Variations of Physiological Parameters and HSP70 and HSP90 Polymorphisms in Water Buffaloes in Taiwan During Cool and Warm Season

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INTRODUCTION
Global warming and climate change have increasingly affected livestock production and related industries; specifically, extreme temperatures, floods, typhoons, and other events associated with global warming and climate change result in damage to livestock facilities, livestock deaths, and decreased reproductive and growth rates [1][2][3].Water buffaloes are found in 129 countries and makeup about 11.1% of the world's cattle population [4].In developing countries, water buffaloes are valuable economic assets that provide milk and meat [5].Nevertheless, they are affected by events associated with climate change.For example, heat stress causes physiological changes in water buffaloes, including rapid increases in body temperature, pulse rate, and respiratory rate (RR), in addition to signs of discomfort.The intensification of such heat stress can impair water buffaloes' growth, reproduction, and productivity, ultimately affecting herd health.Heat stress in water buffaloes can be estimated using the temperaturehumidity index (THI; [6]).By monitoring the THI, farmers can determine the comfort levels of their animals and adjust their livestock management practices or environmental conditions, thus substantially improving the welfare of the animals [6,7].Somparn et al. [8] used the THI to examine heat stress responses in cattle and water buffaloes in Thailand.They determined that a THI of ≤74 can generally be considered to indicate no safety concerns for healthy animals; a THI of 75-78 can be regarded as indicating cautionary conditions, under which water buffaloes experience reduced weight gain; a THI of 79-83 can be considered to indicate dangerous conditions, under which water buffaloes experience a substantial decrease in weight; and a THI of >84, especially if accompanied by inadequate management, can be considered to lead to fatalities among animals.Overall, in water buffaloes, the adverse effects of heat stress, including impaired reproductive performance, begin to appear at a THI of ≥75 [7,9].sequences.Among them, HSP70 is a cellular indicator of heat stress in water buffaloes [10], and HSP90 regulates the cell cycle, signal transduction, stress response, protein folding and degradation, and protein transport.When an animal's body temperature becomes excessively high, heat stress stimuli activate heat-shock transcription factor-1, which enhances the expression of HSPs while reducing the synthesis of other proteins.This activation also induces HSPregulated immune and endocrine systems, altering animals' physiological states to help them adapt to high-temperature environments [11].Bhat et al. [12] analyzed an HSP70 sequence in Tharparkar dairy cattle.They observed that animals with the TT genotype could effectively maintain their rectal temperature (RT) at 38.3 °C during the warm season, indicating that they exhibited considerable heat tolerance.Water buffaloes in Taiwan are of the swamp type and were introduced from Guangdong and Fujian in the 16th century for agricultural purposes.They were initially used as working animals, with their population in Taiwan peaking at 310,000.Nevertheless, this population has been steadily declining because of agricultural mechanization.
Considering the changes mentioned above in the population of water buffaloes in Taiwan and the effects of climate change, the present study investigated the variations in the physiological parameters of water buffaloes between cool and warm seasons.It analyzed the diversity in HSP70 and HSP90.The study findings can help improve water buffalo farming management in response to climate change.

Experimental Animals
This study selected 13 male and 13 female water buffaloes (26 in total) aged 1 to 2 years from a farm in Hualien, Taiwan, for examination in an experiment conducted in February and August of 2020 and 2021.The water buffaloes were raised in a semiopen housing system, with an average exercise field area of 1620 m

Analysis of Temperature and Humidity in Hualien
This study collected and analyzed temperature and humidity records for the 2020-2021 period from the Hualien Observatory Station of the Central Weather Bureau (cwb.gov.twhttps://e-service.cwb.gov.tw/HistoryDataQuery/index.jsp).

Measurement of Ambient Temperature and Humidity at the Experimental Site
A temperature and humidity data collector (THD-8, Jiude Electronics, Taichung, Taiwan) continuously collected ambient temperature and humidity data in the practical barn and exercise field for five consecutive days.Based on the collected data, the THI was calculated using the formula proposed by Kendall and Webster [6]: Tdb represents dry-bulb temperature (°C) and RH represents relative humidity (%).

Measurement of RT and RR
The water buffaloes' RT and RR were measured daily at 13:30-14:30 for five consecutive days in February and August of 2020 and 2021.Moreover, their heat tolerance coefficient (HTC) was calculated using the following formula: (RR/23) + (RT/38.33).An HTC value of 2 indicated maximum adaptability, and an HTC value of >2 indicated a lower adaptability state [13,14].

Analysis of Plasma Protein Expression
Blood collection tubes containing ethylenediaminetetraacetic acid were used to collect blood samples (8-10 mL) from the jugular veins of each animal in February and August.The collected samples were centrifugated at 1,500 g for 15 min (Kubota 5800, Japan).Subsequently, the upper plasma layer was composed for the analysis of HSP70 and HSP90 alpha (HSP90AA1) expression by using bovine HSP70 and HSP90 ELISA kits (Cusabio Technology, Houston, TX 77054, USA) following the manufacturer's instructions.

Extraction of DNA
Blood samples (3-5 mL) were collected from all animals included in the study.Genomic DNA was extracted from the samples.It was then purified using a DNA extraction kit (QIAamp ® DNA Mini Kit, Qiagen GmbH, Germany) and stored at −20 °C.

Analysis of Target Gene Sequences
Specific DNA primers were designed by the Primer3 software (https://primer3.org/) to study the gene fragment sequences of HSP70 and HSP90 in the water buffaloes, as reported in Table 1; these sequences were then aligned for analysis.A polymerase chain reaction (PCR) was performed to amplify the target genes.

Execution of PCR
A PCR was conducted using 100 ng of genomic DNA as a template.The reaction mixture (total volume: 25 µL) contained Fast-Run TM 2X Taq Master mix (Protech, Taiwan), 0.5 µL of forward and reversed primers (10 pmol each), and sterile water.The PCR profile used was 35 cycles at 94°C for 30 s, a primerspecific annealing temperature (see Table 1) for 30 s, and an extension period of 70 s at 72°C with an initial denaturation for 5 min at 94°C and a final extension at 72°C for 10 min.The PCR products were analyzed through electrophoresis on 1.5% agarose gel at 100 V and visualized using ethidium bromide staining and a gel-imaging system.Further sequencing was performed if fragments of the expected length for the target genes were detected.PCR products were then purified with the Gene-Spin™ 1-4-3 DNA extraction kit (Protech, Taiwan).

Alignment of Target Sequences
The BLAST function on the NCBI website was used to determine whether the sequences matched the gene fragments of interest.All target gene fragments from the 26 water buffaloes were sequenced (Genomics company, New Taipei City, Taiwan), and the sequencing results were aligned using the alignment function of MEGA6 software [15] to identify singlenucleotide variations (SNVs).

Statistical Analysis
All statistical analyses were conducted using SPSS version 22 (IBM Corp, Armonk, NY, USA).The data were expressed as mean ± standard deviation (SD) and analyzed using an independent-sample t-test and Pearson's method correlation coefficient.

Ambient Temperature and Humidity, As Well As RT and RR of Water Buffalo
In Hualien, the average temperature in February (cool season) was 19.5 °C, and the corresponding THI was 66.0; the average temperature in August (warm season) was 28.7 °C, and the corresponding THI was 80.6.Significant differences were observed in temperature, humidity, and THI (all P < 0.0001) between cool and warm seasons (Table 2).The results also indicated significant differences in RT (P= 0.0004), RR (P < 0.0001), and HTC (P < 0.0001) between the two seasons (Table 2).A Pearson correlation coefficient analysis indicated moderate to high positive correlations between RT (0.518), RR (0.744), HTC(0.757), and THI (Table 3), suggesting that the water buffaloes physiological parameters increased with temperature and humidity levels.These findings are consistent with those of previous studies [16,17].Manjari et al. [10] compared the RRs, pulse rates, and RTs of 20 Indian Tarai water buffaloes between winter and warm seasons.They observed that all three physiological parameters were significantly higher during the warm season (P < 0.05).Shenhe et al. [18]  found a significant correlation between THI, RR (r = 0.84), and RT (r = 0.81) in crossbred buffaloes.The water buffaloes are homeothermic animals that maintain their body temperature through metabolic heat production and heat dissipation [19,20].In water buffaloes, higher RTs in warm climates indicate inadequate heat dissipation; such insufficient heat dissipation can lead to increased stress levels, elevated pulse rates, respiratory frequency, and discomfort in these animals, which can gradually affect their feed intake and feed conversion efficiency [21,22].However, healthy water buffaloes maintain normal body temperatures through physiological and behavioral adaptation strategies.Physiologically, they shiver to generate heat during cold stress and sweat to reduce body temperature during heat stress; behaviorally, they seek shade to avoid direct sunlight and wallow in water ponds to regulate body temperature [23].Vale [7] demonstrated that water buffaloes with a 3-minute shower of cool water twice daily effectively dissipate excess heat.
The results of this study indicated that in the same season, the RTs of the male water buffaloes were significantly higher than those of the female water buffaloes (Table 4); nevertheless, no significant between-sex differences were observed in RR or HTC.These results are consistent with the findings of Abbaya et al. [24].Factors influencing temperature regulation include sex hormone levels, water balance regulation, physical activity, body weight, body size, muscle mass, body fat content, and behavior.Compared with males, females generally have smaller body sizes and a higher surface-to-volume ratio, causing faster heat dissipation.However, males have more muscle mass than their female counterparts, which increases heat production.The higher RTs of the male can be attributed to their greater muscle mass; specifically, even at rest, muscles contribute to approximately 25% of the body's heat production, resulting in increased body temperatures [25,26].The HTC analysis results revealed that regardless of the season (i.e., cool or warm season), water buffaloes with an HTC of <2 exhibited significantly lower RRs than did those with an HTC of ≥2; nevertheless, no difference in RT was noted between water buffaloes with an HTC of <2 and those with an HTC of ≥2 (Table 5).These findings are consistent with previous studies, which reported a weaker correlation between RT and heat tolerance and a strongly positive correlation  between RR and heat tolerance [17,27].Liu et al. [28] used principal component analysis to classify cattle populations into heat-tolerant and heat-sensitive types; they revealed that heat-tolerant cattle exhibited significantly lower RTs and RRs than heat-sensitive cattle.The sweat glands in the skin of water buffaloes are only one-sixth of those in the skin of other cattle breeds [21]; therefore, water buffaloes are less heattolerant mammals [29].Water buffaloes efficiently regulate body temperature by sweating and panting for evaporative cooling.Accordingly, for water buffaloes, respiration is a more effective mechanism of heat dissipation than sweating [21,29,30].
Water buffaloes have weaker physiological responses to extreme heat and cold than other cattle breeds [7].In summer, water buffaloes must be provided with shelters or shower facilities; in winter, they should be provided with appropriate housing systems to protect them from cold winds [21].Water buffaloes cool themselves by wallowing in mud; alternatively, they can be cooled by being continuously sprayed with water and then allowing the air or wind to dry them off.Water buffaloes cool down rapidly in shaded or dark areas; a possible reason is that their black skin, rich in blood vessels, effectively conducts and dissipates heat.Even without mud baths, water buffaloes can maintain their growth if provided with sufficient shade [21].In addition, Garcia [31] suggested that the introduction of trees in grazing areas can increase local biodiversity, provide shade and resting places for animals, reduce direct sunlight exposure, and improve the comfort and well-being of animals.In Brazil, water buffaloes raised in silvopastoral and rotational grazing systems were reported to achieve a daily weight gain of up to 0.911 kg [32].According to the data collected from the Central Weather Bureau from 2015 to 2019, the average THI in Hualien, Taiwan, was 73.3, and it was >75 (75.8-80.1)from April to September.Therefore, in these months, appropriate adjustments should be made to the management and environment of water buffalo herds in Taiwan; such adjustments can include planting trees and installing simple shade structures or sprinkler systems.

Impact of Environmental Changes during Cool and Warm Seasons on Plasma HSP70 and HSP90 Expression Levels
This study determined that HSP70 expression levels in the plasma of the water buffaloes were significantly higher during the warm season than during the cool season (Table 6).The Pearson correlation coefficient analysis results (Table 3) revealed a strong positive correlation (0.891) between HSP70 levels and the THI but a weak positive correlation (0.352) between HSP90 levels and the THI.Moreover, HSP70 levels exhibited moderate to high positive correlation with RT (0.494), RR (0.743), and HTC (0.751).A weak correlation (0.319) was observed between HSP70 and HSP90 levels.These findings are consistent with those in the literature.For example, Shenhe et al. [18] compared the physiological parameters of crossbred water buffaloes (Nili-Ravi × Murrah) between spring, summer, and winter.They reported that the R.R.s, R.T.s, and plasma HSP70 and HSP90 expression levels were significantly higher in summer compared to spring and winter (P < 0.05).During the warm season, they also reached the physiological parameters of crossbred and purebred Mediterranean water  expression levels increased in response to short-term heat stress, but the increase in HSP90 expression was nonsignificant.The present study observed a lower correlation coefficient between HSP90 expression levels and the other parameters (Table 3), possibly owing to large individual variations in HSP90 expression levels.Studies have also indicated that livestock and poultry exhibited increased HSP70 and HSP90 mRNA expression levels in their organs or peripheral blood mononuclear cells as an adaptation mechanism to dry and warm-humid environments; additionally, HSP70 mRNA expression levels were significantly higher during the warm season than they were during winter and were strongly correlated with RR, pulse rate, and RT [33][34][35][36].
This study compared plasma HSP70 and HSP90 expression levels between the male and female water buffaloes during the same season (Table 7).The results revealed that the plasma HSP70 expression levels were significantly higher in the female water buffaloes during the cool season.Under normal conditions, plasma HSP70 expression in female animals is twice that in male animals [37].Furthermore, this study compared HSP70 and HSP90 expression levels between heat-tolerant and heat-intolerant water buffaloes-grouped based on HTC values-in either season and found no significant difference in these levels between the groups (Table 8).By contrast, Liu et al. [28] reported that plasma HSP70 and HSP90 levels were higher in heat-tolerant water buffaloes than in heat-intolerant water buffaloes.The reason for this discrepancy is unclear.A small group of water buffalo could cause it.Further research is warranted to clarify the differences in HSP70 and HSP90 levels between heat-tolerant and heat-intolerant water buffaloes.

Primer Design for Amplification of Target Gene Fragments
This study designed primer sequences based on the target gene sequences of marsh-type water buffaloes published in GenBank.Thus, the study could successfully amplify HSP70 and HSP90 fragments (Table 1).

Sequencing Results and Genetic Polymorphism Analysis
After sequencing, this study analyzed the sequence diversity of the target gene fragments.The analysis revealed the presence of SNVs at certain loci in HSP70 and HSP90 of the water buffaloes: HSP70 g.24927355 T>C, HSP70 g.24927424 G>A, HSP70 E2 g. 24928813 C>T, HSP90 g.17755706 G>A, and HSP90 g. 17756478 T>C.Their genotypic and allelic frequencies are presented in Table 9.All identified SNV loci in this experiment are in Hardy-Weinberg equilibrium, according to the analysis.
HSPs are crucial molecules involved in animals' response to heat stress.Most HSP genes lack introns, which may contribute to their rapid expression.The HSP40, HSP60, HSP70, and HSP90 family proteins prevent misfolding of denatured proteins, assisting in the refolding of denatured proteins into their native conformations, activating immune and endocrine systems and altering physiological states to help animals adapt to their environments [36].Crossbreeding with heat-tolerant breeds improves the heat tolerance of buffaloes [16,22,41], but breeding may make them more susceptible to stress instead of enhancing their stress resistance [34,42].

CONCLUSIONS
Our results revealed a close relationship between the physiological parameters of water buffaloes in Taiwan and changes in ambient temperature and humidity.These findings can guide management practices to provide a more comfortable environment for water buffaloes, enhancing animal welfare.Furthermore, HSP70 and HSP90 polymorphisms in water buffaloes in Taiwan can serve as references for future research on breeding and relevant molecular markers.

FUNDING
This research received no specific grant from funding agencies in the public, commercial, or not-forprofit sectors.

Table 4 : Mean RTs, RRs, and HTCs of Male and Female Water Buffaloes in Cool and Warm Seasons
Means within the same row in the same season with different superscripts differ significantly (P < 0.05).