changes and neutrophils number were signi cantly high in Dromedarian camels as compared to Bactrian camels (Table   2), whereas the number of lymphocytes were high in native Bactrian camels. These ndings are indicative of free radicals generation and immunosuppression under high altitude stress conditions. (Zongping, 2003;Ouajd and Kamel, 2009). Various studies observed that more neutrophils are released to counter oxidative stress-induced free radicals and microbial infections (Higgins and Kock, 1984). Moreover, neutrophils number increases in acute response to stress conditions, whereas lymphocytes number increases in chronic response conditions (Klokker et al., 1993). This indicates that low lander camels are under the process of adaptation in response to high altitude, while high lander camels have adapted very well in these conditions resulting in lower neutrophils and higher lymphocytes count. Since, these high lander Bactrian camels are bred and reared for several generations under high altitude stress conditions, all these changes are likely to be acquired by them in response to high altitude conditions over several generations.
In the present study, the level of platelets were signi cantly high in both the breeds of camels, which could be due to stimulation of platelets precursors cells in response to hypoxia conditions at high altitude ( Table 2). There is competitive response between stem cells of erythrocyte and platelets precursor cells.

Biochemical and oxidative stress parameters
The biochemical parameters showed signi cantly higher level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and creatinine, whereas signi cant decrease in glucose and triglycerides levels in native Bactrian as compared to Dromedarian camels (p<0.05; Table 3). However, other parameters viz. albumin, total protein (TP), calcium, magnesium, uric acid and iron remain unaffected ( Table 3). The high AST and ALT levels in Bactrian indicates that the liver metabolism is highly active under stress at high altitude region which releases more ALT and AST level into the blood during adaptation (Aragon & Younossi, 2010). Slightly higher creatinine and urea level in native Bactrian camels indicates more active renal metabolism, which might be a adaptive mechanism of water conservation in high altitude cold stress conditions (Samra & Abcar, 2012). Liver metabolism is a vital physiological process to maintain body homeostasis to ful ll cellular function under stressful conditions. The animal susceptible to environmental stress conditions have poor metabolism, and therefore their body temperature is lower than the normal, and they are more prone to stress mediated ailments. In present study, low lander camel has signi cantly low rectal temperature than the Bactrian camels and normal reference values, which is an indicative of poor liver metabolism at high altitude. Hence, our ndings supported the hypothesis of higher liver and kidney metabolic activity required for metabolic adaptation to high altitude.
Interestingly, total serum glucose levels recorded in native Bactrian camels were very low as compared to Dromedarian camels (Table 3). Earlier reports have documented that exposure to high altitude region leads to transient increase in glycemic index initially, this could be an adaptive mechanism to low energy level which is effective in camel at high altitude (Brooks et al., 1991;Larsen et al., 1997). However, prolonged and continuous exposure to high altitude lowers glycemia in serum (Brooks et al., 1991). The acclimatization to high altitude seems to be the main reason for the increase ATP fuel dependency from non-glucose energy sources. Under cold stress, non-glucose energy substrates are more important and generally preferred for ATP generation. At high altitude, the glucose based energy diets are avoided and protein-fat based diets are preferred as adaptation mechanism for cellular energy generation. In present study, low glucose level were observed in native Bactrian, which is an indicative that native Bactrian camels are more dependent on non-glucose energy substrates. Whereas, high triglycerides and glucose levels were present in low lander camels, which shows that the energy metabolism in Dromedarian is more dependent on glucose and triglycerides rather than protein-fat diet. This further support the hypothesis that low lander camels are poorly metabolically adapted to high altitude conditions. All these important ndings indicated the effect of climate on biochemical parameters, which is an The present study observed no signi cant difference in oxidative stress markers, DPPH, and FRAP between low lander and high lander camels from different origins ( Table 3). The high antioxidant levels help in reducing oxidative stress generated cellular damage in camels induced by high-altitude adverse conditions (Kumar et al., 2019). Hence, the oxidative stress reducing ability to protect important biomolecules like DNA, RNA, proteins, carbohydrates and lipids in their functional active state was very well documented in camels serum samples. These results suggests the importance of estimation of antioxidant parameters for monitoring health status and endurance parameters. In conclusion, the current study helped in understanding of morphological, haematological and physio-biochemical adaptation mechanism in camels to high altitude environment. These variations could be due to diversity in genetic makeup, environmental factors and altitude effect.

Conclusion
The present study concluded that the native high lander Bactrian camel has effective liver metabolism, and more dependency on non-glucose energy sources as compared to low lander Dromedarian camel. The change in leukocytes number and neutrophils morphology was observed to counter environmental stress conditions prevalent at high altitude. Further, this study revealed that variation in physio-biochemical and haematological parameters in two breeds of camel is an indicative of difference in stage of adaptation to high altitude. However, further studies are required to demonstrate changes in these physio-biochemical and haematological long term adaptation parameters, which will be helpful in selection of well adapted breed to high altitude stress conditions for endurance performance.