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Heat Shock Protein 70 (HSP70) m-RNA Expression Pattern during Different Seasons in Red Kandhari Cattle of India

M
Maharudra J. Sanap1,*
https://orcid.org/0009-0002-1079-3262
P
Pranesh V. Yeotikar2
https://orcid.org/0000-0003-0316-1486
P
Prabhakar B. Ghorpade2
https://orcid.org/0000-0002-8793-3553
S
S. Sajid Ali2
https://orcid.org/0000-0003-1226-0910
V
Vilas B. Dongre1
https://orcid.org/0000-0002-5681-4789
S
Suhas A. Amrutkar3
https://orcid.org/0009-0006-6487-2037
M
M.F.M.F. Siddiqui2
https://orcid.org/0009-0002-0943-6233
1College of Veterinary and Animal Sciences, Maharashtra Animal and Fishery Sciences University, Udgir-431 402, Maharashtra, India.
2College of Veterinary and Animal Sciences, Maharashtra Animal and Fishery Sciences University, Parbhani-413 517, Maharashtra, India.
3Institute of Veterinary and Animal Sciences, Akola-444 001, Maharashtra, India.

ABSTRACT

Background: Heat shock proteins (HSPs), particularly HSP70, are highly conserved and ubiquitously expressed proteins that are synthesized in response to a variety of stressors. Among these, HSP70 plays a crucial role in cellular protection during heat stress by being rapidly up-regulated, thereby exerting a cytoprotective effect. Acting as molecular chaperones, HSPs help maintain the proper three-dimensional structure of proteins under adverse environmental conditions. As a result, they enhance cellular adaptability and contribute significantly to stress tolerance and thermal regulation. The present study aimed to examine the expression pattern of the HSP70 gene in Red Kandhari cattle across different seasons, with a particular focus on assessing relative thermal stress during summer in comparison to the winter and rainy seasons.

Methods: In the present experiment, apparently healthy, male (n=12) and females (n=12) experimental animals of Red Kandhari cattle with the average age ranging from 1-4 yrs were selected from Livestock Farm Complex, College of Veterinary and Animal Sciences, Parbhani. Blood samples (2 mL) were collected once for the three different seasons viz. summer: April-May (May: THI 83.36), rainy: June-September (September: THI 78.24) and winter: January-February (January: THI 67.84) during 2023. A quantitative Real-Time PCR (qPCR) expression study was undertaken to evaluate the relative m-RNA expression pattern of the HSP70 gene during different seasons.

Result: The study revealed that the relative mRNA expression of the HSP70 gene was significantly higher (p<0.01) during the summer season compared to the winter and rainy seasons in Red Kandhari cattle. Additionally, expression levels were significantly higher (p<0.01) in the rainy season than in winter. These findings suggest that fluctuations in ambient temperature and humidity lead to increased HSP70 gene expression. Notably, during the summer, the elevated expression of heat shock proteins in Red Kandhari cattle appears to support cellular adaptability, enhance stress tolerance and maintain thermal homeostasis.

INTRODUCTION

The Red Kandhari cattle categorized as an indigenous breed believed to be originated from Kandhar Tehsil of the Nanded district of Maharashtra state. Red Kandhari animal are well adapted to agro-climatic conditions of various breeding tracts e.g. Nanded, Parbhani, Hingoli and Beed districts of Maharashtra state and the Bidar district of Karnataka. Red Kandhari breed is used as draft animal for agricultural work. Now a day, Red Kandhari breed is reared as a milch animal for the purpose of milk in Maharashtra state.
       
Among stressors, one of the major environmental stressor is heat stress that affects the animal production (Behera et al., 2022). It can cause disturbances in homeostasis such as physiological, thermoregulatory responses (West, 1994). Cattle are homoeothermic animals which live in thermo-neutral zone ranging from 4oC to 25oC. Animals can maintain their core body temperature by making evaporative and non-evaporative heat losses. It was revealed that, the adverse effects of high ambient temperature are aggravated when heat stress is accompanied by high ambient humidity (Marai et al., 2007).
       
Temperature humidity index (THI), is most extensively used parameter for the determination heat load on the animals which is good indicator of thermal stress of climatic conditions (McDowell et al., 1976; Behera et al., 2018). The thermoregulation mechanism is vital along with thermo neutrality and comfort zone for maintaining body conditions in response to stress (Kadzere et al., 2002). It is governed by stable heat proteins known as Heat Shock Proteins (HSP) in ruminants (Collier et al., 2008).
       
Heat shock proteins comprise a large group of molecular chaperones, classified based on molecular size and similarities in amino acid sequences (Kumar et al., 2015). Amongst members of the HSP family, HSP70 is the most abundant and temperature sensitive essential protein (Beckham et al., 2004) well known to be conserved and ubiquitous in nature (Liu and Steinacker, 2001; Kregel, 2002) produced by cells in response to the stress conditions (Lanneau et al., 2007).
       
Heat shock protein 70 (HSP70) are dynamic, conferring cryoprotection against different stressors. Amongst the stressors, thermal stress triggers a complex mechanism of gene expression as well as adaptive biochemical responses (Lindquist, 1986 and Fujita, 1999). Heat shock proteins can prevent the cells from oxidative stress through its antioxidant and thermo tolerance effect (AL-Jaryan et al., 2023). It is also functioning as molecular chaperon which is attributed to fold, unfold and refold stress-denatured proteins. 
       
Numerous research activities have been conducted based on HSP70 gene expression patterns during different seasons in cross-bred cattle throughout the globe.  However, very meagre studies were found in the Indigenous cattle breeds of India. Therefore, the present study was undertaken to determine the relative mRNA expression pattern of HSP70 gene with special reference to the summer season in response to thermal stress in Red Kandhari cattle.

MATERIALS AND METHODS

The present search was conducted in the department of Veterinary Biochemistry, College of Veterinary and Animal Sciences (MAFSU), Parbhani, total twenty-four (24) apparently healthy animals of either sex with average age ranging from 1-4 yrs. were selected from Livestock Farm Complex, COVAS, Parbhani. They were kept under similar managemental and nutritional regimen throughout the duration (January 2023 to September 2023) of the experimental period as shown in Table 1.

Table 1: Experimental animals for study.


 
Temperature humidity index
 
Temperature and relative humidity was recorded inside the shed with the help of dry bulb/wet bulb thermometer and thermo hygrometer (Mader et al., 2006). Temperature humidity index (THI) of the animal shed was calculated using the formula as follows:
 
THI = (0.81 × Tdb) + {(RH ÷ 100) × (Tdb - 14.4)} + 46.6
 
Where,
Tdb = Average temperature in oC of the dry bulb thermometer
RH = Average relative humidity.
 
Collection of samples
 
The blood samples (2 mL) were collected aseptically by jugular venipuncture with EDTA vacutainer from Red Kandhari cattle during peak summer month (May, THI: 83.36); peak rainy month (September, THI: 78.24); and peak winter (January, THI: 67.84); seasons. During the collection of samples, cold chain was maintained by use of ice cubes and were immediately processed further for the isolation of total RNA. 
 
Reagents, media and kits
 
The glassware and plastic ware procured from various firms such as Borosil, Himedia etc. The reagents or kits obtained namely e.g. Agarose, Ethidium bromide, DEPC, Hi Temp PCR master mix, SYBR green master mix, DNase-I, Amplification grade, Invitrogen life Technologies, California, USA.
 
Isolation of total RNA from whole blood
 
The pure linkTM total RNA blood purification kit (Invitrogen Life Technologies, California, USA) was used for Isolation of total RNA from whole blood by following the recommended manufacturer’s protocol. Due to its Instability, immediately RNA was processed further for the synthesis of cDNA.
 
Synthesis of cDNA
 
The protocol was optimized for cDNA. The quality and quantity of cDNA was recorded (Nanodrop Spectrophotometer, Himedia). ThecDNA samples showing OD: 260/280 more than 1.6  were processed further for RT- qPCR analysis.
 
Primers
 
The cattle specific published primer sequences complementary to HSP70 and GAPDH genes were purchased from Barcode Bioscience, Bangalore (Karnataka).  The primer sequences, annealing temperature and predicted fragment size shown (Table 2).

Table 2: Primer sequence of genes.


 
Optimization of PCR conditions and primers specificity
 
The primers were diluted with double distilled water to make 100 µM stock solutions and were stored at -20oC in Deep freezer until use. PCR conditions were optimized by employing various annealing temperature corresponding to the Tm of the primes in Thermo cycler.
 
The confirmation of cDNA
 
The PCR product revealed distinct bands for HSP70 and GAPDH genes on GelDoc (Bio-rad) by Agarose gel electrophoresis with 1.5% Agarose gel as shown (Fig 1).

Fig 1: Confirmation of cDNA and specificity of primers on 1.5 Agarose gel electrophoresis.


 
Amplification plot for HSP70 and GAPDH gene expression
 
The amplification plot for expression of HSP70 and GAPDH genes was obtained. This was used for examining the unspecific binding, primer dimer or secondary structure formation as shown (Fig 2).

Fig 2: Real time pCR (qPCR) amplification plot of HSP70 and GAPDH gene expression.


 
Confirmation of qPCR product
 
The real time PCR product was confirmed on 1.5% Agarose gel through Gel Doc(Bio-Rad) system as shown (Fig 3).

Fig 3: Confirmation of real time PCR products on 1.5 Agarose gel elctrophoresis.


 
Validation of qPCR product by melt curve analysis
 
The dissociation characteristics of double stranded DNA were assessed during thermal cycles of qPCR as shown (Fig 4).

Fig 4: Melt curve analysis of real time PCR (qPCR) reaction products.


 
Statistical analysis
 
Statistical analysis of data was done by using SPSS statistical software for windows (version 24). One way ANOVA was applied to analyse the variance about mean Ct value for relative mRNA expression of HSP70 gene between seasons.

RESULTS AND DISCUSSION

Temperature humidity index
 
The mean values of THI recorded during the peak months of different (summer, winter and rainy) seasons are presented (Table 3).

Table 3: Temperature humidity index (Mean ± SE) of three different seasons.


       
In the present study, the highest THI (83.36) was recorded in the month of May and the lowest (67.84) in January. However, the intermediate value (78.24) was recorded in the September.
       
The values of THI 74 or less indicating comfort ability, 75-78 showing an alert status, greater than 79 to 83 could be vulnerable, THI equal to or above 84 is an emergency situation (McDowell, 1976 and Kohli, 2014).
       
We observed that temperature and humidity were the factors responsible for increase in m-RNA expression during peak summer (May: THI 83.36) as compared to peak rainy (September: THI 78.24) and peak winter (January: THI 67.84) seasons. Temperature humidity index (THI) is used to estimate the degree of stress on the animals (Armstrong, 1994 and Vaidya et al., 2010).
       
It is worldwide accepted that THI equal to 72 is the threshold for environmental heat stress. However, Thatcher et al. (2010) reported that, if THI above 72, dairy cows could be exposed to heat stress and when it exceeds 88, then animals might be living under severe heat stress conditions. Our findings indicate that the animal may be living under moderate heat stress conditions, particularly during the summer season, where THI is 83.36. Moreover, during the rainy season, animals might be living under heat stress condition, where, THI is 78.24, during these conditions, animal was unable to maintain thermoregulatory responses.
 
HSP70 gene expression
 
The HSP70 gene expression was significantly differed amongst all the three seasons. It was found significantly (P<0.01) higher in the experimental animals during peak summer season followed by rainy season, whereas the HSP70 gene expression was significantly (P<0.01) lower during winter season (Table 4 and Fig 5).

Table 4: The relative quantitative m-RNA expression of HSP70 and GAPDH gene in terms of Ct value (Mean ± SE) amongst different seasons in Red Kandhari cattle.



Fig 5: The relative m-RNA expression pattern of HSP70 gene in Red Kandhari cattle.


       
The HSP70 gene expression corresponds to the low Ct values indicating the higher gene expression during the summer as compared to the other two seasons i.e. rainy  and winter.
       
The results of present study revealed that, the significantly (P<0.01) elevated levels of mRNA expression of HSP70 gene during the peak summer season as compared to rainy and winter seasons against hot dry and hot humid conditions in Red Kandhari cattle. These findings are in accordance with the outcomes of Parmar et al. (2015), who found that the relative mRNA expression of HSP70 gene was increased in peripheral blood mononuclear cells (PBMCs) of Sahiwal cows during the summer season as compared to winter. Further, the findings of the present study corroborated by De Maio (1999), who revealed that the cells can increase the expression of several classes of proteins when exposed to environmental stresses. Yadav et al. (2015) and Kumar et al. (2017) have also observed significantly(P<0.01) increase in mRNA and protein expression of HSP70 and HSP90 during summer (high THI) as compared to thermo-neutral season in all the goat breeds.
       
Red Kandhari cattle are acclimatized to hot and dry breeding tract of Maharashtra region. But during peak summer, ambient temperature was higher reaches to peak 44oC which may exert heat stress on animals. The upregulation of gene expression was recorded which is influenced by rise in THI during the summer and rainy seasons, these observation were collinear with the Bharati et al. (2017), who investigated the mRNA expression of HSP70 gene in cultured PBMCs of Tharparkar cattle on exposure to thermal stress. They reported that the HSP70 expression was increased in temperature and time dependent manner in cultured PBMCs as compared to control. These responses potentiate the protective effect of antioxidant enzymes through its chaperon functions (Uttarani, 2017). The upregulation of HSP70 mRNA reduces the oxidative stress at cellular level (Kalmar and Greensmith, 2009), improving antioxidant capacity and inhibits lipid peroxidation in order to protect the animals from harmful effect of heat stress.
       
Droge (2002) who reported that the body releases the HSPs proteins along with the antioxidants on exposure to heat stress to combat the cellular effects of ROS. However, Han et al. (2016), who observe that the transcription of the genes (HSP27, HSP70 and HSP90) of heat shock protein (HSP) was significantly enhanced under heat stress (HS) wherein, the peak transcription of HSP70 was 14 times higher than the control at 1 h.
       
Banerjee et al. (2014), who explored the effect of temperature sensitivity and seasonal variation on the expression patterns of HSP70 genes in Indian goats. They observed a significant (P<0.01) variation between different seasons for all HSP70 gene expressions, the expression pattern was higher during summer season. They concluded that, the animals exposed to ambient temperature beyond the comfort zones were living under thermal stress during summer than that of winter. Also, the similar results were observed by Lacetera, 2006; Patir and Upadhaya, 2007, 2010, who found that the heat induced stress causes upregulation of HSP70 gene expression.
       
The comparable results were reported by Ambade, 2023, who found that the expression of HSP70 gene was significantly (P<0.05) higher during summer as compared to winter season in Pandharpuri buffaloes. They recorded the upregulation of HSP70 gene during both summer and rainy seasons whereas, during winter down regulation of gene was observed. The higher expression of HSP70 genes during the thermal stress suggested that there was involvement of HSP70 to rearrange the harmful effect of heat stress for maintaining the cellular integrity and homeostasis (Dangi, 2012).
       
Our study was corroborated by Kumar et al. (2019) who were undertaken the profiling of HSP70 gene in Murrah buffalo (Bubalus bubalis) under sub-tropical climate of India. They reported that there was relatively higher mRNA expression in summer, when THI was more than 84 as compare to the spring and winter seasons. Moreover, they also reported that there was fold change increase by 4.5 times in summer than the spring seasons.
       
The present study was supported by Pangaonkar et al. (2023) who estimated seasonal effect of THI on HSP70 gene expression in Deccani sheep, they predicted that HSP70 could be used as a potential bio marker for selecting climate-resilient animals that would show superior thermo-tolerance to enhance livestock productivity.
       
Suhendro et al. (2024) who investigated the association of heat shock protein 70.1 gene with physiological and physical performance in Bali cattle. They reported the higher expression of HSP70.1 that could mitigate the deleterious effect of heat stress.
       
Kumar et al. (2024) carried out the expression and SNP profiling of HSP70 gene associated with thermo tolerance traits in Munjal Sheep.  Sheep were categorized into two groups-heat stress susceptible (HSS) and heat stress tolerant (HST)-based on their heat tolerance coefficient. Significant differences in HSP70 gene expression were observed between the HSS and HST groups.
       
Biradar et al. (2024) who studied the single nucleotide polymorphism in promoter region of HSP70 gene in Deoni cattle. They revealed that HSP70 gene polymorphism in genotype AA could be associated with physiological parameter e.g. respiration rate and rectal temperature.
       
In line with the present results, Kim et al. (2025), who reported that the HSP70 gene expression in hair follicle was increased with increase in temperature humidity index which implies that increase in HSP expression indicating thermo tolerance in diary calves. They also reported that there was genetic variation in stress tolerance that could be attributed to HSP70 gene polymorphism which is affecting the cellular responses and gene expression in animals exposed to heat stress.

CONCLUSION

The present study demonstrated that elevated temperature-humidity index (THI) during the summer season significantly influences HSP70 gene expression, suggesting its vital role in the cellular defence mechanism against thermal stress. In Red Kandhari cattle, the upregulation of HSP70 during summer supports cellular adaptability, stress tolerance and thermal regulation. However, the increased expression also indicates potential cellular vulnerability to heat stress during peak summer. HSP70 may serve as a reliable molecular marker for thermotolerance and could be utilized to identify heat-resilient animals in organized breeding programs. Further studies on genetic polymorphisms, particularly SNPs in the HSP70 gene, may enable the selection of heat-tolerant animals for future breeding strategies.

ACKNOWLEDGEMENT

The author wish to thank Associate Dean, College of Veterinary and Animal Sciences, Parbhani for providing the animals and data for this research. The Author also, thankful to The Director of Research, MAFSU, Nagpur for providing valuable guidance.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
Study was approved by Institutional Animal Ethical Committee (IAEC) of the College of Veterinary and Animal Sciences, Parbhani (Maharashtra Animal and Fishery Sciences University, Nagpur) with vide No. IAEC /100/2022.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest in relation to this publication.

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    Full Research Article

    Heat Shock Protein 70 (HSP70) m-RNA Expression Pattern during Different Seasons in Red Kandhari Cattle of India

    M
    Maharudra J. Sanap1,*
    https://orcid.org/0009-0002-1079-3262
    P
    Pranesh V. Yeotikar2
    https://orcid.org/0000-0003-0316-1486
    P
    Prabhakar B. Ghorpade2
    https://orcid.org/0000-0002-8793-3553
    S
    S. Sajid Ali2
    https://orcid.org/0000-0003-1226-0910
    V
    Vilas B. Dongre1
    https://orcid.org/0000-0002-5681-4789
    S
    Suhas A. Amrutkar3
    https://orcid.org/0009-0006-6487-2037
    M
    M.F.M.F. Siddiqui2
    https://orcid.org/0009-0002-0943-6233
    1College of Veterinary and Animal Sciences, Maharashtra Animal and Fishery Sciences University, Udgir-431 402, Maharashtra, India.
    2College of Veterinary and Animal Sciences, Maharashtra Animal and Fishery Sciences University, Parbhani-413 517, Maharashtra, India.
    3Institute of Veterinary and Animal Sciences, Akola-444 001, Maharashtra, India.

    ABSTRACT

    Background: Heat shock proteins (HSPs), particularly HSP70, are highly conserved and ubiquitously expressed proteins that are synthesized in response to a variety of stressors. Among these, HSP70 plays a crucial role in cellular protection during heat stress by being rapidly up-regulated, thereby exerting a cytoprotective effect. Acting as molecular chaperones, HSPs help maintain the proper three-dimensional structure of proteins under adverse environmental conditions. As a result, they enhance cellular adaptability and contribute significantly to stress tolerance and thermal regulation. The present study aimed to examine the expression pattern of the HSP70 gene in Red Kandhari cattle across different seasons, with a particular focus on assessing relative thermal stress during summer in comparison to the winter and rainy seasons.

    Methods: In the present experiment, apparently healthy, male (n=12) and females (n=12) experimental animals of Red Kandhari cattle with the average age ranging from 1-4 yrs were selected from Livestock Farm Complex, College of Veterinary and Animal Sciences, Parbhani. Blood samples (2 mL) were collected once for the three different seasons viz. summer: April-May (May: THI 83.36), rainy: June-September (September: THI 78.24) and winter: January-February (January: THI 67.84) during 2023. A quantitative Real-Time PCR (qPCR) expression study was undertaken to evaluate the relative m-RNA expression pattern of the HSP70 gene during different seasons.

    Result: The study revealed that the relative mRNA expression of the HSP70 gene was significantly higher (p<0.01) during the summer season compared to the winter and rainy seasons in Red Kandhari cattle. Additionally, expression levels were significantly higher (p<0.01) in the rainy season than in winter. These findings suggest that fluctuations in ambient temperature and humidity lead to increased HSP70 gene expression. Notably, during the summer, the elevated expression of heat shock proteins in Red Kandhari cattle appears to support cellular adaptability, enhance stress tolerance and maintain thermal homeostasis.

    INTRODUCTION

    The Red Kandhari cattle categorized as an indigenous breed believed to be originated from Kandhar Tehsil of the Nanded district of Maharashtra state. Red Kandhari animal are well adapted to agro-climatic conditions of various breeding tracts e.g. Nanded, Parbhani, Hingoli and Beed districts of Maharashtra state and the Bidar district of Karnataka. Red Kandhari breed is used as draft animal for agricultural work. Now a day, Red Kandhari breed is reared as a milch animal for the purpose of milk in Maharashtra state.
           
    Among stressors, one of the major environmental stressor is heat stress that affects the animal production (Behera et al., 2022). It can cause disturbances in homeostasis such as physiological, thermoregulatory responses (West, 1994). Cattle are homoeothermic animals which live in thermo-neutral zone ranging from 4oC to 25oC. Animals can maintain their core body temperature by making evaporative and non-evaporative heat losses. It was revealed that, the adverse effects of high ambient temperature are aggravated when heat stress is accompanied by high ambient humidity (Marai et al., 2007).
           
    Temperature humidity index (THI), is most extensively used parameter for the determination heat load on the animals which is good indicator of thermal stress of climatic conditions (McDowell et al., 1976; Behera et al., 2018). The thermoregulation mechanism is vital along with thermo neutrality and comfort zone for maintaining body conditions in response to stress (Kadzere et al., 2002). It is governed by stable heat proteins known as Heat Shock Proteins (HSP) in ruminants (Collier et al., 2008).
           
    Heat shock proteins comprise a large group of molecular chaperones, classified based on molecular size and similarities in amino acid sequences (Kumar et al., 2015). Amongst members of the HSP family, HSP70 is the most abundant and temperature sensitive essential protein (Beckham et al., 2004) well known to be conserved and ubiquitous in nature (Liu and Steinacker, 2001; Kregel, 2002) produced by cells in response to the stress conditions (Lanneau et al., 2007).
           
    Heat shock protein 70 (HSP70) are dynamic, conferring cryoprotection against different stressors. Amongst the stressors, thermal stress triggers a complex mechanism of gene expression as well as adaptive biochemical responses (Lindquist, 1986 and Fujita, 1999). Heat shock proteins can prevent the cells from oxidative stress through its antioxidant and thermo tolerance effect (AL-Jaryan et al., 2023). It is also functioning as molecular chaperon which is attributed to fold, unfold and refold stress-denatured proteins. 
           
    Numerous research activities have been conducted based on HSP70 gene expression patterns during different seasons in cross-bred cattle throughout the globe.  However, very meagre studies were found in the Indigenous cattle breeds of India. Therefore, the present study was undertaken to determine the relative mRNA expression pattern of HSP70 gene with special reference to the summer season in response to thermal stress in Red Kandhari cattle.

    MATERIALS AND METHODS

    The present search was conducted in the department of Veterinary Biochemistry, College of Veterinary and Animal Sciences (MAFSU), Parbhani, total twenty-four (24) apparently healthy animals of either sex with average age ranging from 1-4 yrs. were selected from Livestock Farm Complex, COVAS, Parbhani. They were kept under similar managemental and nutritional regimen throughout the duration (January 2023 to September 2023) of the experimental period as shown in Table 1.

    Table 1: Experimental animals for study.


     
    Temperature humidity index
     
    Temperature and relative humidity was recorded inside the shed with the help of dry bulb/wet bulb thermometer and thermo hygrometer (Mader et al., 2006). Temperature humidity index (THI) of the animal shed was calculated using the formula as follows:
     
    THI = (0.81 × Tdb) + {(RH ÷ 100) × (Tdb - 14.4)} + 46.6
     
    Where,
    Tdb = Average temperature in oC of the dry bulb thermometer
    RH = Average relative humidity.
     
    Collection of samples
     
    The blood samples (2 mL) were collected aseptically by jugular venipuncture with EDTA vacutainer from Red Kandhari cattle during peak summer month (May, THI: 83.36); peak rainy month (September, THI: 78.24); and peak winter (January, THI: 67.84); seasons. During the collection of samples, cold chain was maintained by use of ice cubes and were immediately processed further for the isolation of total RNA. 
     
    Reagents, media and kits
     
    The glassware and plastic ware procured from various firms such as Borosil, Himedia etc. The reagents or kits obtained namely e.g. Agarose, Ethidium bromide, DEPC, Hi Temp PCR master mix, SYBR green master mix, DNase-I, Amplification grade, Invitrogen life Technologies, California, USA.
     
    Isolation of total RNA from whole blood
     
    The pure linkTM total RNA blood purification kit (Invitrogen Life Technologies, California, USA) was used for Isolation of total RNA from whole blood by following the recommended manufacturer’s protocol. Due to its Instability, immediately RNA was processed further for the synthesis of cDNA.
     
    Synthesis of cDNA
     
    The protocol was optimized for cDNA. The quality and quantity of cDNA was recorded (Nanodrop Spectrophotometer, Himedia). ThecDNA samples showing OD: 260/280 more than 1.6  were processed further for RT- qPCR analysis.
     
    Primers
     
    The cattle specific published primer sequences complementary to HSP70 and GAPDH genes were purchased from Barcode Bioscience, Bangalore (Karnataka).  The primer sequences, annealing temperature and predicted fragment size shown (Table 2).

    Table 2: Primer sequence of genes.


     
    Optimization of PCR conditions and primers specificity
     
    The primers were diluted with double distilled water to make 100 µM stock solutions and were stored at -20oC in Deep freezer until use. PCR conditions were optimized by employing various annealing temperature corresponding to the Tm of the primes in Thermo cycler.
     
    The confirmation of cDNA
     
    The PCR product revealed distinct bands for HSP70 and GAPDH genes on GelDoc (Bio-rad) by Agarose gel electrophoresis with 1.5% Agarose gel as shown (Fig 1).

    Fig 1: Confirmation of cDNA and specificity of primers on 1.5 Agarose gel electrophoresis.


     
    Amplification plot for HSP70 and GAPDH gene expression
     
    The amplification plot for expression of HSP70 and GAPDH genes was obtained. This was used for examining the unspecific binding, primer dimer or secondary structure formation as shown (Fig 2).

    Fig 2: Real time pCR (qPCR) amplification plot of HSP70 and GAPDH gene expression.


     
    Confirmation of qPCR product
     
    The real time PCR product was confirmed on 1.5% Agarose gel through Gel Doc(Bio-Rad) system as shown (Fig 3).

    Fig 3: Confirmation of real time PCR products on 1.5 Agarose gel elctrophoresis.


     
    Validation of qPCR product by melt curve analysis
     
    The dissociation characteristics of double stranded DNA were assessed during thermal cycles of qPCR as shown (Fig 4).

    Fig 4: Melt curve analysis of real time PCR (qPCR) reaction products.


     
    Statistical analysis
     
    Statistical analysis of data was done by using SPSS statistical software for windows (version 24). One way ANOVA was applied to analyse the variance about mean Ct value for relative mRNA expression of HSP70 gene between seasons.

    RESULTS AND DISCUSSION

    Temperature humidity index
     
    The mean values of THI recorded during the peak months of different (summer, winter and rainy) seasons are presented (Table 3).

    Table 3: Temperature humidity index (Mean ± SE) of three different seasons.


           
    In the present study, the highest THI (83.36) was recorded in the month of May and the lowest (67.84) in January. However, the intermediate value (78.24) was recorded in the September.
           
    The values of THI 74 or less indicating comfort ability, 75-78 showing an alert status, greater than 79 to 83 could be vulnerable, THI equal to or above 84 is an emergency situation (McDowell, 1976 and Kohli, 2014).
           
    We observed that temperature and humidity were the factors responsible for increase in m-RNA expression during peak summer (May: THI 83.36) as compared to peak rainy (September: THI 78.24) and peak winter (January: THI 67.84) seasons. Temperature humidity index (THI) is used to estimate the degree of stress on the animals (Armstrong, 1994 and Vaidya et al., 2010).
           
    It is worldwide accepted that THI equal to 72 is the threshold for environmental heat stress. However, Thatcher et al. (2010) reported that, if THI above 72, dairy cows could be exposed to heat stress and when it exceeds 88, then animals might be living under severe heat stress conditions. Our findings indicate that the animal may be living under moderate heat stress conditions, particularly during the summer season, where THI is 83.36. Moreover, during the rainy season, animals might be living under heat stress condition, where, THI is 78.24, during these conditions, animal was unable to maintain thermoregulatory responses.
     
    HSP70 gene expression
     
    The HSP70 gene expression was significantly differed amongst all the three seasons. It was found significantly (P<0.01) higher in the experimental animals during peak summer season followed by rainy season, whereas the HSP70 gene expression was significantly (P<0.01) lower during winter season (Table 4 and Fig 5).

    Table 4: The relative quantitative m-RNA expression of HSP70 and GAPDH gene in terms of Ct value (Mean ± SE) amongst different seasons in Red Kandhari cattle.



    Fig 5: The relative m-RNA expression pattern of HSP70 gene in Red Kandhari cattle.


           
    The HSP70 gene expression corresponds to the low Ct values indicating the higher gene expression during the summer as compared to the other two seasons i.e. rainy  and winter.
           
    The results of present study revealed that, the significantly (P<0.01) elevated levels of mRNA expression of HSP70 gene during the peak summer season as compared to rainy and winter seasons against hot dry and hot humid conditions in Red Kandhari cattle. These findings are in accordance with the outcomes of Parmar et al. (2015), who found that the relative mRNA expression of HSP70 gene was increased in peripheral blood mononuclear cells (PBMCs) of Sahiwal cows during the summer season as compared to winter. Further, the findings of the present study corroborated by De Maio (1999), who revealed that the cells can increase the expression of several classes of proteins when exposed to environmental stresses. Yadav et al. (2015) and Kumar et al. (2017) have also observed significantly(P<0.01) increase in mRNA and protein expression of HSP70 and HSP90 during summer (high THI) as compared to thermo-neutral season in all the goat breeds.
           
    Red Kandhari cattle are acclimatized to hot and dry breeding tract of Maharashtra region. But during peak summer, ambient temperature was higher reaches to peak 44oC which may exert heat stress on animals. The upregulation of gene expression was recorded which is influenced by rise in THI during the summer and rainy seasons, these observation were collinear with the Bharati et al. (2017), who investigated the mRNA expression of HSP70 gene in cultured PBMCs of Tharparkar cattle on exposure to thermal stress. They reported that the HSP70 expression was increased in temperature and time dependent manner in cultured PBMCs as compared to control. These responses potentiate the protective effect of antioxidant enzymes through its chaperon functions (Uttarani, 2017). The upregulation of HSP70 mRNA reduces the oxidative stress at cellular level (Kalmar and Greensmith, 2009), improving antioxidant capacity and inhibits lipid peroxidation in order to protect the animals from harmful effect of heat stress.
           
    Droge (2002) who reported that the body releases the HSPs proteins along with the antioxidants on exposure to heat stress to combat the cellular effects of ROS. However, Han et al. (2016), who observe that the transcription of the genes (HSP27, HSP70 and HSP90) of heat shock protein (HSP) was significantly enhanced under heat stress (HS) wherein, the peak transcription of HSP70 was 14 times higher than the control at 1 h.
           
    Banerjee et al. (2014), who explored the effect of temperature sensitivity and seasonal variation on the expression patterns of HSP70 genes in Indian goats. They observed a significant (P<0.01) variation between different seasons for all HSP70 gene expressions, the expression pattern was higher during summer season. They concluded that, the animals exposed to ambient temperature beyond the comfort zones were living under thermal stress during summer than that of winter. Also, the similar results were observed by Lacetera, 2006; Patir and Upadhaya, 2007, 2010, who found that the heat induced stress causes upregulation of HSP70 gene expression.
           
    The comparable results were reported by Ambade, 2023, who found that the expression of HSP70 gene was significantly (P<0.05) higher during summer as compared to winter season in Pandharpuri buffaloes. They recorded the upregulation of HSP70 gene during both summer and rainy seasons whereas, during winter down regulation of gene was observed. The higher expression of HSP70 genes during the thermal stress suggested that there was involvement of HSP70 to rearrange the harmful effect of heat stress for maintaining the cellular integrity and homeostasis (Dangi, 2012).
           
    Our study was corroborated by Kumar et al. (2019) who were undertaken the profiling of HSP70 gene in Murrah buffalo (Bubalus bubalis) under sub-tropical climate of India. They reported that there was relatively higher mRNA expression in summer, when THI was more than 84 as compare to the spring and winter seasons. Moreover, they also reported that there was fold change increase by 4.5 times in summer than the spring seasons.
           
    The present study was supported by Pangaonkar et al. (2023) who estimated seasonal effect of THI on HSP70 gene expression in Deccani sheep, they predicted that HSP70 could be used as a potential bio marker for selecting climate-resilient animals that would show superior thermo-tolerance to enhance livestock productivity.
           
    Suhendro et al. (2024) who investigated the association of heat shock protein 70.1 gene with physiological and physical performance in Bali cattle. They reported the higher expression of HSP70.1 that could mitigate the deleterious effect of heat stress.
           
    Kumar et al. (2024) carried out the expression and SNP profiling of HSP70 gene associated with thermo tolerance traits in Munjal Sheep.  Sheep were categorized into two groups-heat stress susceptible (HSS) and heat stress tolerant (HST)-based on their heat tolerance coefficient. Significant differences in HSP70 gene expression were observed between the HSS and HST groups.
           
    Biradar et al. (2024) who studied the single nucleotide polymorphism in promoter region of HSP70 gene in Deoni cattle. They revealed that HSP70 gene polymorphism in genotype AA could be associated with physiological parameter e.g. respiration rate and rectal temperature.
           
    In line with the present results, Kim et al. (2025), who reported that the HSP70 gene expression in hair follicle was increased with increase in temperature humidity index which implies that increase in HSP expression indicating thermo tolerance in diary calves. They also reported that there was genetic variation in stress tolerance that could be attributed to HSP70 gene polymorphism which is affecting the cellular responses and gene expression in animals exposed to heat stress.

    CONCLUSION

    The present study demonstrated that elevated temperature-humidity index (THI) during the summer season significantly influences HSP70 gene expression, suggesting its vital role in the cellular defence mechanism against thermal stress. In Red Kandhari cattle, the upregulation of HSP70 during summer supports cellular adaptability, stress tolerance and thermal regulation. However, the increased expression also indicates potential cellular vulnerability to heat stress during peak summer. HSP70 may serve as a reliable molecular marker for thermotolerance and could be utilized to identify heat-resilient animals in organized breeding programs. Further studies on genetic polymorphisms, particularly SNPs in the HSP70 gene, may enable the selection of heat-tolerant animals for future breeding strategies.

    ACKNOWLEDGEMENT

    The author wish to thank Associate Dean, College of Veterinary and Animal Sciences, Parbhani for providing the animals and data for this research. The Author also, thankful to The Director of Research, MAFSU, Nagpur for providing valuable guidance.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    Study was approved by Institutional Animal Ethical Committee (IAEC) of the College of Veterinary and Animal Sciences, Parbhani (Maharashtra Animal and Fishery Sciences University, Nagpur) with vide No. IAEC /100/2022.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest in relation to this publication.

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