Thermoregulatory and Adaptive Responses of Adult Buffaloes (bubalus Bubalis) during Hyperthermia: Physiological, Behavioral, and Metabolic Approach

Aim: The study was planned to evaluate the indigenous animal adaptive capabilities during optimum temperature versus heat stress (HS). Materials and Methods: Four adult buffaloes were exposed at 25°C, 30°C, 35°C, and 40°C for 21 days at every treatment in environmentally controlled chamber and physio-biochemical variation and animal behavior was observed. Conclusion: The results reflect the impact of hyperthermia both acute and chronic, on the animals forcing various physio-biochemical, endocrinal, and behavioral changes for acclimatization during a stressful period aimed at maintaining homeothermy.


Introduction
The accumulation of greenhouse gases in the Earth's atmosphere has led to predictions that global surface temperatures will increase between 1 and 6°C during the 21 st century (Intergovernmental Panel on Climate Change, 2007).A 25% loss in animal productivity in developing countries has been estimated due to global warming [1].The global warming scenario coupled with the subtropical harsh environment in Indian subcontinent exposes the livestock naturally to thermal stress for a prominent part of the year affecting production and economy.
The buffalo which is mainly reared for its milk, meat, and drought purpose is an integral part of the Indian agricultural system.Buffaloes have poor heat tolerance than cattle [2] due to many physiological and genetic reasons (less sweat glands, black colored skin) and so is more pronounced to thermal stress.During HS, heat increment exceeds heat loss modifying the homeostatic functions.As per Gudev et al. [3] heat stress (HS) elicits an integrative physiological and endocrinal modulation altering overall metabolism and helping the animal sustain during the stressful period.Various in-depth studies on HS ruminants have made observations indicating severely compromised thermoregulatory functions and an overall negative effect of high temperature [4][5][6].
Hence, the study was planned to observe the indigenous buffalo's adaptive capabilities during HS versus optimum environmental conditions and to correlate with other studies to quantify the impact of stress.

Ethical approval
The experimental design and procedure were carefully assessed and approved by the Institute's Ethical Committee ensuring that no malpractice or potential harm toward animal welfare was done, and no subject was jeopardized during the entire exploratory period.

Animal management
The study was conducted at the Division of Physiology and Climatology during 2011 to 2012 on four adult dry buffaloes (9.00±0.40years, 492.14±9.58 kg).Animals were randomly selected and exposed at 25°C, 30°C, 35°C and 40°C for 21 days at each treatment in the environmentally controlled chamber (7.5 m × 7.5 m × 2.5 m) equipped with individual tie stall, feeders, and waterers.Daily exposure period was from 10:00 to 15:00 h.Basal diet of wheat straw ad libitum along with the required amount of concentrate mixture to meet the maintenance requirement [7] was offered daily at 08:30 and 15:15 h.Water was provided in graduated containers and intake was recorded accordingly.All the physiological readings were made daily at 15:05 h, immediately after exposure.Rectal temperature (RT, °C) was measured by inserting a digital thermometer 5 cm deep per rectum and reading was only made after 1 min.For recording respiration rate (RR, breaths/min) the flank movement was observed for 1 min, heart rate (HR) (pulse rate [PR], beats/min) was monitored by palpating the coccygeal artery for 1 min.Behavioral signs like restlessness, panting, rumination, etc., were monitored for the entire experimental period (during and after exposure).For biochemical studies, blood samples were collected on every 5 th day starting from day one from jugular veni-puncture and serum was harvested within 1 h by centrifuging the sample at 3000 g for 15 min.The samples were frozen (−80°C) until the end of the trial and analyzed together.All experimental procedures were reviewed and approved by the animal Ethics Committee at the Institute.

Parameter investigation
All the samples were analyzed at Nuclear Research Laboratory of the division.In each serum sample, sodium, potassium, chloride, calcium, aspartate aminotransferase (AST), alanine aminotransferase (ALT), were measured on double beam UV-vis spectrophotometer (uv5704ss, Electronics Corporation of India, Ltd.) using commercial kits (Crest Biosystems; SPAN Diagnostic).All measurements were made according to the manufacturer's instructions.Serum superoxide dismutase (SOD) and reactive oxygen metabolites were measured using established protocols [8,9].Hormones assay for thyroxine (T 4 ), triiodothyronine (T 3 ), cortisol, and aldosterone, were determined by radioimmunoassay procedure on Sr-3000 Stratec Counter (Stratec Biomed Systems, Birkenfeld, Germany).

Statistical analyses
Data were analyzed by the one-way analysis of variance model using SPSS 16.0 software [10] and indicated by their probability value (p).The general model used was Y ij =μ+A i +B j +e ij , where Y ij is the dependent variable, μ is the least square mean, A i is the effect of temperature, B j is the animal effect and e ij is the residual error.Differences among treatments were determined using Tukey's b test [10] and indicated by the superscripts a, b, c, d.All data were presented as means ± standard error of the mean and level of significance was set at p<0.05 for all the parameters.

Results
The changes in water intake (WI), RT, RRs, PR and biochemical variables at different treatments are presented in Tables-1 and 2, respectively.RT differed with the increasing exposure temperature and was higher (p<0.001) at 35°C and 40°C as compared to 25°C and 30°C, respectively.Likewise, the RRs also increased during thermal stress period and were significantly high (p<0.001)at 35°C and 40°C than at lower treatments.Contrastingly, the PR decreased significantly (p<0.001) during heat exposition, and highest values were recorded at 25°C and 30°C, respectively.WI was significantly higher (p<0.01) at 35°C than at optimum conditions and 30°C, but no variation was observed at 40°C.During the thermically stressful period at 35°C and 40°C, sweating, panting, restlessness, and nervousness was more pronounced, while rumination and urination were reduced.Significant rise (p<0.05) was noted for sodium at 40°C than at 30°C but no variation (p>0.05) was observed for potassium, calcium, and chloride at any treatment during entire experimental period.The reactive oxygen radicals levels were significantly high at 40°C as compared to any other treatment.SOD did not vary significantly (p>0.05) at 25°C, 30°C, and 35°C, neither was any difference observed for the enzyme at peak temperature of 40°C.Gradual rise was observed for the enzymes AST and ALT with the heat increment and significant differences (p<0.05) were observed at 35°C for AST and both 35°C and 40°C for ALT as compared to optimum treatments.
The highest concentration for thyroid hormones was seen at optimum conditions which dropped significantly (p<0.001) with the increasing thermal stress.T 4 decreased at 30°C and 40°C while, T 3 was significantly low at all the treatments as compared to that at 25°C, respectively.Glucocorticoid activity was significantly higher (p<0.05) at 35°C as compared to 25°C but we observed no difference for cortisol at 30°C and 40°C treatment.For aldosterone no significant variation (p>0.05) was recorded for the entire experimental period.

Discussion
The significant rise in RT during HS (37.54°C vs. 38.01,38.77°C) indicate increasing heat load on the buffaloes and inability to normally dissipate the accumulating heat, consequently raising the body temperature.Significant increase in RT (p<0.001) was also seen in heat-exposed buffalo calves as compared to cool comfortable conditions [11].Similarly, higher RTs (°C) were also seen in heat-exposed Tharparkar (38.74±0.11vs. 38.96±0.05)and Karan Fries heifers (38.84±0.06 vs. 39.19±0.24)than at thermoneutral treatments validating our findings [12].To increase the evaporative cooling panting is commonly seen in HS animals in an attempt to maintain normal body temperature.Banerjee and Ashutosh [13] observed significant rise in respiratory frequencies in Tharparkar and Karan Fries heifers at higher environmental temperatures.During thermically stressful period, the respiratory frequencies and RT in Alentejana, Frisian, Limousine and Mertolenga cattle breeds increased significantly by 2.7, 2.8, 2.5, 2.9% and 1.1, 2.0, 1.8 and 0.2%, respectively, in accordance to our findings [14].Present findings completely agree with previous works [13,14] and higher RR in buffaloes was probably adopted to increase the evaporative cooling.The cardiovascular systems response during HS is varied, so the decrease in HR in buffaloes during thermal stress could be mediated by low epinephrine and nor-epinephrine levels [15], circulatory adjustments like increase in peripheral resistance, increased resistance to venous return as an attempt to reduce metabolic heat generation [16].Similar decrease in HRs was also observed in buffalo calves as essential cardiovascular adjustments during HS, which support present results [12].The HR of 2-3 months old male Bos taurus calves showed a decline when the temperatures were raised from 18 to 40.5°C at 50% RH.The mean HR of 114.5/min at 18°C fell to 110.5/min and continued to fall to 95/min at 40.5°C in the climate chamber [17].The cardiovascular and respiratory adjustments in accordance to increasing stress indicate the importance of physiological adaptations acting as the first line of defense in maintaining thermoregulation to prevent drastic metabolic alterations.
Water is directly involved in cooling of reticulo-rumen and also serves as the primary vehicle for heat transfer.Increased sweating and panting might had resulted into osmoconcentration of the extracellular fluid activating hypothalamic thirst center leading to higher WI in the acclimating buffaloes to HS.Similarly, Beatty et al. [18] observed significant increase (p<0.001) in daily WI during thermically stressful period in B. taurus and Bos indicus cattle.Significant (p<0.01)increase in total WI (28.5 and 48.3%) and free WI (25.2 and 56.4%) was also noted in 12-month-old buffalo and cattle calves due to chronic heat exposure further validating present findings [19].Although no difference in WI was observed at 40°C still the WI was higher signifying the important role of water in heat dissipation.Animal behavior changed during HS, and they were more nervous, restless, and profuse salivation accompanied with panting was observed.Panting is seen when the normal heat dissipation mechanism is compromised, culminating into evaporative cooling as means of heat loss.We also observed lower frequency of rumination and urination at the higher treatments which possibly indicated changes in rumino-reticular environment, changing fermentation patterns and water conservation mechanism, respectively.Nardone et al. [20] also reported increase panting, lowered saliva production and decreased rumination during thermal stress in livestock.Furthermore, other workers [21] have reported reduction in average rumination time in cattle exposed to higher temperatures temperature (24±4 vs. 17±4 min) supporting present findings.The behavioral changes help in the adaptation to a stressful period and can be used to assess the impact of thermal stress in ruminants.
HS results in negative mineral balance and also reduces electrolyte absorption, so the significant rise (p<0.05) in sodium at 40°C than at 30°C is unique finding of the present study.Aboul-Naga et al. [22] recorded a significant 3% decrease in serum sodium in Friesian calves exposed at 36°C.During HS sodium concentrations (mEq/L) decreased in Tharparkar (136.25±1.75 vs. 132.88±2.09)and Karan Fries heifers (133.33±2.44 vs. 128.39±2.00)[13] but, present observations in buffaloes contradict them.An isotonic expansion of extracellular fluid volume or enhanced renal reabsorption or conservation might have resulted in higher sodium levels [16] in present study, but further exploration in ruminants will be needed to confirm the present finding observed during HS.Potassium concentrations did not varied at any treatment (p>0.05) although there are many studies reporting significant reduction in potassium levels in HS buffaloes which they attributed to losses in sweat and decreased retention [23,24].The low levels of aldosterone combined with increased renal conservation might had resulted optimum potassium concentrations in present study, similar to that observed in heat-tolerant Romosinuano (4.27 vs. 4.22 mEq/L) and heat-susceptible Angus (4.31 vs. 4.50 mEq/L) cattle during thermoneutral against HS conditions [25].Lack of variation for chloride and calcium in buffaloes was probably due to optimum retention for the minerals during HS period.Similar nonsignificant variation for chloride (109.50 vs. 116.50mmol/l) was also noted in Omani sheep [26] and Holstein cattle [27] during hot environment than in thermoneutral conditions supporting present findings.
Cells continuously produce free radicals and reactive oxygen species (ROS) as part of normal metabolic processes but hyperthermia has been reported to enhance numerous forms of ROS in different cells and tissues [28].This increased ROS activity during HS (0.06±0.150 mg H 2 O 2 /dL at 40°C) can be attributed to changing cellular metabolism, leading to faster free radical production than that could be neutralized by the cellular antioxidant system.Present findings totally agree with observations made in dairy cows with similar increased free radicals (thiobar-bituric acid reactive substances during summer (8.8±0.4 nmol/ml) than in spring season (7.6±0.4 nmol/ml) [29].SOD and catalase (CAT) provide the first line of defense against ROS-induced damage [30].Kumar et al. [31] observed significant (p<0.05)increased erythrocyte SOD activity with days of exposure in hot dry (40°C) and hot humid (37°C) stressful conditions in buffaloes.Our findings contradict other works reporting increased SOD activity in HS cattle and buffaloes [29,32] as no variation was seen for SOD during thermal stress which might be due to collaborative contributions by other antioxidant systems (CAT, glutathione peroxidase, vitamins A, E, and C and flavonoids) in neutralizing the free radicals [33].The enzymes AST and ALT have a key role in gluconeogenesis hence are essential for stress adaptations [34].The significantly higher enzyme levels during thermal stress were probably due to stimulation of gluconeogenesis by glucocorticoids to fulfill the increasing energy demands [35].Similarly, Marai et al. reported significant increase in enzymes AST (613 vs. 758 units/L) and ALT (179 vs. 228 units/L) in the summer as compared to winter in Friesian calves [36].A positive effect (increase) of THI on AST activity was also observed in lactating dairy cows in other study [37] which validate the present observations of high enzyme activity during thermal stress.
Thyroid hormones are known to play an important role in animal's adaptation to environmental changes, and T 3 is more concerned with thermogenesis in farm animals.In the present study, T 3 and T 4 declined consistently with increasing temperatures probably mediated by higher glucocorticoid levels and lowered thyroid gland function.The T 3 levels were significantly lower (p<0.05)at 33°C (0.95±0.05 ng/mL) than at 20°C and 28°C (1.45±0.05 and 1.32±0.05ng/mL), respectively, in prepubertal Holstein heifers [38].The decrease in thyroid hormones as an adaptive stress response is established by many workers in cattle confirming present observations [14,39].Cortisol also known as the "stress hormone" was significantly higher at 35°C than at optimum treatments possibly due to the activation of hypothalamo-pituitary-adrenal axis increasing the cortisol concentration enabling the animals for necessary adaptation during stressful period [40].In Friesian calves exposed to direct solar radiation of the hot summer, cortisol concentration increased from 11 to 29 ng/ml [41].Similar increase in cortisol hormone during summer months as compared to the winter period was also reported in Tuli, Senepol, and Brahman-sired Angus heifers [42].No notable difference for cortisol at 40°C reflected gradual adaptation by the buffaloes, which make the corticoid hormone pertinent for both short-and long-term adaptation to HS [43].The decrease in thyroid hormones with a concurrent increase in the glucocorticoid indicates indispensible role of the endocrine system essential to maintain energy and normal cellular homeostasis during a stressful period.
Acute thermal stress results in increasing concentrations of aldosterone in non-ruminants and decreasing concentrations during chronic thermal stress in ruminants [44].Lack of variation for aldosterone is quiet surprising and unique finding of the study as higher sodium levels were noted during HS.These low hormone levels might have helped in essential potassium ion conservation which is known to be lost both in urine and sweat [17].Further extensive explorations on aldosterone action and sodium conservation are essential to confirm these surprising observations made in HS buffaloes.

Conclusion
Native animals have some genetic and physiological advantages due to evolution and selection strategies but they still are vulnerable to both acute and prolonged thermal stress and hyperthermia has drastic impact on the behavior, normal physiology, and metabolism as was observed in present study.Sustenance during thermal stress is a synergistic function of different body systems, diverting all resources toward maintaining thermoregulation.
RT=Rectal temperature; PR=Pulse rate; WI=Water intake; RR=Respiration rate.Means bearing different superscript a, b, c, d within a row differ significantly at (p<0.05), SEM=Standard error of the mean Table-2: Mean±SEM of different biochemical and endocrinal variants at 25°C, 30°C, 35°C, and 40°C in buffaloes.SEM=Standard error of the mean; AST=Aspartate aminotransferase; ALT=Alanine aminotransferase; T 4 =Thyroxine; T 3 =Triidothyronine; ROS=Reactive oxygen species, SOD=Superoxide dismutase.Means bearing different superscript a, b within a row differ significantly at (p<0.05)