Thermoregulatory responses in riverine buffaloes against heat stress: An updated review.

Journal of Thermal Biology 96 (2021) 102844 Available online 9 January 2021

0306-4565/© 2021 Elsevier Ltd. All rights reserved.

Thermoregulatory responses in riverine buffaloes against heat stress: An updated review

S.R. Mishra Department of Veterinary Physiology, C.V.Sc & A.H., O.U.A.T, Bhubaneswar, 751003, India

A R T I C L E I N F O Keywords:

Thermoregulatory responses Heat stress Buffaloes A B S T R A C T

High heat and humidity stress have been a perpetual perilous for the buffalo’s production and productivity in tropics and subtropics including India. Productive potential of livestock’s species including buffaloes is maximum with in thermo-neutral zone (TNZ) and if ambient temperature exceeds TNZ and upper critical temperature expose livestock’s to heat stress conditions. For decades, heat stress has been the prime factor to plummet buffalo’s growth, development, reproduction and production in tropics and subtropics including India. In gen­ eral, buffaloes are homeotherms and known as temperature regulators as they resist the variations in ambient temperatures. Generally, buffaloes like other livestock’s display amalgamation of thermoregulatory responses to withstand the changes occurred in their micro and macro environment. These thermoregulatory responses are behavioural, physiological, neuro-endocrine and molecular responses acting synergistically to counteract the deleterious effects of heat stress. Amidst all responses, molecular responses play major role to confer thermo- tolerance through expression of highly conserved family of proteins known as heat shock proteins (HSPs).

Despite of these thermoregulatory responses, heat stress prodigiously muddles buffalo’s production and pro­ ductivity. The present review highlights the thermoregulatory responses manifested by riverine buffaloes against heat stress.

1. Introduction Intergovernmental panel on climate change (IPCC) anticipates a rise in Earth’s surface temperature by 0.2 ◦C per decade and thus, it might lead to an overall spike of around 1.8 ◦C to 4.0 ◦C by 2100 (IPCC, 2007).

Basically, thermo-neutral zone (TNZ) is the range of ambient tempera­ tures where homeotherms survive without any expenditure of energy to maintain body homeostasis. Any deviation in the ambient temperature which goes beyond the upper critical temperature could terminate in heat stress (Bharati et al., 2017; Sahu et al., 2019). Amongst all the climatic variables, ambient temperature plays a paramount role affecting livestock’s reproduction and production (Ayo et al., 2008; Das et al., 2011; Mishra et al., 2013; Singh et al., 2014). In addition, increase in ambient temperature by more than 4 ◦C than average atmospheric temperature during scorching summer, severely upsets buffalo’s pro­ duction and productivity in tropical and subtropical regions including

India (Upadhyay et al., 2010). Furthermore, ambient temperature in the tropics and sub-tropics reach around 44 ◦C or even more thereby expose buffaloes to the hostile effects of heat stress (Hassan et al., 2019). On the other hand, temperature humidity index (THI) has been predominantly used to quantify the intensity of thermal stress across domestic species (Bharati et al., 2017a). Various studies reported that, THI below 72, between 72 and 80, within 80–85 and beyond 85 is recognised as no stress, mild stress, severe stress and lethal stress respectively (Akyuz et al., 2010; Kohli et al., 2014). Payne (1990) had discussed regarding the ultimate climatic conditions for growth, development, reproduction and production in buffaloes. According to Payne (1990), buffaloes are best suited to an ambient temperature ranges between 13 and 18 ◦C in combination with relative humidity around 55–65% along with wind velocity of 5–8 km/h. Payne (1990) suggested that, THI above 72 are considered as stressful while TH that exceeds 78 is regarded as severe stress in buffaloes. Marai and Haeeb (2010) also illustrated that high environmental temperature coupled with high humidity would be very lethal to the buffaloes growth, reproduction and production. However,

THI does not include solar radiations and wind velocity. Later on, Black globe temperature humidity index (BGTHI), equivalent temperature index (ETI) and heat load index (HLI) have been identified which in­ cludes solar radiations and wind velocity (Lenis Sanin et al., 2015; Silva and Passini, 2017).

Buffaloes are multipurpose domestic ruminants, reared for milk,

E-mail address: smruti.mishra1983@gmail.com.

Contents lists available at ScienceDirect Journal of Thermal Biology journal homepage: http://www.elsevier.com/locate/jtherbio https://doi.org/10.1016/j.jtherbio.2021.102844

Received 26 December 2020; Received in revised form 4 January 2021; Accepted 4 January 2021

Journal of Thermal Biology 96 (2021) 102844 2 meat, and draught power (Mishra et al., 2016; Rajesh et al., 2017).

Buffaloes are distributed throughout the Asian and Mediterranean countries including India (Mishra et al., 2015, 2016a; Reshma et al.,

2016). Buffalo population in India is around 96.9 million out of 170.4 million of the world’s total buffalo population (Mishra et al., 2016c;

Rajesh et al., 2018). Thus, India contributes around 57.8% of the world’s total buffalo population (Mishra and Sarkar, 2018) thereby considered as prime ecological niche for buffaloes (Mishra et al., 2016b; Rajesh et al., 2017). In addition, India is quite fortunate to be at zenith vis a vis buffalo milk production in the world as Indian buffaloes contribute more than half of total milk production in the world (Mishra et al., 2017). It has also been shown that, highly producing dairy animals are more sensitive to heat stress than meat producing animals as the former de­ velops more metabolic heat during heat stress conditions (Bernabucci et al., 2010). Earlier studies indicated that, buffaloes have meagre thermo-tolerance capacity as well as lower immunity and thus their production and productivity is immensely affected by under heat stress (Koga et al., 2004; Marai and Haeeb, 2010). Amongst domestic animals, buffaloes are highly vulnerable to the menace of heat stress due to poorly developed sweat glands, more thicker and dark coloured skin along with sparse hairs on their body surface which preclude evapora­ tive heat loss thereby incur major loss in production under extreme environmental conditions (Das et al., 1999; Koga et al., 1999; Vo and

Wang, 2007). Moreover, buffaloes possess one-eighth of the sweat glands compared to cattle thereby become more susceptible to get affected by the adverse effects of heat stress (Kishore et al., 2016).

Additionally, buffaloes absorb profound quantity of solar radiations due to their dark skin and sparse coat or hair (Kapila et al., 2016). Buffaloes by default respond to heat stress by expressing various thermoregulatory responses such as behavioural, physiological, neuro-endocrine and molecular responses (Fig. 1). Albeit buffaloes are quite productive under summer heat load in most of the tropical and subtropical countries including India but they exhibit summer anoestrus which deeply upsets their fertility rate. In this present climate change scenario, it is quite indispensable to understand the basic mechanisms by which buffaloes are acclimatized to the adverse environmental conditions during sum­ mer heat stress. Therefore, the present review highlights the details of thermoregulatory responses exhibited by riverine buffaloes under heat stress.

1.1. Behavioural responses shown by buffaloes against heat stress

Behavioural responses are the immediate responses manifested by buffaloes on exposure to heat stress. Different behavioural responses such as change in dry matter and water intake, change in duration of lying down and standing including wallowing are exhibited by buffaloes during heat stress (Table 1), which are described in this section.

1.2. Dry matter intake In general, dry matter intake gets reduced during summer season in all livestock species (Habeeb et al., 2018). Dry matter intake as well as dry matter digestibility was found to be significantly declined in summer heat stress in lactating Murrah buffaloes (Verma et al., 2000). Ashour et al. (2007) reported up to 40% decline in dry matter intake in buffaloes on exposure to heat stress during peak summer than winter season.

Likewise, dry matter intake was reduced by 8–10% in heat stressed buffalo heifers on heat exposure at 40 ◦C (Hooda and Singh, 2010).

Identically, dry matter intake was noted to be greater in buffaloes kept under modified roof than buffaloes kept under normal roof systems during heat stress (Khongdee et al., 2013). Uniformly, dry matter intake was reduced to less than 9.5 kg/day in Egyptian buffaloes exposed to heat stress (Hady et al., 2018). Consequently, reduction in dry matter intake tends to decrease the body weight gain in heat stressed buffaloes.

Daily body weight gain was significantly reduced by 16.5 and 22.6% in buffaloes during heat exposure at 32 and 36 ◦C respectively compared to control at 18 ◦C (Habeeb et al., 2007). Uniformly, daily body weight gain was decreased by 18.1, 17.41 and 8.65% in buffalo calves during 1st,

Fig. 1. Impact of heat stress on behavioural, physiological, neuro-endocrine and molecular responses in buffaloes.

S.R. Mishra Journal of Thermal Biology 96 (2021) 102844

3 2nd and 3rd months of summer season respectively (Habeeb et al.,

2012). Meanwhile, Das et al. (2011) experimented on effect of washing frequency on physiological responses shown by Nili-Ravi buffalo calves exposed to hot environment. Das et al. (2011) suggested that, four times washing could increase average dry matter utilization and daily body weight gain young Nili-Ravi buffalo calves during summer heat stress under tropical climate compared to either three or two times washing.

Dry matter intake was significantly higher in buffaloes allowed to wallow in pond than buffaloes kept under water showers during hot dry and hot humid seasons, suggesting the advantage of wallowing over water showers (Aggarwal and Singh, 2010). Reduction in dry matter intake could be due to depression of lateral hypothalamus by higher ambient temperature. Wankar et al. (2014) reported lower rumination rate in adult buffaloes exposed to heat strain at 35 ◦C and 40 ◦C. Dry matter intake was significant increased in buffaloes housed with foggers or fans or foggers plus fans than control group buffaloes without any cooling system during summer months (Seerapu et al., 2015). Dry matter intake was highest in buffaloes kept under shade with fan and sprinkler (14.73 kg/d) followed by buffaloes kept under shade with fan (14.56 kg/d) and only shade (13.24 kg/d) on exposure to hot humid conditions (Ahmad et al., 2017). Additionally, total time spent in feed intake was maximum in buffaloes offered combined treatment of fan and sprinkler (309.50 min/24 h) followed by buffaloes under fans (246.33 min/24 h) and only roof shade (280.33 min/24 h) hot humid conditions (Ahmad et al., 2017). Furthermore, time spent in rumination was maximum in buffaloes offered combined treatment of fan and sprinkler (399.00 min/24 h) followed by buffaloes under fans (385.17 min/24 h) and only roof shade (360.83 min/24 h) hot humid conditions (Ahmad et al., 2017).

1.3. Water intake Nessim (2004) noticed an increase in water intake in 12 months old buffalo calves exposed to chronic heat stress. Buffaloes kept under modified roof (normal roof fitted with woven polypropylene shade cloth) consumed less water (29.71 ± 0.86 L/day) than buffaloes kept under normal roof (34.14 ± 1.06 L/day) under similar climatic condi­ tions in hot humid conditions (Khongdee et al., 2013). Water intake was found to be highest on heat exposure at 35 ◦C and did not differ there­ after during heat exposure at 40 ◦C (Wankar et al., 2014) . Increase water intake in buffaloes could be attributed to dehydration due to massive sweating to maintain thermoregulation which might induce thirst centre in hypothalamus during excessive heat load (Wankar et al.,

2014). In another study, daily and total water intake was significantly increased by 56.7 and 16.2% lactating Murrah buffaloes exposed to summer heat stress compared to winter (Sharma et al., 2016). Water intake was significantly lowest in buffaloes kept under shade with fan and sprinkler (112.74 lit/d) followed by buffaloes kept under shade with fan (122.61 lit/d) and only shade (139.38 lit/d) on exposure to hot humid conditions (Ahmad et al., 2017). Further, total time spent in feed intake was maximum in buffaloes offered combined treatment of fan and sprinkler (19.50 min/24 h) followed by buffaloes under fans (22.50 min/24 h) and only roof shade (24.67 min/24 h) hot humid conditions (Ahmad et al., 2017). Lower urination and defecation were detected in buffalo heifers provided with either fan or fan with sprinklers compared to control during summer season (Kumar, 2005).

1.4. Laying down and standing behaviour It was reported that, use of sprinklers and fans tend to reduce the effects of heat stress in buffalo heifers thereby increase their laying down duration during summer season (Vijayakumar et al., 2011). Lying duration was maximum in buffaloes offered combined treatment of fan and sprinkler (236.83 min/24 h) followed by buffaloes under fans (197.67 min/24 h) and only roof shade (193.00 min/24 h) hot humid conditions (Ahmad et al., 2017). However, standing time was lowest in buffaloes housed in roof shade with fan plus sprinkler (281.33 min/24 h) followed by buffaloes under fans (294 min/24 h) and only roof shade (306.83 min/24 h) hot humid conditions (Ahmad et al., 2017).

1.5. Wallowing Wallowing is a process of evaporative heat loss and serves as the major heat loss mechanism in buffaloes under high heat and humidity stress. Generally buffaloes have dark skin and sparse sweat glands and therefore prefer to wallow than sweat to counteract the negative effects of summer stress. According to Somparn et al. (2006), buffaloes prefer to wallow during daytime when intensity of solar radiation is high. In addition, wallowing attenuates the negative effects of summer stress in buffaloes thereby allow them to spend more time in grazing during daytime in summer months (Somparn et al., 2006). It has been shown that, wallowing significantly reduces rectal temperature and respiration rate than either showering or shading during high ambient temperature (Aggarwal and Singh, 2010).

1.6. Physiological responses shown by buffaloes against heat stress

Rectal temperature, respiration rate, heart rate and skin temperature are considered as the major physiological parameters which alter in livestock’s species during thermal stress (Table 2). Significant positive correlation was observed between ambient temperature, respiration rate and pulse rate in heat exposed lactating Murrah buffaloes (Radadia et al., 1980). Moreover, buffaloes adapt to acute heat stress via different physiological responses like rectal temperature, respiration rate and pulse rate (Sethi et al., 1994). In their review, Marai and Haeeb (2010) described on sudden elevation in rectal temperature, respiration rate and pulse rate in buffalo’s upon heat stress.

Table 1 Behavioural responses in buffaloes during heat stress.

Behavioural responses Heat stress References Buffalo breed

Dry matter intake Decrease Verma et al. (2000) Murrah

Hooda and Singh (2010) Aggarwal and Singh (2010) Wankar et al. (2014)

Seerapu et al. (2015) Ashour et al. (2007) Egyptian buffaloes

Habeeb et al. (2007) Habeeb et al. (2012) Hady et al. (2018)

Das et al. (2011) Nili-Ravi Ahmad et al. (2017) Khongdee et al. (2013)

Thai swamp buffalo Water intake Increase Wankar et al. (2014)

Murrah Sharma et al. (2016) Murrah Nessim (2004) Egyptian buffaloes

Khongdee et al. (2013) Thai swamp buffalo Ahmad et al. (2017)

Nili-Ravi Urination and defecation Decrease Kumar (2005)

Murrah Laying down duration Decrease Vijayakumar et al. (2011)

Murrah Ahmad et al. (2017) Nili-Ravi Standing time

Increase Ahmad et al. (2017) Nili-Ravi Wallowing duration

Increase Somparn et al. (2006) Thai swamp buffalo Aggarwal and Singh (2010)

Murrah S.R. Mishra Journal of Thermal Biology 96 (2021) 102844

4 1.7. Rectal temperature Rectal temperature has been used as a sensitive marker to evaluate the intensity of thermal stress. Chikamune and Shimizu (1983) noted high correlation between buffalo’s core body temperature and ambient temperature. Initially, Mullick (1960) indicated that, buffaloes experi­ ence slight lower rectal temperature than cattle on exposure to high and low humidified conditions. Mullick (1964) detected higher rectal tem­ perature in buffaloes exposed to summer heat stress. Kamal et al. (1978) reported an expansion in rectal temperature from 37.8 to 38.0 ◦C in buffalo heifers on exposure to summer season. Heavy workloads for 3 h had elevated rectal temperature in buffaloes in hot dry conditions (Upadhyay and Rao, 1985). Identically, Verma and Husain (1986) noticed greater rectal temperature in buffaloes exposed to extreme ambient temperature. Likewise, Joshi and Tripathy (1991) recorded an up-surge in rectal temperature from 102.0 ◦F to 103.8 ◦F in buffalo calves exposed to prolonged heat stress at 40.5 ◦C for 3 months. Uni­ formly, Sethi et al. (1994) found noticeable increase in rectal tempera­ ture by 2.6 ◦C in buffalo calves on exposure direct solar radiations in hot summer months during June and July. Consistent with previous studies,

Verma et al. (2000) noted an up-regulation in rectal temperature in lactating Murrah buffaloes during summer stress. Akin to earlier find­ ings in buffaloes, Koga et al. (2004) found greater rectal temperature on exposure to high ambient temperature compared to tropical and temperate cattle. During summer season, Kumar (2005) observed lower

Table 2 Physiological responses in buffaloes during heat stress.

Physiological responses Heat stress References Buffalo Breed

Rectal temperature Increase Upadhyay and Rao (1985)

Murrah Verma and Husain (1986) Verma et al. (2000)

Kumar (2005) Korde et al. (2007) Aggarwal and Singh (2008)

Aggarwal and Singh (2010) Hooda and Singh (2010) Rahangdale et al. (2011)

Ambulkar et al. (2011) Haque et al. (2012) Kumar and Kumar (2013)

Singh et al. (2014) Wankar et al. (2014) Seerapu et al. (2015)

Yadav et al. (2016) Kumar et al. (2018) Lakhani et al. (2018)

Singh et al. (2005) Nili-Ravi Das et al. (2011) Das et al. (2014)

Ahmad et al. (2017) Gudev et al. (2007) Bulgarian Murrah

Hafez et al. (2011); Hady et al. (2018) Egyptian buffalo

Khongdee et al. (2013) Thai swamp buffalo Manjari et al. (2015)

Tarai buffalo Shenhe et al. (2018) Nili-Ravi × Murrah

Brcko et al. (2020) Murrah × Mediterranean Decrease

Liu et al. (2019) Nili-Ravi × Murrah No change Salem (1980)

Egyptian buffalo Chaudhary et al. (2015) Surti Li et al. (2020)

Nili-Ravi Respiration rate Increase Mishra et al. (1963)

Murrah Upadhyay and Rao (1985) Joshi and Tripathy (1991)

Das et al. (1997) Das et al. (1999) Verma et al. (2000)

Kumar (2005) Aggarwal and Singh (2008) Dandage and Thesis submitted to (2009)

Aggarwal and Singh (2010) Hooda and Singh (2010) Singh et al. (2011)

Rahangdale et al. (2011) Ambulkar et al. (2011) Haque et al. (2012)

Singh et al. (2014) Wankar et al. (2014) Seerapu et al. (2015)

Yadav et al. (2016) Kumar et al. (2018) Lakhani et al. (2018)

Salem (1980) Egyptian buffalo Hafez et al. (2011) Singh et al. (2005)

Nili-Ravi Das et al. (2011) Das et al. (2014) Ahmad et al. (2017)

Li et al. (2020) Gudev et al. (2007) Bulgarian Murrah

Table 2 (continued) Physiological responses Heat stress

References Buffalo Breed Manjari et al. (2015) Tarai buffalo

Chaudhary et al. (2015) Surti Shenhe et al. (2018)

Nili-Ravi × Murrah Brcko et al. (2020) Murrah × Mediterranean

Decrease Liu et al. (2019) Nili-Ravi × Murrah Heart rate

Increase Joshi et al. (1982) Murrah Upadhyay and Rao (1985)

Kumar (2005) Aggarwal and Singh (2008) Hooda and Singh (2010)

Singh et al. (2011) Haque et al. (2012) Seerapu et al. (2015)

Yadav et al. (2016) Kumar et al. (2018) Lakhani et al. (2018)

Salem (1980); Hafez et al. (2011) Egyptian buffalo

Singh et al. (2005) Nili-Ravi Das et al. (2011) Das et al. (2014)

Ahmad et al. (2017) Li et al. (2020) Manjari et al. (2015)

Tarai buffalo Decrease Singh et al. (2014); Wankar et al. (2014)

Murrah No change Chaudhary et al. (2015) Surti Skin temperature

Increase Das et al. (1997) Murrah Aggarwal and Singh (2008)

Ambulkar et al. (2011) Haque et al. (2012) Kumar and Kumar (2013)

Kumar et al. (2018) Singh et al. (2005) Nili-Ravi Ahmad et al. (2017)

Li et al. (2020) Hafez et al. (2011) Egyptian buffalo

Shenhe et al. (2018) Nili-Ravi × Murrah S.R. Mishra

Journal of Thermal Biology 96 (2021) 102844 5 rectal temperature in buffalo heifers provided with either fan or fan with sprinklers compared to control. Rectal temperature was found to be lower in buffaloes offered water splashing and wallowing than control buffaloes during hot summer months (Singh et al., 2005). Similarly,

Korde et al. (2007) observed an increase in rectal temperature in buffalo calves under hot environment than under cool environment. In another study, greater rectal temperature was observed in lactating buffaloes at

15.00 Hrs under direct sunlight at THI 77.83 compared to those housed under the barn (Gudev et al., 2007). Gudev et al. (2007a) documented that, buffaloes kept under the barn had a steady rectal temperature within the TNZ amidst higher respiration rate. Rectal temperature was significantly lower in wallowing group buffaloes than showering group during evening hours of hot summer months (Aggarwal and Singh,

2008). In another study, wallowing group buffaloes (100.5 + 0.1 and

100.7 + 0.1 ◦F) had significantly lower rectal temperature than show­ ering buffaloes (101.2 + 0.1 and 102.3 + 0.1 ◦F) during evening hours of hot dry and hot humid months respectively (Aggarwal and Singh, 2010).

El-Kaschab et al. (2009) reported higher rectal temperature in buffaloes housed in tie stall barn (38.33 ◦C) than those in loose housing barn system (38.13 ◦C). Hooda and Singh (2010) found an increment in rectal temperature in buffalo heifers during exposure to summer stress at 40

◦C. Rectal temperature was found to be increased in Egyptian buffaloes under elevated THI (Hafez et al., 2011). Marked reduction in rectal temperature was observed in Nili-Ravi buffalo calves after washing four times than either three or two times during summer heat stress under tropical climate (Das et al., 2011). In a study conducted in Murrah buffaloes, deep body temperature was highest (100.54 ◦F) at 2 p.m. and lowest (98.93 ◦F) at 6 a.m. during hot summer (Rahangdale et al., 2011).

Ambulkar et al. (2011) reported that, Murrah buffaloes subjected with high pressure fogger System (HPFF) had lower (37.52 ◦C) body tem­ perature than control group (37.83 ◦C) during summer heat stress.

Rectal temperature was up-regulated in young and adult buffaloes exposed to heat stress at 40, 42 and 45 ◦C for 4 h than control buffaloes within TNZ at 22 ◦C (Haque et al., 2012). In another study, Khongdee et al. (2013) detected lower mean rectal temperature (39.14 ± 0.07 ◦C) in young male buffaloes kept under modified roof (normal roof fitted with woven polypropylene shade cloth) than buffaloes kept under standard roof (40.00 ± 0.10 ◦C) during hot humid stress suggesting the fact that modified roof diminishes the negative impacts of heat stress on buffaloes. Lactating Murrah buffaloes had four times more heat storage during exposure to hot humid and hot dry seasons than spring season (Kumar and Kumar, 2013). Consequently, rectal temperature was found to be highest in lactating Murrah buffaloes under hot humid season followed by hot dry and spring season (Kumar and Kumar, 2013).

Likewise, Singh et al. (2014) detected greater rectal temperature in

Murrah buffaloes during exposure to summer (102.52 ± 0.25 ◦C) than winter season (100.68 ± 0.19 ◦C). In another study, rectal temperature was found to be declined in lactating Nili-Ravi buffaloes in both hot dry and hot humid seasons supplemented with nutrients like niacin, yeast, edible oil and modified micro-environment with curtains, ceiling fans and mist fans in the shed than those buffaloes in control group deprived of everything (Das et al., 2014). Rectal temperature was escalated in buffaloes subjected to heat stress at 35 ◦C and 40 ◦C than at 25 ◦C and 30

◦C (Wankar et al., 2014). Rectal temperature was markedly declined in buffaloes housed with foggers (101.6 ± 0.02 ◦F) or fans (102.1 ± 0.06

◦F) or foggers plus fans (101.5 ± 0.02 ◦F) than control (102.5 ± 0.06 ◦F) group buffaloes without any cooling system (Seerapu et al., 2015).

Identically, Manjari et al. (2015) reported higher rectal temperature during summer season (39.50 ◦C) than winter season (38.42 ◦C) in Tarai buffaloes. Yadav et al. (2016) revealed that, misting and wallowing significantly lowered rectal temperature in lactating Murrah buffalo during hot dry and hot humid seasons and therefore suggested that rectal temperature could be considered as the gold standard to under­ stand the magnitude of heat stress in buffaloes. Ahmad et al. (2017) reported lowest rectal temperature in buffaloes kept under shade with fan and sprinkler (101.05 ± 0.9 ◦F) followed by buffaloes kept under shade with fan (101.69 ± 0.08 ◦F) and only shade (102.06 ± 0.07 ◦F) on exposure to hot humid conditions. Similarly, rectal temperature was lesser in buffaloes kept under modified shed (100.94 ± 0.12 ◦F) than those under normal loose housing system (101.56 ± 0.06 ◦F) during autumn season (Kumar et al., 2018). Recently, Hady et al. (2018) detected up to 1 ◦C elevation in rectal temperature in Egyptian buffaloes during exposure to THI around 79.74 to 90.4 indicating that buffaloes are more susceptible to the deleterious effects of high THI. Similarly, rectal temperature was significantly higher in Murrah buffaloes during hot dry and hot humid seasons compared to TNZ (Lakhani et al., 2018).

Rectal temperature was markedly higher in crossbred buffaloes during summer (39⋅24 ◦C) than spring (38⋅34 ◦C) and winter (38⋅13 ◦C) season (Shenhe et al., 2018). However, mean rectal temperature of crossbred buffaloes (39⋅12 ◦C) was significantly lower than Mediterranean buf­ faloes (39⋅38 ◦C) during summer season (Shenhe et al., 2018). Rectal temperature was noted highest (39.01 ◦C) at 3 p.m. on exposure of fe­ male buffaloes to direct sunlight in a hot and humid climate (Brcko et al.,

2020). Increase in rectal temperature in buffaloes subjected to summer stress could be attributed to the fact that buffaloes are impuissant to dissipate excess body heat generated at the time of thermal load of summer months (Marai and Habeeb, 2010). In contrast, rectal temper­ ature did not vary in Surti buffaloes among hot dry (THI = 81.70), hot humid (THI = 80.60) and control (THI = 68.72) (Chaudhary et al.,

2015). Identically, rectal temperature did not change significantly in

Nili-Ravi buffaloes among summer, spring, autumn and winter season (Li et al., 2020). Similarly, Salem (1980) did not notice any variation in rectal temperature in buffaloes under different environmental condi­ tions i.e. hot humid, warm and cold seasons. Liu et al. (2019) found lower rectal temperature in heat stressed buffaloes (38.72 ◦C) than control ones (39.67 ◦C).

1.8. Respiration rate High ambient temperature increases respiration rate in cattle and buffaloes (Bianca and Findlay, 1962). Ambient temperature and respi­ ration rate are highly correlated and respiration rate was found to be increased in buffaloes with increase in ambient temperature (Mishra et al., 1963). Identically, respiration rate was significantly increased from 20.5 to 22.4 cycles/min during heat stress (Kamal et al., 1978).

Beccari et al. (1978) found greater respiration rate in heat stressed buffaloes compared to those in TNZ. In another study, Salem (1980) observed highest respiration rate during hot humid followed by warm and cold seasons. Similarly, significant increase in respiration rate was noticed in buffaloes due to heavy workloads for 3 h in hot dry conditions (Upadhyay and Rao, 1985). In Murrah buffalo calves, Joshi and Tripathy (1991) observed a gradual increase in respiration rate from 29 to 59 breaths/min under heat stress conditions. Likewise, respiration rate in buffaloes was increased by 3–4 fold at high ambient temperature (Chaiyabutr, 1993). In another study, respiration rate was elevated by

5–6 fold (Das et al., 1997) and 2.5 fold (Das et al., 1999) in Murrah buffalo calves on exposure to solar radiation. Verma et al. (2000) found an increment in respiration rate up to 32.75 breaths/min and suggested that both rectal temperature and respiration rate could be considered as the most sensitive indicator to evaluate the intensity of thermal stress.

Kumar (2005) found lower respiration rate in buffalo heifers provided with either fan or fan with sprinklers compared to control group buf­ faloes during summer season. Respiration rate was significantly declined in buffaloes offered water splashing and wallowing than control buf­ faloes during hot summer months (Singh et al., 2005). Higher respira­ tion rate was detected in lactating buffaloes on exposure to direct solar radiations (THI = 77.83) at 15.00 h compared to those kept under the barn (Gudev et al., 2007). In another study, buffaloes offered wallowing had lower respiration rate than buffaloes undergone showering during hot dry months (Aggarwal and Singh, 2008). Consistently, higher respiration rate was observed in Murrah buffaloes on heat exposure to 4 h at 40 ◦C with 50% RH in climatic chamber (Dandage and Thesis

S.R. Mishra Journal of Thermal Biology 96 (2021) 102844

6 submitted to, 2009). In another study conducted by El-Kaschab et al. (2009), respiration rate was significantly higher in buffaloes housed in loose housing barn (38.21 breaths/min) than in tie stall barn (32.21 breaths/min). Buffaloes have undergone wallowing (16.3 + 0.2 and

15.2 + 0.2 breaths/min) had shown lower respiration rate than buf­ faloes undergone showering (21.2 + 0.2 and 17.8 + 0.2 breaths/min) during evening hours of hot dry and hot humid months respectively (Aggarwal and Singh, 2010). Uniformly, respiration rate in buffalo heifers was significantly elevated during summer stress at 40 ◦C (Hooda and Singh, 2010). Respiration rate was significantly elevated in Egyp­ tian buffaloes during exposure to THI at 94 (Hafez et al., 2011). Respi­ ration rate was noticeably declined after supplementation of yeast in buffaloes during heat stress (Singh et al., 2011). In another study, sig­ nificant reduction in respiration rate was noticed after washing Nili-Ravi buffalo calves for four times than either three or two times during summer heat stress (Das et al., 2011). Likewise, respiration rate was altered in time dependant manner and found to be highest (33.54 breaths/min) at 2 p.m. and lowest (24.44 breaths/min) at 6 a.m. (Rahangdale et al., 2011). In addition, respiration rate was found to be

28.78, 28.49 and 29.68 breaths/min in wallowing, splashing and control group Murrah buffaloes respectively, suggesting significant effect of wallowing and splashing on respiration rate (Rahangdale et al., 2011).

Furthermore, wallowing and splashing induced docile temperament in

20 and 33.33 percent of buffaloes respectively than control group buf­ faloes without any cooling practice (Rahangdale et al., 2011). In an experiment conducted by Ambulkar et al. (2011), respiration rate was significantly declined in buffaloes subjected with high pressure fogger

System (21.61 breaths/min) than control group (23.01 breaths/min).

Higher respiration rate was observed in buffaloes on exposure to heat stress at 40, 42 and 45 ◦C for 4 h than control buffaloes within TNZ at 22

◦C (Haque et al., 2012). Singh et al. (2014) reported greater respiration rate in buffaloes exposed to summer (18.77 ± 1.25 breaths/min) than winter season (12.29 ± 1.97 breaths/min). Respiration rate was found to be lower in treatment group (supplemented with nutrients and modified shade) buffaloes than control group during exposure to either hot dry or hot humid seasons (Das et al., 2014). Wankar et al. (2014) reported significant elevation in respiration rate during hyperthermia at 35 ◦C and 40 ◦C than at 25 ◦C and 30 ◦C in adult buffaloes. Significant increase in respiration rate was observed during summer season (36.18 breath­ s/min) than winter season (27.88 breaths/min) in Tarai buffaloes (Manjari et al., 2015). In Surti buffaloes, respiration rate was highest during hot dry (THI = 81.70) followed by hot humid (THI = 80.60) and control (THI = 68.72) season (Chaudhary et al., 2015). Significant decline in respiration rate was observed in buffaloes housed with foggers (22.15 ± 0.26 breaths/min) or fans (28.32 ± 0.58 breaths/min) or foggers plus fans (22.50 ± 0.23 breaths/min) than control (37.81 ± 0.37 breaths/min) group buffaloes without any cooling system (Seerapu et al., 2015). Buffaloes offered misting and wallowing have shown lower respiration rate than control group buffaloes during hot dry and hot humid months (Yadav et al., 2016). Ahmad et al. (2017) detected lowest respiration rate in buffaloes kept under shade with fan and sprinkler (26.16 ± 1.12 breaths/min) followed by buffaloes kept under shade with fan (33.8 ± 0.82 breaths/min) and only shade (38.48 ± 0.84 breath­ s/min). In another study, buffaloes kept under modified shed had shown lower respiration rate (30.99 ± 1.21 breaths/min) than those under normal loose housing system (37.39 ± 1.02 breaths/min) during autumn season (Kumar et al., 2018). Crossbred buffaloes had higher respiration rate during summer (69⋅74 breaths/min) than spring (15⋅76 breaths/min) and winter (10⋅49 breaths/min) season (Shenhe et al.,

2018). However, mean respiration rate of crossbred buffaloes (60⋅82 breaths/min) was significantly lower than Mediterranean buffaloes (76⋅84 breaths/min) during summer season (Shenhe et al., 2018).

Likewise, respiration rate was significantly greater in Murrah buffaloes on exposure to hot dry and hot humid seasons compared to TNZ (Lakhani et al., 2018). Uniformly, respiration rate was significantly higher in Nili-Ravi buffaloes during summer season compared to spring, autumn and winter season (Li et al., 2020). Similarly, respiration rate was found to be was noted highest (36.08 breaths/min) at 1.28 p.m. on exposure of female buffaloes to direct sunlight in a hot and humid climate (Brcko et al., 2020). Higher respiration rate could be attributed to increase heat loss via evaporative cooling. Higher respiration rate during summer heat load could enhance the evaporative heat loss thereby triggers cooling to heat stressed buffaloes. Moreover, spike in respiration rate could be due to increase demand of oxygen for various tissues during summer heat stress. Contrarily, Liu et al. (2019) detected lower respiration rate in heat stressed buffaloes (41.67 breaths/min) than non heat stressed group (100.12 breaths/min).

1.9. Heart rate and pulse rate Salem (1980) reported higher pulse rate in buffaloes during warm season followed by cold and hot humid seasons. Joshi et al. (1982) examined higher pulse rate in Murrah buffaloes during hyperthermia.

Heart rate was significantly increased in buffaloes due to heavy work­ loads for 3 h in hot dry conditions (Upadhyay and Rao, 1985). During summer months, buffalo calves kept inside cool shaded area had lower pulse rate than those under direct sunlight (Chauhan et al., 1999).

Similarly, buffalo heifers provided with either fan or fan with sprinklers had lower pulse rate compared to control group buffaloes during sum­ mer season (Kumar, 2005). Buffaloes subjected to water splashing and wallowing had shown lower pulse rate than control non-cooled buf­ faloes during summer months (Singh et al., 2005). In another study, pulse rate in wallowing buffaloes (46.0 ± 0.3 beats/min) was signifi­ cantly lower than showering buffaloes (53.8 ± 0.2 beats/min) during the evening hours of hot dry season (Aggarwal and Singh, 2008).

El-Kaschab et al. (2009) observed higher pulse rate in buffaloes hosed in loose housing barn (64.39 beats/min) than in tie stall barn (61.50 beats/min). Hooda and Singh (2010) reported significant increase in pulse rate in buffalo heifers subjected to summer stress at 40 ◦C.

Respiration rate was significantly elevated in Egyptian buffaloes during exposure to THI at 94 (Hafez et al., 2011). In another study, supple­ mentation of yeast during heat stress resulted in significant reduction of pulse rate in buffaloes (Singh et al., 2011). According to Das et al. (2011), four times washing during summer heat stress could reduce pulse rate in Nili-Ravi buffalo calves than three or two times washing.

Haque et al. (2012) documented higher pulse rate in young and adult buffaloes after 4 h of thermal exposure at 40, 42 and 45 ◦C than those within TNZ at 22 ◦C. Lactating Nili-Ravi buffaloes supplemented with nutrients (niacin, yeast and edible oil) and kept under modified shade (ceiling fans and mist fans) had lower pulse rate than control group during hot dry and hot humid seasons (Das et al., 2014). Pulse rate was significantly elevated during summer season (76.57 beats/min) than winter season (72.47 beats/min) in Tarai buffaloes (Manjari et al.,

2015). In another study, Seerapu et al. (2015) found significant reduc­ tion in pulse rate in buffaloes housed with foggers (51.39 ± 0.32 beats/min) or fans (57.12 ± 0.40 beats/min) or foggers plus fans (52.00

± 0.26 beats/min) than control (67.86 ± 0.41 beats/min) group buf­ faloes without any cooling system. Lower pulse rate was observed in wallowing buffaloes than control buffaloes in hot humid months of July (Yadav et al., 2016). Ahmad et al. (2017) detected lowest pulse rate in buffaloes kept under shade with fan and sprinkler (54.30 ± 1.09 beats/min) followed by buffaloes kept under shade with fan (64.72 ±

3.96 beats/min) and only shade (69.80 ± 1.52 beats/min) during hot humid months. Pulse rate was significantly reduced in buffaloes housed under modified shed (52.52 ± 1.44 beats/min) than those under normal loose housing system (60.91 ± 1.17 beats/min) during autumn season (Kumar et al., 2018). Comparably, pulse rate was significantly greater in

Murrah buffaloes on exposure to hot dry and hot humid seasons compared to TNZ (Lakhani et al., 2018). Taken together, up-surge in pulse rate in buffaloes could increase blood flow towards peripheral circulation to enhance heat loss to the surrounding environment thereby maintain homeostasis during heat stress. On the contrary, pulse rate was

S.R. Mishra Journal of Thermal Biology 96 (2021) 102844

7 reduced in buffaloes during heat stress at 35 ◦C and 40 ◦C than at 25 ◦C and 30 ◦C (Wankar et al., 2014). Singh et al. (2014) noticed higher heart rate during winter season (73.43 ± 4.21 beats/min) in comparison to summer season (67.65 ± 2.8 beats/min) in Murrah buffaloes. Addi­ tionally, pulse rate did not change in Surti buffaloes during exposure to hot dry (THI = 81.70) or hot humid (THI = 80.60) season than control (THI = 68.72) (Chaudhary et al., 2015).

1.10. Skin temperature Generally skin temperature of buffaloes increases with increase in intensity of solar radiation (Das et al., 1997). Significantly higher skin temperature was observed in buffaloes compared to their counter-parts tropical and temperate cattle, with increase in environmental temper­ ature (Koga et al., 2004). Skin temperature was reduced in buffaloes offered water splashing and wallowing than control buffaloes during hot summer months (Singh et al., 2005). Skin temperature was found to be greater in showering group compared to wallowing group buffaloes during evening time of hot dry season (Aggarwal and Singh, 2008).

El-Kaschab et al. (2009) found significantly higher skin temperature in buffaloes housed in tie stall barn (35.57 ◦C) than in loose housing barn (34.91 ◦C). Ambulkar et al. (2011) reported lower respiration rate in buffaloes kept under high pressure fogger System (37.52 ◦C) than con­ trol group (37.83 ◦C). Skin temperature was found to be increased in

Egyptian buffaloes during exposure to THI at 94 (Hafez et al., 2011).

Skin temperature was significantly higher in both young and adult buffaloes following 4 h of heat exposure at 40, 42 and 45 ◦C than those kept in TNZ at 22 ◦C (Haque et al., 2012). Uniformly, skin temperature was noticed highest in lactating Murrah buffaloes on exposure to hot humid season followed by hot dry and spring season (Kumar and Kumar,

2013). Significantly lowest skin temperature was observed in buffaloes kept under shade with fan and sprinkler (32.38 ± 0.15 ◦C) followed by buffaloes kept under shade with fan (33.55 ± 0.04 ◦C) and only shade (34.66 ± 4.77 ◦C) during hot humid months (Ahmad et al., 2017). Body surface temperature was reported to be higher in crossbred buffaloes during summer (38⋅01 ◦C) than spring (35⋅44 ◦C) and winter (24⋅51 ◦C) season (Shenhe et al., 2018). However, mean body surface temperature of crossbred buffaloes (37⋅81 ◦C) was found to be lower than Mediter­ ranean buffaloes (38⋅23 ◦C) during summer season (Shenhe et al., 2018).

In another study, significant reduction in skin temperature was noticed in buffaloes stayed under modified shed (93.01 ± 0.57 ◦F) those under normal loose housing system (95.19 ± 0.61 ◦F) during autumn season (Kumar et al., 2018). Identically, body surface temperature was signif­ icantly higher in Nili-Ravi buffaloes during summer season compared to spring, autumn and winter season (Li et al., 2020). Higher skin tem­ perature could be due to increase in blood flow to peripheral circulation thereby increase heat loss via skin surface during elevated ambient temperature.

1.11. Neuro-endocrine responses shown by buffaloes against heat stress

Onset of neuro-endocrine responses are marked by alternations in secretion of various circulating hormones and considered as one of the mechanism by which livestock achieve thermo-tolerance. Generally, heat stress triggers hypothalamo-pituitary adrenal (HPA) axis and sympathetic adrenal medullary (SAM) axis to produce cortisol and cat­ echolamines into systemic circulation to regulate body metabolism thereby maintain energy homeostasis in livestock’s species. Major neuro-endocrine hormones responsible for thermal adaptation are cortisol, thyroid hormone, prolactin, insulin growth hormone and aldosterone (Table 3).

1.12. Cortisol Cortisol is the chief glucocorticoid hormone and considered as the major neuro-endocrine stress marker in domestic ruminants (Marai and

Haeeb 2010; Wankar et al., 2014; Kamal et al., 2018). Heat stress acti­ vates hypothalamo-pituitary adrenal (HPA) axis to secrete cortisol into systemic circulation. In particular, heat stress provokes para-ventricular nuclei (PVN) of hypothalamus to secrete corticotrophin releasing hor­ mone (CRH) which acts on corticotrophs of adenohypophysis to secrete adreno-corticotropic hormone (ACTH) which finally stimulates zona fasciculata of adrenal cortex to secrete cortisol into systemic circulation.

Roy and Prakash (2007) investigated higher plasma cortisol level in buffalo heifers subjected to summer heat load. Plasma cortisol level was significantly increased and found to be 12.53 ng/ml in heat stressed buffaloes (Marai and Haeeb, 2010). Meanwhile, plasma cortisol level was significantly up-regulated in buffaloes exposed to heat stress at 35

◦C (Wankar et al., 2014). Likewise in Surti buffaloes, Chaudhary et al. (2015) determined higher plasma cortisol level subjected to hot dry season (THI = 81.70) compared to control (THI = 68.72). Plasma cortisol level was significantly lower in buffalo heifers provided with

Table 3 Neuro-endocrine responses in buffaloes during heat stress.

Hormone Heat stress Author Breed Cortisol Increase

Kumar (2005) Murrah Roy and Prakash (2007) Kumar et al. (2010)

Aggarwal and Singh (2010) Vijayakumar et al. (2011)

Wankar et al. (2014) Silva et al. (2014) Yadav et al. (2016)

Kumar et al. (2018) Lakhani et al. (2018) Khongdee et al. (2013)

Thai swamp buffalo Chaudhary et al. (2015) Surti Das et al. (2014); Li et al. (2020)

Nili-Ravi Shenhe et al. (2018) Nili-Ravi × Murrah Liu et al. (2019)

Decrease Dwaraknath et al. (1984) Murrah No change

Hafez et al. (2011) Egyptian buffalo T3 Decrease Habeeb et al. (2000)

Egyptian buffalo Nessim (2004) Korde (2004) Murrah

Aggarwal and Singh (2010) Wankar et al. (2014) Lakhani et al. (2018)

Chaudhary et al. (2015) Surti Li et al. (2020) Nili-Ravi

Increase Hafez et al. (2011) Egyptian buffalo Silva et al. (2014)

Murrah No change E1-Masry and Habeeb (1989) Egyptian buffalo

Das et al. (2014) Nili-Ravi Dixit et al. (1984) Murrah

Yadav et al. (2016) T4 Decrease Khurana (1983) Murrah

Dwaraknath et al. (1984) Korde (2004) Aggarwal and Singh (2010)

Wankar et al. (2014) Lakhani et al. (2018) E1-Masry and Habeeb (1989)

Egyptian buffalo Nessim (2004) Increase Silva et al. (2014)

Murrah Mayahi et al. (2014) Khuzestan buffalo Li et al. (2020)

Nili-Ravi No change Dixit et al. (1984) Murrah Yadav et al. (2016)

Hafez et al. (2011) Egyptian buffalo Das et al. (2014)

Nili-Ravi Prolactin Increase Roy and Prakash (2007)

Murrah Yadav et al. (2016) Insulin Decrease Aggarwal and Singh (2010)

Murrah Li et al. (2020) Nili-Ravi GH Decrease Li et al. (2020)

Nili-Ravi Aldosterone No change Wankar et al. (2014)

Murrah S.R. Mishra Journal of Thermal Biology 96 (2021) 102844

8 either fan or fan with sprinklers compared to control group buffaloes during summer season (Kumar, 2005). Serum cortisol level was noted to be lower in buffaloes supplemented with electrolyte and ascorbic acid than control buffaloes during heat stress, depicting the ameliorative nature of ascorbic acid during heat stress (Kumar et al., 2010). Cooling conditions reduced plasma cortisol concentration in lactating buffaloes under compared to those under direct sunlight (El-Khashab, 2010). In another study, plasma cortisol level was found to be higher in showering group (4.80 + 0.14 ng/ml) compared to wallowing group (2.60 + 0.08 ng/ml) buffaloes during hot dry season, indicating the fact that wal­ lowing alleviate heat stress much more than showering (Aggarwal and

Singh, 2010). Plasma cortisol level was also higher in showering group (4.33 + 0.16 ng/ml) compared to wallowing group (2.64 + 0.32) buf­ faloes during hot humid season (Aggarwal and Singh, 2010). Vijaya­ kumar et al. (2011) observed significant reduction plasma cortisol level in buffalo heifers offered both fan and sprinklers than those offered only fan or control group without fan and sprinkler. On exposure to hot humid conditions, young male buffaloes housed under modified roof (normal roof fitted with woven polypropylene shade cloth) have shown significant reduction in plasma cortisol level (2.14 ± 0.24 ng/ml) compared to those under normal roof (3.38 ± 0.37 ng/ml), suggesting the advantage of modified roof system over traditional normal roof system in reducing the negative impacts of hot humid conditions on buffaloes (Khongdee et al., 2013). Silva et al. (2014) also found highest plasma cortisol levels in female buffaloes on direct exposure to sun rays during rainy and less rainy seasons than those housed under shed in eastern amazon region of Brazil suggesting the advantage of silvopas­ toral system over traditional housing systems. In lactating Murrah buf­ falo, Yadav et al. (2016) reported an elevation in plasma cortisol level during hot humid stress in the month of July. Yadav et al. (2016) also found that misting and wallowing significantly reduce plasma cortisol in lactating Murrah buffalo during hot humid month. In another study, plasma cortisol level was significantly lower in buffaloes kept under modified shed (3.31 ± 0.21 ng/ml) than those under normal loose housing system (4.04 ± 0.23 ng/ml) during autumn season (Kumar et al., 2018). Plasma cortisol level was found to be highest in Murrah buffaloes on exposure to hot humid seasons followed by hot dry season and TNZ (Lakhani et al., 2018). Plasma cortisol level was significantly higher in crossbred buffaloes than purebred Mediterranean buffaloes (Shenhe et al., 2018). Comparably, Liu et al. (2019) observed higher plasma cortisol level in heat stressed buffaloes (251.64 pg/ml) than non heat stressed group (121.46 pg/ml). Uniformly, Li et al. (2020) found higher plasma cortisol level in Nili-Ravi buffaloes during summer season (THI = 82) compared to spring, autumn and winter season. However, plasma ACTH level was found to be lower in summer compared to other seasons (Li et al., 2020). Higher cortisol might inhibit the corticotrophs of adenohypophysis via negative feedback mechanism to reduce ACTH level during summer season. In contrast, lower plasma cortisol con­ centrations were reported in buffalo calves exposed to extreme hot environment (Dwaraknath et al., 1984). In addition, Hafez et al. (2011) did not notice any significant variation in plasma cortisol in Egyptian buffaloes on exposure to high THI at 94. All together in buffaloes, plasma cortisol level was increased and decreased during acute and chronic heat stress respectively (Marai and Haeeb, 2010). The quick rise in plasma cortisol could be explained by the fact that cortisol triggers gluconeogenesis to adapt the buffaloes in acute heat stress while chronic down fall in cortisol level could be attributed to reduction in animal body thermogenesis to restore metabolic heat production during hy­ perthermia (Marai and Haeeb 2010). However, plasma cortisol levels did not vary in buffaloes supplemented with nutrient (niacin, yeast, and edible oil) or housed in modified micro-environment (curtains, ceiling fans and mist fans) than control group during hot dry and hot humid seasons (Das et al., 2014). The authors believed that, temperature around the treatment group buffaloes might not be adequate to secrete the concerned hormone from adrenal cortex.

1.13. Thyroid hormone Heat stress stimulates hypothalamo-pituitary thyroid (HPT) axis to produce thyroid hormones which plays important role in body meta­ bolism thereby regulates energy homeostasis in livestock’s species.

Hypothalamic paraventricular nuclei (PVN) synthesizes thyrotropin releasing hormone (TRH) which activates thyrotrophs of adenohy­ pophysis to produce thyroid stimulating hormone (TSH) which finally stimulates thyroid follicle to produce tri-iodo thyronine (T3) and thryroxine (T4). It has been shown that, heat stress influences thyroid gland activity and functions in domestic species (Rasooli et al., 2004).

Moreover, thyroid hormones play pivotal role in adaptation of livestock species against heat stress. Khurana (1983) detected lower plasma T4 level in buffaloes on exposure to hot-dry season (39.10 ng/ml) than hot-humid season (41.44 ng/ml). Dwaraknath et al. (1984) detected lower plasma T4 level in buffalo bulls on exposure to high air temper­ ature. In another study, T3 uptake (%) did not change between summer and winter seasons whereas plasma T4 level was significantly reduced in summer compared to winter season in Egyptian buffaloes (E1-Masry and

Habeeb, 1989). Habeeb et al. (2000) reported significant reduction in plasma T3 level by 17.2% in lactating buffaloes when ambient temper­ ature increased from 17.5 to 37.1 ◦C. In another study, Nessim (2004) found a reduction in T3 and T4 by 35.25 and 17.59% in buffaloes during summer heat stress. Similarly, Korde (2004) in his treatise observed reduction in plasma T3 and T4 levels in buffalo calves subjected to heat strain. In another study, higher plasma T4 concentration was noted in wallowing group (52.57 + 0.67 ng/ml) than showering group buffaloes (50.65 + 0.50 ng/ml) while plasma T3 concentration did not differ be­ tween both the groups during hot dry season (THI = 80.3) (Aggarwal and Singh, 2010). However, during hot humid season (THI = 83.6), both plasma T3 and T4 levels were noticed to be higher in wallowing group (1.99 + 0.03 and 50.57 + 0.61 ng/ml) than showering group (1.83 +

0.04 and 48.25 + 0.54 ng/ml) buffaloes (Aggarwal and Singh, 2010).

Identically, plasma T3 was found to be decreased at all the heat treat­ ments (30 ◦C, 35 ◦C and 40 ◦C) in comparison to control at 25 ◦C while plasma T4 was declined during heat exposure at 30 ◦C and 40 ◦C but not at 35 ◦C (Wankar et al., 2014). In tropical climate of Eastern Amazon in

Brazil, T3 and T4 concentrations were found to be highest in female buffaloes on direct exposure to sunlight during rainy season compared to buffaloes kept under shade (Silva et al., 2014). Plasma T3 concentration was found to be lowest in Surti buffaloes during exposure to hot dry (THI = 81.70) followed by hot humid (THI = 80.60) season and control (THI = 68.72) (Chaudhary et al., 2015). Likewise, plasma T3 and T4 levels were significantly reduced in Murrah buffaloes on exposure to hot dry and hot humid seasons compared to TNZ (Lakhani et al., 2018).

Plasma T3 level was significantly lower in Nili-Ravi buffaloes during summer and autumn than spring and winter whereas Plasma T4 level was significantly higher during summer and autumn than spring and winter season (Li et al., 2020). Consistent with Li et al. (2020), plasma T4 level was found to be higher during the summer than winter season in

Khuzestan buffalo bulls (Mayahi et al., 2014). In contrast, plasma T3 and

T4 levels and their ratio did not vary during different summer, monsoon and winter seasons in Murrah buffalo bulls (Dixit et al., 1984). Anti­ thetically, Das et al. (2014) did not notice any variation in plasma T3 and

T4 level between treatment (supplemented with either nutrients or modified shade) and control group buffaloes during hot dry and hot humid seasons. Contrarily, Yadav et al. (2016) did not find any signifi­ cant change in plasma T3 and T4 levels in lactating Murrah buffalo across control, misting and wallowing group during hot dry and hot humid seasons. In contrast, Hafez et al. (2011) found higher plasma T3 while did not notice any variation in plasma T4 in Egyptian buffaloes on exposure to high THI at 94. Reduction in plasma thyroid hormones level could reduce body metabolism thereby lowers thermogenesis to accli­ matize buffaloes to summer stressful conditions.

S.R. Mishra Journal of Thermal Biology 96 (2021) 102844

9 1.14. Prolactin Down-regulation of prolactin inhibiting hormone or dopamine in­ duces lactotroph cells of adenohypophysis to produce prolactin (Matteri et al., 1994; Alamer, 2011). Plasma prolactin level was significantly elevated in Murrah buffalo heifers on exposure to summer than winter months (Roy and Prakash, 2007). Similarly, significant elevation in serum prolactin level was noticed in lactating Murrah buffalo during hot humid month of July than hot dry month of May, suggesting that hot humid was more stressful to lactating Murrah buffaloes (Yadav et al.,

2016). Interestingly, misting and wallowing reduced serum prolactin level during hot humid month of July and hot dry month of May (Yadav et al., 2016).

1.15. Insulin Plasma insulin level was significantly higher in wallowing (10.86 +

0.27 and 9.62 + 0.30 μU/ml) than showering group buffaloes (8.30 +

0.26 and 7.86 + 0.33 μU/ml) during exposure to hot dry and hot humid seasons respectively (Aggarwal and Singh, 2010). This could be due to higher dry matter intake in wallowing buffaloes compared to showering buffaloes. Plasma insulin level was significantly lower in Nili-Ravi buf­ faloes on exposure to summer season than spring, autumn and winter season (Li et al., 2020).

1.16. Growth hormone Growth hormone releasing hormone (GHRH) acts on somatotroph cells of adenohypophysis to produce growth hormone, responsible for thermoregulation in buffaloes during heat stress. Plasma GH level was significantly lower in Nili-Ravi buffaloes during summer and autumn than spring and winter season (Li et al., 2020).

1.17. Aldosterone Hyperkalemia triggers zona glomerulosa of adrenal cortex to pro­ duce aldosterone, responsible for mineral homeostasis in domestic species. Therefore, aldosterone is considered as the major mineralo­ corticoid hormone in domestic species. Plasma aldosterone level neither changed at 35 ◦C nor at 40 ◦C of heat stress (Wankar et al., 2014).

1.18. Molecular responses shown by buffaloes against heat stress

It is now well established fact that, heat strain expedites the pro­ duction of innumerable conserved family of proteins known as heat shock proteins (HSPs) across the livestock species (Kishore et al., 2014;

Mishra, 2020). Accumulated evidences suggest that, HSPs behaves as molecular chaperones to promote proper folding of nascent proteins, re-folding of misfolded proteins, prevent protein aggregation and denature misfolded proteins thereby play pivotal role to confer thermo-tolerance in livestock species (Sodhi et al., 2013; Zhang et al.,

2016). It has also been shown that, HSPs curb apoptosis in different cellular systems thereby serve as a cyto-protective molecule during heat stress (Parsell and Lindquist, 1993; Sonna et al., 2002; Deb et al., 2015).

In general, HSPs are classified according to their molecular weight i.e. small HSPs such as HSP10 and HSP27 and large HSPs like HSP40,

HSP60, HSP70 and HSP90 (Mishra and Palai 2014; Kumar et al., 2015;

Kishore et al., 2016). Peripheral blood mononuclear cells (PBMCs) have been approved as the most genuine cellular system to quantify the in­ tensity of heat stress across livestock species as it serves as the store­ house for the generation of copious HSPs in response to heat stress (Romero et al., 2013; Kishore et al., 2014; Bharati et al., 2017). Despite the fact that PBMCs serve as phenomenon cellular model to comprehend molecular responses against thermal stress across domestic animals, several cellular systems have also been emerged to express HSPs namely skeletal myocyte (Gutierrez and Guerriero, 1991), hepatocyte (Joo et al.,

2005), lung cell (Sun et al., 2007), kidney cell (Zulkifli et al., 2010), adipocyte (Qu et al., 2015) and myocardial cells (Sahu et al., 2019).

Furthermore, HSPs maintains cellular integrity and homeostasis against the thermal stress. In this section, impacts of heat stress on transcrip­ tional and translational abundance of various HSPs in buffaloes is dis­ cussed vividly (Table 4).

1.19. HSF-1 Heat shock factors (HSF) are considered as the transcription factors which regulate the expression of HSPs inside the cellular systems. Out of all HSFs, HSF-1 is the chief regulator of HSPs inside cellular system during heat stress. Heat stress activates HSF which forms trimer and translocates into nucleus. Inside the nucleus, HSF binds with heat shock response element (HSE) located in the promoter region of DNA thereby regulates transcription of HSPs (Mishra, 2020). In an interesting study,

Pawar et al. (2014) reported significant up-regulation in HSF1 mRNA expression by 0.43 and 9.46 folds during summer 2 (end of August) and

3 (mid September) compared to summer 1 (beginning of August). In another study, Kumar et al. (2015) found significantly higher HSF1 mRNA expression in Murrah buffalo (5.68 folds) followed by Sahiwal (4.53 folds) and Tharparkar cattle (4.38 folds) exposed to summer heat stress.

1.20. HSP10 Kumar et al. (2015) experimented on the outcome of summer and winter stress on the mRNA expression pattern of some major HSPs in

Table 4 Molecular responses in buffaloes during heat stress.

HSPs Expression Author Breed HSF-1 Increase Pawar et al. (2014)

Murrah Kumar et al. (2015) HSP10 Increase Kumar et al. (2015)

Murrah HSP27 Increase Kapila et al. (2013) Murrah Kapila et al. (2016)

Kishore et al. (2016) HSP40 Increase Kapila et al. (2013)

Murrah Kapila et al. (2016) Kishore et al. (2014) Kishore et al. (2016)

Shandilya et al. (2020) HSP60 Increase Kapila et al. (2013)

Murrah Kapila et al. (2016) Kishore et al. (2014) Kumar et al. (2015)

Kishore et al. (2016) Shandilya et al. (2020) HSP70

Increase Mishra et al. (2011) Murrah Lallawmkimi et al. (2012)

Haque et al. (2012) Pawar et al. (2012) Kapila et al. (2013)

Kishore et al. (2014) Pawar et al. (2014) Kumar et al. (2015)

Kapila et al. (2016) Kishore et al. (2016) Priyadarshini and Aggarwal (2018)

Shandilya et al. (2020) Manjari et al. (2015) Tarai buffalo

Shenhe et al. (2018) Nili-Ravi × Murrah Liu et al. (2019)

HSP90 Increase Kapila et al. (2013) Murrah Kishore et al. (2014)

Kapila et al. (2016) Kumar et al. (2015) Kishore et al. (2016)

Shandilya et al. (2020) Shenhe et al. (2018) Nili-Ravi × Murrah

Liu et al. (2019) S.R. Mishra Journal of Thermal Biology 96 (2021) 102844

10 Murrah buffalo, Tharparkar and Sahiwal cattle. HSP10 mRNA abun­ dance was significantly higher in summer stress than winter stress and

TNZ (spring season). HSP10 mRNA abundance was found to be increased by 6.25, 6.59 and 7.02 folds in Murrah buffalo, Tharparkar and Sahiwal cattle respectively during summer heat stress.

1.21. HSP27 HSP27 is the most abundant small heat shock proteins across the farm animal species. In buffalo MECs, highest expression of HSP27 mRNA (8.20 fold) was registered at 16 h post heat stress at 42 ◦C (Kapila et al., 2013). Later on Kapila et al. (2016) conducted another experiment to document the impact of thermal stress on transcriptional abundance of various HSPs in heat stressed buffalo MECs. They noticed an imme­ diate induction in HSP27 mRNA expression at 30 min and maximum induction between 2 and 4 h post heat stress at 42 ◦C. In another study,

Kishore et al. (2016) aimed to determine the effect of seasonal variations on expression patterns of HSP27 in PBMCs of Murrah buffaloes, Holstein

Friesian and Sahiwal cows. Expression of HSP27 transcript was lower in

Murrah buffaloes during summer than winter season but failed to show significant modulation.

1.22. HSP40 Highest induction in HSP40 transcription (20.45 fold) was noticed between 2 and 4 h post heat stress on cultured buffalo MECs at 42 ◦C (Kapila et al., 2013). Later on, Kapila et al. (2016) observed a similar trend in HSP40 transcription in heat stressed buffalo MECs. Kishore et al. (2014) had undertaken an experiment to investigate the expression dynamics of different HSPs in Murrah buffaloes, Holstein Friesian and

Sahiwal cows on exposure to heat stress at 42 ◦C. They exposed the cultured PMBCs of Murrah buffaloes and both cattle breeds to heat challenge at 42 ◦C for 12 h and then determined the induction in tran­ scription of different HSPs at different incubation periods. They observed that, HSP40 transcript was hyper-expressed at 2 h of post heat shock at 42 ◦C and then reduced till 12 h of heat challenge. Moreover, expression of HSP40 transcript was noted greatest in Murrah buffaloes (15.27 folds) followed by Holstein Friesian (3. folds) and Sahiwal (1.87 folds) cows after 2 h of heat load at 42 ◦C. In another study, HSP40 mRNA expression was increased in Murrah buffaloes during summer than winter season but found to be non significant (Kishore et al., 2016).

HSP40 mRNA expression was significantly higher in heat stressed buf­ falo fibroblast than cattle fibroblasts at 0, 4 and 16 h of post heat stress (Shandilya et al., 2020).

1.23. HSP60 The expression of HSP60 transcript was found to be maximum (1.97 fold) in cultured bubaline MECs at 4 h post heat stress at 42 ◦C (Kapila et al., 2013). In another study conducted by Kapila et al. (2016) in heat stressed bubaline MECs, HSP60 transcription pattern was consistent with their previous study by Kapila et al. (2013). HSP60 mRNA abun­ dance was significantly highest in PBMCs of Murrah buffaloes (15.2 folds) followed by Holstein Friesian (9.14 folds) and Sahiwal (8.04 folds) cows at 2 h post heat stress at 42 ◦C (Kishore et al., 2014). In another study, HSP60 mRNA abundance was found to be elevated in Murrah buffaloes (4.87 folds), Tharparkar (6.58 folds) and Sahiwal (7.64 folds) cattle during summer season compared to winter season and TNZ (Kumar et al., 2015). In another study, a non significant increase in

HSP60 mRNA expression was observed in Murrah buffaloes during summer than winter season (Kishore et al., 2016). In a recent study,

HSP60 mRNA expression was significantly greater in buffalo fibroblast than cattle fibroblasts at all time durations of post heat stress except at 8 h, suggesting the fact that buffalo fibroblasts are more heat responsive as compared to cattle fibroblasts (Shandilya et al., 2020).

1.24. HSP70 HSP70 has been considered as the predominant HSP amongst all the

HSPs and express in almost all the cellular systems upon heat stress (Mishra and Palai, 2014; Mishra, 2020). In addition, HSP70 is the most extensively studied protein amongst all the HSPs hitherto. Patir and

Upadhyay (2010) for the first time purified HSP70 protein from buffalo lymphocytes and documented an increase in HSP70 protein concentra­ tion in the PBMCs after first 2 h followed by a dip after 3 and 4 h of heat exposure 45 ◦C. Mishra et al. (2011) tried to investigate the impact of induced heat stress on abundance of HSP70 transcript in Murrah buffalo calves. In the in vivo study, they exposed buffalo calves to hot-dry con­ ditions at 42 ◦C with 30% relative humidity (RH) and hot humid con­ ditions at 35 ◦C with 70% RH inside the psychometric chamber for 4 h daily continuously for 12 days. Mishra et al. (2011) detected more than

200 fold increase in serum HSP70 levels in both hot-dry as well as hot humid conditions than control. Moreover, hot dry condition had induced more HSP70 expression than hot humid condition. On the other hand in the in vitro model, they exposed cultured lymphocytes at 42 ◦C and reported a 2.5 fold increase in HSP70 concentration in compared to control at 37 ◦C. Thus Mishra et al. (2011) suggested that, serum HSP70 concentration could be considered as sensitive biomarker for heat stress management to mitigate the wrath of heat stress in Murrah buffaloes. In another study, buffalo lymphocytes were exposed to heat stress at 38 ◦C with 50 RH and then as 42 ◦C with 40 RH inside the psychometric chamber (Lallawmkimi et al., 2012). HSP72 transcription was signifi­ cantly up-regulated after 2 h and then reduced after 3 h of heat exposure at 38 ◦C and 42 ◦C. Further, HSP72 transcription was found to be greater during heat exposure at 42 ◦C than 38 ◦C, suggesting that HSP72 tran­ scription was positively correlated with temperature. In their study,

Haque et al. (2012) planned to evaluate the optimum temperature for induction of HSP70 in Murrah buffaloes. In their in vitro model, HSP70 registered highest concentration in cultured lymphocytes following 3 h of heat exposure at 40, 42 and 45 ◦C. Additionally, in their in vivo experiment, plasma HSP70 concentration were reported to be higher in young compared to adult buffaloes after 4 h of heat exposure at 45 ◦C indicating the fact that young buffaloes are more prone to heat stress compared to adult ones. Haque et al. (2012) concluded that, induction in

HSP70 in Murrah buffaloes begins when ambient temperature is 2–3 ◦C higher than core body temperature. Pawar et al. (2012) carried out an experiment to explore the expression pattern of HSP70 in buffalo PBMCs exposed to different temperatures and incubation periods i.e. at 39 ◦C for

24 h (control), 41 ◦C for 4 h and 43 ◦C for 4 h. Expression of HSP70 transcripts was significantly increased by 1.09 and 2.47 fold at 41 ◦C and

43 ◦C respectively than control at 39 ◦C. In a study conducted in riverine buffalo, quick induction in HSP70 transcription was found in MECs at

30 min, continued till 8 h and then declined up to 48 h post heat stress at

42 ◦C (Kapila et al., 2013). Moreover, induction in HSP70 transcription was highest (72.54 fold) between 2 and 4 h post heat stress. In their next study on buffalo MECs, Kapila et al. (2016) noted an identical pattern of induction in HSP70 mRNA expression like Kapila et al. (2013). More­ over, after an initial peak in HSP70 transcription at 2–4 h followed by sharp fall suggest the role of HSP70 as a molecular marker of acute heat stress responses in cattle and buffaloes (Kapila et al., 2016). In another study, Pawar et al. (2014) attempted to determine the mRNA expression dynamics of HSP70 in buffalo leukocytes exposed to summer stress. In their experiment, they divided both the summer as well as winter sea­ sons into 3 phases i.e. summer 1 (beginning of August), summer 2 (end of August) and summer 3 (mid September). Pawar et al. (2014) reported a spike in HSP70 mRNA expression by 0.22 and 9.01 fold during summer

2 and 3 compared to summer 1. In another study in heat stressed PBMCs at 42 ◦C, expression of HSP70 transcript was found to be highest after 2 h in Murrah buffaloes (80.00 fold) followed by Holstein Friesian (52.68 fold) and Sahiwal cows (35.64 fold). Interestingly, HSP70 transcription was highest in Murrah buffalo amongst all the HSPs where fold change of HSP70 (80.00) was significantly ahead of HSP90 (18.75) followed by

S.R. Mishra Journal of Thermal Biology 96 (2021) 102844

11 HSP40 (15.27) and HSP60 (15.20) in cultured PBMCs exposed to heat stress at 42 ◦C for 2 h. Due to the prominent high induction of HSP70 after 2 h of post heat stress amongst all the HSPs across buffalo and cattle breeds, Kishore et al. (2014) suggested to consider HSP70 as the most sensitive biomarker to measure the quantum of heat stress. Later on,

Manjari et al. (2015) had undertaken an experiment to elucidate the expression trend of HSP70 transcript in PBMCs of Tarai buffaloes during different seasons. They found significant up-regulation in expression of

HSP70 transcript during summer (2.37 fold) than winter season (0.29 fold). In addition, Manjari et al. (2015) identified positive correlation of

HSP70 transcript expression with respiration rate. Thus they suggested that, induction in expression of HPS70 transcript and respiration rate could be regarded as the cellular and physiological marker respectively to quantify the intensity of thermal stress in Tarai buffaloes. Kumar et al. (2015) investigated on HSP70 family of genes (HSPA1A, HSPA1B and

HSPA8) in buffalo and cattle during different season under tropical conditions. They reported highest HSPA1A transcription in Murrah buffalo (14.48 fold) than Tharparkar (9.70 fold) and Sahiwal (10.65 fold) cattle during summer season. However, expression of HSPA1B transcript in Murrah buffalo (13.55 fold) was higher than Tharparkar (9.51 fold) but lower than Sahiwal (14.81) during summer heat load.

Identical to HSPA1A, relative abundance of HSPA8 transcript in Murrah buffalo (6.01 fold) was highest followed by Tharparkar (5.56 fold) and

Sahiwal (5.17 fold) during exposure to summer heat strain. In another study, non significant elevation in HSP70 mRNA expression (1.73 fold) was observed in Murrah buffaloes during summer than winter season (Kishore et al., 2016). Plasma HSP70 concentration was significantly higher in crossbred buffaloes during summer (421⋅28 pg/ml) than spring (263⋅65 pg/ml) and winter (174⋅11 pg/ml) season (Shenhe et al.,

2018). However, mean plasma HSP70 concentration of crossbred buf­ faloes (375⋅12 pg/ml) was found to be higher than Mediterranean buf­ faloes (202⋅11 pg/ml) during summer season (Shenhe et al., 2018).

Identically, Liu et al. (2019) found higher plasma HSP70 level in heat stressed buffaloes (454.26 pg/ml) than control ones (142.86 pg/ml). In another study, HSP70.1, HSP70.2 and HSP70.8 mRNA expression was significantly higher in buffaloes exposed to summer than winter season (Priyadarshini and Aggarwal, 2018). In particular, HSP70.1, HSP70.2 and HSP70.8 mRNA expression was gradually increased and found to be highest on the day of parturition and then declined till days 21 of post-partum in control and treatment group buffaloes during summer season. Highest expression of HSP70.1, HSP70.2 and HSP70.8 mRNA on the day of parturition indicates the intensity of calving stress on buf­ faloes. HSP70.1, HSP70.2 and HSP70.8 mRNA expression was signifi­ cantly lower in buffaloes treated with astaxanthin (3.58 ± 0.03, 2.11 ±

0.02, 1.97 ± 0.02) than control group (3.84 ± 0.03, 2.40 ± 0.02, 2.25 ±

0.02) throughout the experimental period during summer season, sug­ gesting the fact that astaxanthin decreases the HSPs expression by reducing the oxidative stress and improves the immunity level in peri­ parturient buffaloes during summer season (Priyadarshini and Aggar­ wal, 2018). Kaur et al. (2018) reported substantial induction in ATP1A1 and ATP1A2 isoforms in heat stressed buffalo MECs and indicated a positive correlation between ATP1A1 and ATP1A2 isoforms with

HSP60, HSP70 and HSP90. Immediate induction in ATP1A1 mRNA was observed at 30 min, reached zenith at 4 h (4.659 fold) and then grad­ ually declined till 48 h post heat stress. Identically, sudden induction in

ATP1A2 mRNA was noted at 30 min, reached highest at 4 h (1.84 fold) followed by gradually declined till 48 h post heat stress. Thus immediate induction in transcription of ATP1A1 and ATP1A2 isoforms indicate their possible involvement in heat shock response and maintenance of proteostasis during heat stress. Finally Kaur et al. (2018) compared the expression levels of ATP1A1 and ATP1A2 isoforms with the best known molecular chaperone HSP70 and documented that HSP70 was the most sensitive heat response gene followed by ATP1A1 and ATP1A2.

Recently, Shandilya et al. (2020) designed an experiment to evaluate the consequences of heat stress on expression dynamics of HSPs in cultured dermal fibroblast of cattle and buffaloes exposed to heat load at 42 ◦C for

1 h. The induction in HSP70 transcription in buffalo fibroblasts was noted at various incubation periods during post heat stress. In particular,

HSP70 transcription was up-regulated by 8.88, 8.67, 7.05, 5.74, 6.12 and 7.12 at 0, 2, 4, 8, 16 and 24 h of post heat stress respectively.

Moreover, induction in HSP70 transcription was significantly higher in buffalo fibroblast than cattle fibroblasts at all incubation periods of post heat stress, indicating that buffalo fibroblasts are more thermo-sensitive compared to cattle fibroblasts. Finally Shandilya et al. (2020) suggested that dermal fibroblasts could be regarded as a cellular model to deter­ mine the magnitude of thermal stress in cattle and buffaloes. There are literatures regarding the effects of polymorphism in regulation of heat stress response in buffaloes. Sodhi et al. (2013) reported an association between the SNP in 5′UTR region of HSP70 with thermal stress, milk production and disease vulnerability in riverine buffalo. Kumar et al. (2017) detected seven SNP (three transitions and four transversions) at

5′ untranslated (UTR) region of HSP70. The authors also found close association between the SNP at 5′ UTR of HSP70 with post partum anestrus in Murrah buffaloes. Then, Kumar et al. (2017) indicated that the SNP in 5′ UTR of HSP70 could be considered as marker to diagnose the condition of post partum anoestrus in Murrah buffaloes.

1.25. HSP90 Highest induction in HSP90 transcription was achieved in cultured buffalo MECs at 4 h following heat stress at 42 ◦C (Kapila et al., 2013).

Uniformly, Kapila et al. (2016) found similar pattern of HSP90 tran­ scription in buffalo MECs on exposure to heat stress at 42 ◦C. In another study, induction in HSP90 transcription was greatest in PBMCs of

Murrah buffaloes (18.75 fold) followed by Sahiwal (7.32 fold) and

Holstein Friesian (3.14 fold) cows after 2 h of heat stress at 42 ◦C (Kishore et al., 2014). In another study, significant elevation in HSP90 mRNA expression was observed in Murrah buffaloes (2.53 fold), Thar­ parkar (2.87 fold) and Sahiwal (3.77 fold) cattle during summer season compared to winter season and TNZ (Kumar et al., 2015). In addition,

Kishore et al. (2016) found a non significant induction in HSP90 mRNA expression was observed in Murrah buffaloes during summer than winter season. Plasma HSP90 concentration was found to be signifi­ cantly higher in crossbred buffaloes during summer (3348⋅48 pg/ml) than spring (1311⋅08 pg/ml) and winter (947⋅21 pg/ml) season (Shenhe et al., 2018). However, mean plasma HSP90 concentration of crossbred buffaloes (2938 pg/ml) was found to be higher than Mediterranean buffaloes (1381⋅61 pg/ml) during summer season (Shenhe et al., 2018).

Similarly, Liu et al. (2019) reported higher plasma HSP90 level in heat stressed buffaloes (3972.53 pg/ml) than control ones (845.42 pg/ml). In another study conducted by Shandilya et al. (2020), HSP90 mRNA expression was significantly greater in dermal fibroblasts of buffalo than cattle at all time points post heat stress, depicting that cellular tolerance of buffalo dermal fibroblasts in quite lower than that of cattle dermal fibroblasts.

1.26. Role of miRNAs in thermo-tolerance in buffaloes

MicroRNAs (miRNAs) are family of highly conserved single-stranded non-coding RNA comprising around 22 nucleotides which depresses post-transcription by base pairing with their target mRNAs of respective genes (Bartel, 2004; Mishra, 2020). It is well known that, miRNAs reg­ ulates various physiological processes such as cellular proliferation and differentiation, apoptosis, development, focal adhesion and biosynthesis of secondary metabolites (Sengar et al., 2018). Generally, miRNAs target various molecules like HSPs, toll like receptors (TLRs) along with mul­ tiple ligands via several signaling pathways to regulate heat stress response, immune response, oxidative stress response and cellular apoptosis amongst livestock species (Sengar et al., 2018). Literatures on expression dynamics of miRNAs in heat stressed buffaloes are sparse.

Recently, Liu et al. (2019) identified 418 miRNAs in buffaloes under heat stress and control group by using miRNA-Seq data, out of which 16

S.R. Mishra Journal of Thermal Biology 96 (2021) 102844

12 miRNAs were differentially expressed (05 mature miRNAs and 11 novel miRNAs). Liu et al. (2019) found that bta-miR-1246 targeted ABCC4 gene thereby form mRNA-miRNA network which could regulate heat stress response in buffaloes. In another study, Shandilya et al. (2020) investigated expression kinetics of multiple miRNAs in heat exposed dermal fibroblasts of cattle and buffaloes. They found quick induction in the expression of various miRNAs such as miR-27 b, miR-19a, miR-19 b, miR-345–3p, miR-30a-5p, miR-146a, miR-146 b and miR-199a-3p in buffalo dermal fibroblasts exposed to heat stress at 42 ◦C. Expression of miR-30a-5p and miR-146a were found to be elevated at all the incuba­ tion periods from 0 to 24 h after heat stress at 42 ◦C. In addition, expression of miR-199a-3p was significantly up-regulated in buffalo dermal fibroblasts at 0, 2, 4 and 8 h and returned to basal level at 24 h of post heat stress at 42 ◦C. Likewise, expression of miR-146 b and miR-345–3p were escalated at 0, 2 and 4 h followed by a dip at 8 and 24 h of post heat stress at 42 ◦C. However, expression of miR-26a and miR-1246 was down regulated in buffalo dermal fibroblasts exposed to heat stress at 42 ◦C compared to control. These miRNAs might target various molecules such as HSPs, TLRs, PLA2R1 and PICEN, therefore could modulate heat, immune and oxidative stress responses in buffaloes.

2. Conclusion This present review could be very helpful for the researchers to comprehend the fundamentals in thermoregulatory responses in buf­ faloes. Alternations in behavioural, physiological, neuro-endocrine and molecular responses confer thermo-tolerance in buffaloes. As literatures regarding the significance of miRNAs in buffaloes in response to heat stress are sparse, thus future research works are warranted to explore on expression dynamics, specific target molecules and network analysis of various miRNAs to unmask the exact molecular mechanism and signaling pathways of miRNAs in heat stressed buffaloes. Furthermore, future research works should be targeted to unveil deep insight on the cellular and molecular responses vis a vis molecular chaperones and other cytokines in buffaloes which could possibly assist to ameliorate the negative impacts of thermal stress in buffaloes thereby augmenting their production and productivity.

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