Endocrine response of crossbred calves to acute heat stress in a climate-controlled environment

Introduction

Heat stress in cattle associated with global climatic change affects their overall health and production. Cattle are homeothermic animals and initiate thermoregulatory responses to heat stress (HS). Livestock maintains thermal homeostasis by adopting various thermo-regulatory mechanisms that significantly promote their welfare under stress (Indu and Pareek, 2015) [6] . When the endogenous heat production of cattle exceeds its capacity to lose heat, it results in HS (Bernabucci et al., 2010) [2] . The physio-biochemical functions of cattle will remain normal in thermo-neutral zone (TNZ) or comfort zone. In their comfort zone, there won't be any energy expenditure for thermoregulation. When the ambient temperature exceeds the upper critical limit of TNZ, cattle experience HS (Bagath et al., 2019) [1] . The hypothalamopituitary-adrenal axis (HPA axis) act as one of the principal endocrine regulators of stress responses. The activation of the HPA axis as a response to stress might lead to enhanced production and release of cortisol into circulation which is associated with immune suppression in cattle. Hence serum cortisol is considered a most reliable and primary physiological index for determining stress response in animals (Binsiya et al., 2017) [4] . Cortisol secretion during acute stress acted as a stimulus for the immune system and its secretion during chronic stress was associated with immune suppression (Ju et al., 2014) [7] . Generally, serum cortisol concentration will be elevated during short-term heat stress acclimation (STHSA) and it will be gradually decreased during long-term heat stress acclimation (LTHSA) due to negative glucocorticoid feedback to HPA axis as an adaptation to prolonged HS in cattle (Bharati et al., 2017) [3] . In view of this, the present study was conducted with the objective of assessing the endocrine response of crossbred calves exposed to acute heat stress (Treatment) and thermo-neutral zone (Control) in a climate-controlled environment. in animal holding facility (Fig. 1) and inside the climatecontrolled chamber (Fig. 2). It was followed by experimental period of 10 days each under TNZ and acute HS in the chamber. In TNZ, the chamber was maintained at temperature of 27 °C. For acute HS, maximum temperature of 40 °C was simulated for three hours a day (11 A.M. to 2 P.M.) for period of 10 days. For both TNZ and HS, the relative humidity was maintained as 55-65 per cent. The temperature humidity index (THI) for the chamber was calculated based on the standard formula of Mader et al.

Materials and Methods

Ethical approval

(2006) [9] . After the experiment, the animals were re-shifted to the farm for rehabilitation and reuse, as per the recommendations of CCSEA.

Collection of Venous Blood samples

Blood samples were collected in both the experimental groups on days 0, 1, 5 and 10 of the experiment into clot activator vacutainers between 1 P.M. and 2 P.M. through jugular venipuncture. Blood clotting was ascertained after one hour of incubation at a controlled temperature of 5 °C to ensure optimal serum preparation and were subjected for centrifugation at 2700×g at 4 °C for 15 minutes. Sera were separated and were stored at-20 °C for cortisol estimation.

Estimation of endocrine response

Cortisol levels were estimated in serum by using Cortisol C. T. Radio immuno-assay (RIA) kit (M/s Beckman Coulter, United States) using gamma counter (Perkin Elmer model Wizard, Fig. 3). 1. 2. The tubes were placed in the gamma counter (Fig. 3) for counting radiations.

Calculation of results

Results were obtained from standard curve by interpolation. The results were calculated using a semi-logarithmic curve fit with B (bound)/T (total %) on vertical axis and the cortisol concentration of the calibrators on the horizontal axis (nM/L). The sample values were directly read from the standard curve as nM/L and converted into ng/mL (1 nM/L = 0.3627 ng/mL).

Statistical analysis

The results of the experiment were expressed as Mean ± Standard Error. The statistical significance was assessed by linear mixed model analysis of variance (ANOVA) using SPSS version 24.0 statistical software.

Results and Discussion

The overall serum cortisol level of calves under acute heat stressed condition was significantly higher (p<0.001) when compared to TNZ (Table 2). The mean values differed significantly between different days of both the groups. In TNZ, the highest mean value of serum cortisol was noticed on day 0 (before exposure under TNZ). There was significant decline in serum cortisol levels throughout the experimental days of TNZ. The mean serum cortisol level was significantly higher (p<0.01) on day one of sudden heat exposure when compared to day zero. It was further increased on day five of heat stress. There was a highly significant dip (p<0.01) in serum cortisol level on day 10 of HS when compared to all other days of HS (Fig. 4). Furthermore, high significant difference (p<0.01) was observed in serum cortisol values of day 0, 1 and 5 between the TNZ and HS groups. On day 10, there was no significant difference found between the groups. The current findings of significantly higher (14.64 ± 0.78 ng/mL; p<0.001) serum cortisol level in acute heat stressed calves when compared to thermo-neutral condition (8.28 ± 0.38 ng/mL) was consistent with the results reported by Woo et al. (2024) [10] . They found a significantly increased (p<0.05) serum cortisol values with an increase in THI. In their study, serum cortisol value was lowest at comfort zone (11.96 ng/mL) and highest in HS (17.17 ng/mL) which further validating the present findings. Similar findings were also reported by Kim et al. (2018) [8] who found a higher serum cortisol level in calves exposed to THI of 87.72 (17.1 ng/mL) than those exposed to THI of 76.51 (4.6 ng/mL). Hence the rise in serum cortisol concentrations was temperature-dependent which is in accordance with the findings of Yadav et al. (2015) [11] . According to Ju et al. (2014) [7] , the increase in cortisol level in calves during acute stress might have acted as a stimulus for the immune system. In the present study, the mean serum cortisol level in calves was significantly higher (p<0.01) on the first day of sudden heat exposure when compared to before exposure or day zero. It was further increased on day 5 of HS and there was a significant dip (p<0.01) on day 10 of HS when compared to all other days of treatment. This is consistent with Yadav et al. (2021) [12] who evidenced a significantly higher serum cortisol level in crossbred cattle on day 1 of heat exposure at 40 °C than under TNZ at 25 °C followed by a gradual dip from day 6 to day 21 with a significant dip on day 16 of heat exposure at 40 °C. The initial peak in serum cortisol level might be attributed to sudden activation of the HPA axis. The gradual reduction from the day 5 to significant reduction on the day 10 might be due to the cortisol acclimation pathway in heat-stressed crossbred calves. Similar to the current findings, Bharati et al. (2017) [3] found that serum cortisol concentration was significantly elevated (p<0.05) during short-term heat stress acclimation (STHSA) and it was gradually decreased during long-term heat stress acclimation (LTHSA). They opined that the decline in cortisol levels during LHTSA was attributed to negative glucocorticoid feedback to HPA axis, as an adaptation to prolonged HS in cattle. Garner et al. (2017) [5] found a contradictory finding of no significant difference in mean cortisol values between thermoneutral (3.7 ng/mL) and temperature challenged (3.2 ng/mL) cows kept in a climate-controlled chamber for four days. Similarly, Zarina (2016) [13] also documented that there was no significant correlation between serum cortisol levels and THI.

Conclusion

The present study elucidated the impact of acute heat stress on HPA axis of crossbred calves under climate-controlled environmental conditions. From the study, it was precise and evident that rise in serum cortisol was temperaturedependent. Furthermore, the results demonstrated cortisol acclimation pathway in heat-stressed crossbred calves (STHSA and LTHSA) under controlled climatic conditions. The decline in serum cortisol levels under TNZ in crossbred calves gave a focus on maintaining them in comfort zone for their welfare, production and productivity.

Acknowledgement

We acknowledge Kerala Veterinary and Animal Sciences University (KVASU) and the facilities availed in the Department of Veterinary Physiology, Centre for Animal Adaptation to Environment and Climate Change Studies (CAADECCS) and Central Instrumentation Laboratory (CIL) of College of Veterinary and Animal Sciences, Mannuthy for successful conduct of the research. Conflict of InterestThe authors declared no conflict of interest.

References

1. Bagath M, Krishnan G, Devaraj C, Rashamol VP, Pragna P, Lees AM, et al. The impact of heat stress on the immune system in dairy cattle: a review. Res Vet Sci. 2019;126:94-102.
2. Bernabucci U, Lacetera N, Baumgard LH, Rhoads RP, Ronchi B, Nardone A. Metabolic and hormonal acclimation to heat stress in domesticated ruminants. Animal. 2010;4(7):1167-1183.
3. Bharati J, Dangi SS, Mishra SR, Chouhan VS, Verma V, Shankar O, et al. Expression analysis of toll-like receptors and interleukins in Tharparkar cattle during acclimation to heat stress exposure. J Therm Biol. 2017;65:48-56.
4. Binsiya TK, Sejian V, Bagath M, Krishnan G, Hyder I, Manimaran A, et al. Significance of hypothalamicpituitary-adrenal axis to adapt to climate change in livestock. Int Res J Agri Food Sci. 2017;2:1-20.
5. Garner JB, Douglas M, Williams SR, Wales WJ, Marett LC, DiGiacomo K, et al. Responses of dairy cows to short-term heat stress in controlled-climate chambers. Anim Prod Sci. 2017;57(7):1233-1241.
6. Indu S, Pareek A. Growth and physiological adaptability of sheep to heat stress under semi-arid environment: a review. Int J Emerging Trends Sci Technol. 2015;2(9):3188-3198.
7. Ju XH, Xu HJ, Yong YH, An LL, Jiao PR, Liao M. Heat stress upregulation of toll-like receptors 2/4 and acute inflammatory cytokines in peripheral blood International Journal of Advanced Biochemistry Research https://www.biochemjournal.com mononuclear cells of Bama miniature pigs: an in vivo and in vitro study. Anim. 2014;8:1462-1468.
8. Kim WS, Lee JS, Jeon SW, Peng DQ, Kim YS, Bae MH, et al. Correlation between blood, physiological and behavioral parameters in beef calves under heat stress. Asian-Australas J Anim Sci. 2018;31:919-926.
9. Mader TL, Davis MS, Brown-Brandl T. Environmental factors influencing heat stress in feedlot cattle. J Anim Sci. 2006;84:712-719.
10. Woo JS, Lee NK, Lee HG, Park KK. Effects of heat stress on performance, physiological parameters, and blood profiles of early-fattening Hanwoo steers in climate chambers. Anim Biosci. 2024;37(1):142-150.
11. Yadav B, Singh G, Wankar A. Adaptive capability as indicated by redox status and endocrine responses in crossbred cattle exposed to thermal stress. J Anim Res. 2015;5:67-73.
12. Yadav B, Singh G, Wankar A. Acclimatization dynamics to extreme heat stress in crossbred cattle. Biol Rhythm Res. 2021;52:524-534.
13. Zarina A. Adaptability profile of male cattle and buffalo calves to varying temperature humidity index in Kerala [PhD thesis]. Pookode (India): Kerala Veterinary and Animal Sciences University; 2016. 135 p.