Indian J. Anim. Res., 53 (3) 2019: 307-310 AGRICULTURAL RESEARCH COMMUNICATION CENTRE Print ISSN:0367-6722 / Online ISSN:0976-0555 www.arccjournals.com/www.ijaronline.in Evaluation of different physio-biochemicals indices of exercise performance in Zanskar ponies Prince Vivek*, Vijay K Bharti, Sahil Kalia, Arup Giri, Kapil Nehra, Rohit Kumar, Om Prakash Chaurasia and Bhuvnesh Kumar1 Defence Institute of High Altitude Research, Defence Research and Development Organisation, Leh-Ladakh- 194 101, Jammu and Kashmir, India. Received: 25-09-2017 Accepted: 08-02-2018 DOI: 10.18805/ijar.B-3501 ABSTRACT Zanskar pony is a native breed of high altitude region of Ladakh namely Zanskar valley and Nubra valley. These ponies are commonly used for load carrying by locals and defence personnel due to their adaptability in rugged terrain and rocky track. Therefore, high enduring ponies are in great demand for ride and load carrying at low maintenance cost. So far to the best of our knowledge no studies have made attempt to evaluate different physio-biochemical indices of exercise performance in Zanskar pony, so that selection of elite pony could be made based on these indices. Hence, present study was conducted to evaluate the physiological responses, biochemical, cytokines, hormones, and antioxidant related parameters to performance in Zanskar pony at two geographic areas namely Leh (3500 meter msl) and Nubra valley (3291 meter msl) with different altitude. For this study, total of 6 adult mares ponies were selected for sampling at Leh and 8 adult mare ponies were selected at Partapur. Ponies at different altitude did not exhibit significant difference in physiological responses, and biochemical, cytokines, hormones, and antioxidant related parameters, except aspartate aminotransferase (AST). Though the all the parameters were within the normal ranges towards higher side. These findings indicated that there was no effect of altitude within the entire home tracks of Zanskar pony namely Zanskar Valley and Nubra of Ladakh region. Hence, these parameters may be useful if evaluated during load carrying trials and associated with the performance. Key words: Blood biochemical, Cold desert, High altitude, Zanskar Ponies. INTRODUCTION Zanskar pony is a native breed of Zanskar valley of Zanskar valley of Ladakh region, Jammu and Kashmir State of India. The Zanskar ponies are considered as one of hardiest breed among all the pony breeds and can work comfortably in the extremes of altitude and temperature. This breed is found extensively in the cold desert of high altitude where temperature ranges from +30°C to -40°C. These ponies are known for their hardiness and ability to move on hilly and rocky rough terrain (Venkatesan et al. 2011). This region is characterized by extreme climatic conditions which are characterized by hypobaric hypoxia, high wind velocity, severe cold, and UV- radiation. These high altitude environmental stress factors make animals more susceptible and alter their physiologic and metabolic functions. At high altitude lower partial pressure of oxygen make diffusion of oxygen (O 2) difficult to vascular system resulting in deprivation of oxygen which is known as hypobaric hypoxia. The most important stress factor at high altitude is hypobaric hypoxia which is considered as an acute physiological stress which leads to increase in oxidative stress (Maxwell, 1995). These ponies are distributed to different valley of Ladakh, viz. Nubra, Leh, Changthang and Zanskar at varied altitude. High altitude causes increase in metabolic stress which leads to increase in catabolic rate that result compromised performance and endurance (Mazzeo et al. 2001). Immune and metabolic status is an important index of stress during exercise (Facco et al. 2005). When faced with the added stress during physical activity at high altitude, physiologic and metabolic responses are exacerbated (Mazzeo et al. 2000). So, it was hypothesized that their exercise performance may have different in these valleys due to variation in altitude. So far no studies have been done to evaluate their physiological responses and other physiobiochemical parameters related to exercise performance, which can be helpful in selection of elite ponies from different valley. Hence, evaluation of different physiobiochemical indices in ponies from two altitudes may give clue to this hypothesis. Therefore, the goal of this study is to evaluate the effect of high altitude stress on physiological and biochemical parameters in Zanskar ponies at two different geographic areas of high altitudes. Outcome of this study may be helpful in equine medicine and to examine the fitness level and then deployment for logistic support. *Corresponding author’s e-mail: prince.v68@gmail.com 1 Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organisation DRDO, Timarpur, Delhi-110 054, India. 308 INDIAN JOURNAL OF ANIMAL RESEARCH MATERIALS AND METHODS Animals and sampling procedure: This study was carried out in two different geographic areas located in high altitude region of Ladakh (L-sector), J&K, India. First, study was carried at DIHAR (DRDO), Leh located at 3500 meter mean sea level (msl) and second was carried at Zanskar Ponies Breeding Unit (RVC), at Partapur, located at 3291 meter msl during summer period. Total six mares (N=6) of 5-6 years at Leh and eight mares (N=8) of 4-5 years at Partapur were selected randomly for blood collection. The basic geographical and environmental data for these two studied places were recorded during sampling periods and presented in the Table 1. Diets were fed to all ponies considering a daily consumption @2% of body weight, as per the standard ration scale formulated by Defence Institute of High Altitude Research (DIHAR) for high altitude, whereas water was given ad libitum. All the ponies were healthy and in good body condition during the sampling period. These ponies were made to walk first before the sampling to ensure the well being and fitness of ponies and physiological responses were recorded at rest. Total 10 ml of blood samples were collected from the jugular vein during 11:00-13:00 hrs for isolation of serum and analysis of different serum biochemical, oxidative stress related parameters, metabolic enzymes, hormones, and cytokines. Determination serum biochemical and metabolic enzymes parameters: Glucose (Catalogue no. 93DP10080), Total protein (Catalogue no. TP0102), Albumin (Catalogue no. ALB0102), Globulin, LDH (Catalogue no. 74LS100-25), Creatinine (Catalogue no. 85MB100-66), ALP (Catalogue no. 75DP200-50), ALT (Catalogue no. 76LS20060), AST (Catalogue no. 77LS200-60) and Uric acid (Catalogue no. 82MB100-66) were estimated using commercially available kits as per the manufacturer’s protocol (Span Divergent Ltd.) with a Microplate reader. Determination of serum oxidative stress parameters: Oxidative stress parameters were assayed for lipid peroxidation through the formation of thiobarbituric acid reactive substances (TBARS) in the reaction mixture as described by Ohkawa et al. (1979). The total antioxidant status were measured by FRAP assay as given by Benzie and Strain (1996) with slight modifications. DPPH assay was measured as described by the method of Blios (1958) with a slight modification. Superoxide dismutase (SOD) was measured as described by Das et al. (2000). Catalase was Table 1: The geographical and environmental data of experimental location (Partapur and Leh). Meteorological Condition Altitude (meters) Latitude Longitude Relative humidity (%) temperature (shade) (°C) Partapur 3291.84 m 34.09°8.56’ 74.79°36.87’ 22.4 12.3 Leh 3500 m 34.08°267’ 077°34.378’ 18.6 7.2 estimated by the degradation of hydrogen peroxide (6 mM) as described by Aebi (1984). Determination of hormonal and cytokines parameters: T3 (Catalogue no. CEA453Ge) and Cortisol (Catalogue no. CEA462Ge) were estimated using commercially available ELISA kits as per the manufacturer’s (M/S USCN Life Science Inc, Wuhan, China) protocol. IL-2 (Catalogue no. 431007) and IL-6 (Catalogue no. 431307) were estimated using commercially available ELISA kits with pre-coated plates as per the manufacturer’s (M/S BioLegend Inc. San Diego, US) protocol. Statistical analysis: Paired‘t’-test was applied to determine the effects of two geographic areas of different levels of altitude on oxidative stress, antioxidant, cytokine and on patho-biochemica l properti es, p-val ue <0.05 was considered statistically significant using statistical software SPSS 22 version. RESULTS AND DISCUSSION Change in physiological parameters: Physiological parameters viz. heart rate, pulse rate, respiration rate, and rectal temperature were unaffected at both Partapur and Leh (Table 2). However, these physiological parameters, except rectal temperature were higher than the normal range of adult working horse from the plain areas. These findings indicate the prevalent stressful condition of high altitude that resulting high pulse, heart, and respiration rate to perform well under hypobaric hypoxic condition. Whereas, rectal temperature was within the normal range which indicate these ponies are not in stress, other parameters at higher level is an indicative of adaptive changes in native animals (Table 2). The physiological parameters increase in response to stress imposed by the adverse environmental condition at high altitude during acclimatization and adaptation (Palmer, 2010). Whereas, in this study no any significant change in physiological parameters in Zanskar ponies might indicate that pony has well adapted and acclimatized at this conditions. Therefore, performance evaluation should be based on rate of change in these parameters from the base Table 2: Physiological responses in ponies at different altitude (msl). Parameters Heart Rate (beats/min.) Pulse (pulse/min.) Respiration (cycle/min.) Rectal Temp. (°F) Values are presented as Mean ± SE. Partapur (3291.84 m) 52.20 ± 0.80 48.60 ± 0.98 44.80 ± 0.98 99.53 ± 0.10 Leh (3500m) 55.75 ± 0.86 48.8 ± 0.46 44 ± 2.14 100.10 ± 0.32 Normal range 28-44 28-44 8-16 99.5-101.3 Volume 53 Issue 3 (March 2019) line or normal range, minimal changes on higher load or exercise performance was indicative of better endurance. Change in serum biochemicals and metabolic enzymes: The results showed significant difference in liver AST level between the experimental places, whereas other parameters were unaltered, viz. ALT, ALP, glucose, total proteins, albumin, globulin, uric acid, creatinine, and LDH (Table 3). These changes might be due to altitudinal variation in well adapted ponies, because altitude of Leh region is more as compared to Partapur, which attributed to increase in ROS production and led to oxidative stress resulting higher AST enzymes (Harris et al., 1990). However, these parameters were towards higher range as compared to value of horses from plain areas. Therefore, these parameters are essentially to be within the normal range in elite ponies of high exercise performance. Changes in oxidative stress parameters: The present study did not observed any significant changes in lipid peroxidation (LPO), 2, 2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing ability of plasma (FRAP) and antioxidant enzymes like catalase and superoxide dismutase (SOD) between two group of ponies from different altitudes (Table 4). These changes were in agreement as per reported finding in horses on duty by Kruljc et al., 2014. However, higher trend in these parameters were observed in ponies from Leh valley, which is higher altitude than the other one. These findings were indicative of importance of evaluation of oxidative stress related parameters for performance endurance and health monitoring (Fisher-Wellman and Bloomer, 2009; Gondim et al., 2009; Kruljc et al., 2014). Changes in hormonal and cytokines parameters: These parameters are essential basic and important parameters helped us to assess the physical stamina and inflammatory changes during the exercise performance of the ponies. The present study did not observed any significant variation in hormone cortisol and T3 level as well as serum cytokines interleukin-2 and IL-6 level at both the altitude (Table 4). These biomarkers might indicate that these ponies are well adapted to high altitude under prevalent husbandry conditions (Esquivel and Ramirez, 2016; Fazio et al., 2017; Facco et al., 2005). These observation may indicate that Zanskar ponies has the ability to do hard work and has good stamina without any adverse change in physiological parameters at high altitude. This is the first report on physiological and biochemical biomarkers in Zanskar ponies at two different geographic areas of high altitude within the L-sector of India. CONCLUSION In conclusion, this study shows that these physiobiochemical parameters are essential indicative of their performance and native ponies are physiologically fit and well adapted to harsh high altitude conditions. The findings 309 Table 3: Change in biochemical parameters and metabolic enzyme in ponies at different altitude (msl) Parameters Partapur (3291.84 m) Leh (3500m) ALP (IU/L) 163.7 ± 4.41 165.4 ±7.04 ALT (IU/L) 7.96 ± 7.52 9.72 ± 4.8 AST (IU/L) 235.53 ± 5.8 262.47* ± 4.72 Glucose (g/dL) 90.8 ± 7.8 97.5 ± 5.3 Total Protein (mg/dL) 7.66 ± 1.6 7.15 ± 2.23 Albumin (mg/dL) 3.175 ± 0.5 2.886 ± 1.3 Globulin (mg/dL) 4.525 ± 1.54 4.3286 ± 1.4 Uric Acid (mg/dL) 0.28 ± 0.09 0.38 ± 0.07 Creatinine (mg/dL) 0.94 ±0.28 1.24 ± 0.15 LDH (IU/L) 244.6 ± 8.3 252.1 ± 7.97 Values are presented as Mean ± SE; *indicates significant difference (p<0.05) within the same row. ALP- Alkaline phosphatase; ALT- Alanine aminotransferase; ASTAspartate aminotransferase; LDH- Lactate dehydrogenase; Table 4: Variation in oxidative stress parameters, cytokines, and hormones level in ponies at different altitude (msl). Parameters Partapur (3291.84 m) Leh (3500m) Lipid Peroxidation 7.04 ± 1.3 10.24 ± 2.52 (nMoles MDA/mg protein) DPPH (% inhibition) 12.3 ± 6.36 14.4 ± 9.1 FRAP (M Fe(II)/L) 388.45 ± 7.7 394.24 ± 5.9 SOD (Units/mg protein) 2.32 ± 0.85 1.9 ± 0.84 Catalase (nMoles/mg protein/sec)0.33 ± 0.15 0.42 ± 0.34 Interleukin-2 (pg/mL) 5.35 ± 1.23 6.575 ± 3.75 Interleukin-6 (pg/mL) 15.875 ± 1.99 19.15 ± 3.11 T3 (pg/mL) 25.59 ± 4.6 26.62 ± 1.5 Cortisol (ng/mL) 7.9 ± 1.36 9.32 ± 4.1 Values are presented as Mean ± SE; *indicates significant difference (p<0.05) within the same row. DPPH- 2, 2-diphenyl-1-picrylhydrazyl; FRAP- Ferric reducing ability of plasma; SOD-Superoxide dismutase; T 3 : Triiodothyronine. revealed that no significant variation in different physiological and biochemical parameters of exercise performance in Zanskar ponies from two different geographical locations of their home track. However, further studies are required to demonstrate changes in these physiobiochemical parameters with level of load carrying and exercise performance, which will be helpful in selection of elite ponies adapted to high altitude stress condition. ACKNOWLEDGMENT Authors, would like to thank CSIR-HRDG, New Delhi for providing financial support in the form of Junior Research Fellowship (JRF) to first author (P. Vivek). Research facilities provided by DRDO-Defence Institute of High Altitude Research (DIHAR), C/o- 56APO, India and financial support of Defence Research & Development Organization, India for conducting this study is duly acknowledged. 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