Cloning of heat shock protein genes (hsp70, hsc70 and hsp90) and their expression in response to larval diapause and thermal stress in the wheat blossom midge,Sitodiplosis mosellana

Sitodiplosis mosellana Géhin, one of the most important pests of wheat, undergoes obligatory diapause as a larva to survive unfavorable temperature extremes during hot summers and cold winters. To explore the potential roles of heat shock proteins (hsp) in this process, we cloned full-length cDNAs of hsp70, hsc70 and hsp90 from S. mosellana larvae, and examined their expression in response to diapause and short-term temperature stresses. Three hsps included all signature sequences of corresponding protein family and EEVD motifs. They showed high homology to their counterparts in other species, and the phylogenetic analysis of hsp90 was consistent with the known classification of insects. Expression of hsp70 and hsp90 were highly induced by diapause, particularly pronounced during summer and winter. Interestingly, hsp70 was more strongly expressed in summer than in winter whereas hsp90 displayed the opposite pattern. Abundance of hsc70 mRNA was comparable prior to and during diapauses and was highly up-regulated when insects began to enter the stage of post-diapause quiescence. Heat-stressed over-summering larvae (⩾30 °C) or cold-stressed over-wintering larvae (⩽0 °C) could further elevate expression of these three genes, but temperature extremes i.e. as high as 45 °C or as low as −15 °C failed to trigger such expression patterns. Notably, hsp70 was most sensitive to heat stress and hsp90 was most sensitive to cold stress. These results suggested that hsp70 and hsp90 play key roles in diapause maintenance and thermal stress; the former may be more prominent contributor to heat tolerance and the latter for cold tolerance. In contrast, hsc70 most likely is involved in developmental transition from diapause to post-diapause quiescence, and thus may serve as a molecular marker to predict diapause termination.     

The effects of increased constant incubation temperature and cumulative acute heat shock exposures on morphology and survival of Lake Whitefish (Coregonus clupeaformis) embryos

Increasing incubation temperatures, caused by global climate change or thermal effluent from industrial processes, may influence embryonic development of fish. This study investigates the cumulative effects of increased incubation temperature and repeated heat shocks on developing Lake Whitefish (Coregonus clupeaformis) embryos. We studied the effects of three constant incubation temperatures (2 °C, 5 °C or 8 °C water) and weekly, 1-h heat shocks (+3 °C) on hatching time, survival and morphology of embryos, as these endpoints may be particularly susceptible to temperature changes. The constant temperatures represent the predicted magnitude of elevated water temperatures from climate change and industrial thermal plumes. Time to the pre-hatch stage decreased as constant incubation temperature increased (148 d at 2 °C, 92 d at 5 °C, 50 d at 8 °C), but weekly heat shocks did not affect time to hatch. Mean survival rates and embryo morphometrics were compared at specific developmental time-points (blastopore, eyed, fin flutter and pre-hatch) across all treatments. Constant incubation temperatures or +3 °C heat-shock exposures did not significantly alter cumulative survival percentage (~50% cumulative survival to pre-hatch stage). Constant warm incubation temperatures did result in differences in morphology in pre-hatch stage embryos. 8 °C and 5 °C embryos were significantly smaller and had larger yolks than 2 °C embryos, but heat-shocked embryos did not differ from their respective constant temperature treatment groups. Elevated incubation temperatures may adversely alter Lake Whitefish embryo size at hatch, but weekly 1-h heat shocks did not affect size or survival at hatch. These results suggest that intermittent bouts of warm water effluent (e.g., variable industrial emissions) are less likely to negatively affect Lake Whitefish embryonic development than warmer constant incubation temperatures that may occur due to climate change.     

Combined effects of temperature and cadmium on developmental parameters and biomarker responses in zebrafish (Danio rerio) embryos

To determine the interactions between temperature and cadmium on zebrafish (Danio rerio) development, fertilized eggs were exposed to combinations of three temperature levels (21 °C, 26 °C, and 33 °C) and six cadmium concentrations (0, 0.25, 0.5, 2.0, 5.0, and 10.0 mg/L). Endpoints used included LC₅₀ value (48 h), developmental rate, mortality, heart rate, hatching success, liver histopathology, embryo abnormalities, and heat shock protein (hsp) induction. Results showed a significant acceleration in the developmental rate with increasing temperature and irrespective of the presence of cadmium. Data on LC₅₀ and ELS-test revealed that simultaneous exposure to both cadmium ions and cold stress (21 °C) was highly detrimental to growing embryos, causing a pronounced mortality and a significant reduction in average heart rate and embryo hatchability. In contrast, no similar reactions to cadmium were observed in pre-hatched embryos exposed to both control (26 °C) and high temperature (33 °C), and this can be explained by the significantly higher expression of hsp (hsp70) in embryos at these temperatures. Upon hatching, however, the larvae showed increased sensitivity to cadmium. The severity of malformations in the post-hatched larvae was in the order: hot cadmium stress>cold cadmium stress>cadmium stress alone>no stress at all. Liver histopathology as well as depletion in glycogen reserves exhibited greater severity with increasing cadmium concentration, irrespective of temperature. The present study confirms that temperature effectively confounds cadmium toxicity and needs to be considered for the accurate prediction and assessment of cadmium-induced toxicity in fish.     

Combined effect of temperature and zinc on Caenorhabditis elegans wild type and daf-21 mutant strains

Heavy metal pollution in aquatic ecosystems is a far reaching environmental problem. The possible influences of heavy metal exposure and the potential harm to organisms when combined with other environmental stressors such as temperature have been largely unexplored. An aquatic toxicity test of Caenorhabditis elegans was performed to estimate the 24 h median lethal concentration (LC₅₀) of different zinc concentrations at different temperatures (15 °C, 20 °C, 25 °C, and 30 °C). We also examined the time course thermotolerance on wild type (N2) and daf-21 null (JT6130) adults exposed to 6.1 mM zinc at 37 °C. Hsp90 protein expression level in response to the combined effect of temperature and zinc toxicity was also investigated by both Western blots and ELISA. Our results show that C. elegans wild type nematodes exhibit severe lethal toxicity after a 24 h exposure to zinc at higher temperatures. In addition, the expression level of Hsp90 was highly inhibited in adult worms subjected to zinc stress. This toxicity assay at different temperatures provides insight into organism response to combined effects of temperature and zinc toxicity.     

Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta)

The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34 °C and 36 °C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36 °C, 3 h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events.     

The effect of acclimation temperature on thermal activity thresholds in polar terrestrial invertebrates

In the Maritime Antarctic and High Arctic, soil microhabitat temperatures throughout the year typically range between ?10 and +5 °C. However, on occasion, they can exceed 20 °C, and these instances are likely to increase and intensify as a result of climate warming. Remaining active under both cool and warm conditions is therefore important for polar terrestrial invertebrates if they are to forage, reproduce and maximise their fitness. In the current study, lower and upper thermal activity thresholds were investigated in the polar Collembola, Megaphorura arctica and Cryptopygus antarcticus, and the mite, Alaskozetes antarcticus. Specifically, the effect of acclimation on these traits was explored. Sub-zero activity was exhibited in all three species, at temperatures as low as ?4.6 °C in A. antarcticus. At high temperatures, all three species had capacity for activity above 30 °C and were most active at 25 °C. This indicates a comparable spread of temperatures across which activity can occur to that seen in temperate and tropical species, but with the activity window shifted towards lower temperatures. In all three species following one month acclimation at ?2 °C, chill coma (=the temperature at which movement and activity cease) and the critical thermal minimum (=low temperature at which coordination is no longer shown) occurred at lower temperatures than for individuals maintained at +4 °C (except for the CTmin of M. arctica). Individuals acclimated at +9 °C conversely showed little change in their chill coma or CTmin. A similar trend was demonstrated for the heat coma and critical thermal maximum (CTmax) of all species. Following one month at ?2 °C, the heat coma and CTmax were reduced as compared with +4 °C reared individuals, whereas the heat coma and CTmax of individuals acclimated at +9 °C showed little adjustment. The data obtained suggest these invertebrates are able to take maximum advantage of the short growing season and have some capacity, in spite of limited plasticity at high temperatures, to cope with climate change.     

Behavioral and mortality responses of the bivalves Scrobicularia plana and Cerastoderma edule to temperature,as indicator of climate change's potential impacts

Temperature is one of the most important abiotic factors affected by climate change. It determines physiological processes, ecological patterns and establishes the limits of geographic distribution of species. The induced thermal stress frequently results in physiological and behavioral responses and, in extreme cases, may lead to mortality episodes. Scrobicularia plana and Cerastoderma edule behavioral and mortality responses to temperature were evaluated. Specimens were sampled in the Mondego estuary (Portugal), acclimated and exposed to different temperature treatments (5–35 °C). Individual activity and mortality were registered during 120 h laboratory assays. Both species showed a thermal optimum for their activity (S. plana: 15–23 °C; C. edule: 20–23 °C), and survival was mainly affected by high temperature (S. plana: LC50_120 h = 28.86 °C; C. edule: LC50_120 h = 28.01 °C), with 100% mortality above critical values (≥32 °C). Results further indicated that both species are more affected the higher the temperature and the longer the exposure time. This study indicates that the occurrence of extreme climatic events, especially heat waves, may be particularly impairing for these species.     

Temperature-dependent physiological and biochemical responses of the marine medaka Oryzias melastigma with consideration of both low and high thermal extremes

This study aimed to investigate temperature effect on physiological and biochemical responses of the marine medaka Oryzias melastigma larvae. The fish were subjected to a stepwise temperature change at a rate of 1 °C/h increasing or decreasing from 25 °C (the control) to six target temperatures (12, 13, 15, 20, 28 and 32 °C) respectively, followed by a 7-day thermal acclimation at each target temperature. The fish were fed ad libitum during the experiment. The results showed that cumulative mortalities were significantly increased at low temperatures (12 and 13 °C) and at the highest temperature (32 °C). For the survivors, their growth profile closely followed the left-skewed ‘thermal performance curve’. Routine oxygen consumption rates of fish larvae were significantly elevated at 32 °C but suppressed at 13 and 15 °C (due to a high mortality, larvae from 12 °C were not examined). Levels of heat shock proteins and activities of malate dehydrogenase and lactate dehydrogenase were also measured in fish larvae exposed at 15, 25 and 32 °C. The activities of both enzymes were significantly increased at both 15 and 32 °C, where the fish larvae probably suffered from thermal discomfort and increased anaerobic components so as to compensate the mismatch of energy demand and supply at these thermal extremes. Coincidently, heat shock proteins were also up-regulated at both 15 and 32 °C, enabling cellular protection. Moreover, the critical thermal maxima and minima of fish larvae increased significantly with increasing acclimation temperature, implying that the fish could develop some degrees of thermal tolerance through temperature acclimation.     

Effect of cyclical cold stress during embryonic development on aspects of physiological responses and HSP70 gene expression of chicks

The objective of the present study was to evaluate the effects of cyclical lower incubation temperature at different embryonic ages on the hatchability, body and organs weights, thyroid hormones, and liver HSP70 gene expression of newly hatched chicks. In a completely randomized design, fertile eggs of a broiler breeder (34 weeks of age) were assigned to three treatment groups with six replicates and 145 eggs per each. The treatment groups were as: control group (C) that eggs were incubated at 37.6 °C during the whole incubation period; incubation temperature was decreased to 36 °C for 3 h per day at embryonic age from 12 to 14 (T1); and incubation temperature was decreased to 36 °C for 3 h per day at embryonic age from 15 to 17 (T2). No significant difference was found among treatments for hatchability (P>0.05). There were no differences (P>0.05) among treatments for body weight and liver weight, while heart weight of chicks in T1 and T2 groups were significantly higher than the control group (P<0.05). There were no differences (P>0.05) among treatments for the levels of thyroid hormones, however, the levels of both hormones tended to increase in chicks exposed to cold stress (T1 and T2). Chicks in T2 group had higher liver HSP70 gene expression compared with those in T1 and the control group (P<0.05). Cold stress in both incubation periods had no significant effect on the plasma levels of aspartate aminotransferase and alanine aminotransferase. Treatments had no effect on the plasma levels of glucose, cholesterol and triglyceride. The results of this study suggest that cyclical lower incubation temperatures (36 °C) at the embryonic age from day 15–17 could induce the liver HSP70 gene expression, without negative effects on the hatchability and body weight of hatched chicks.     

Immobilization of glycolate oxidase from Medicago falcata on magnetic nanoparticles for application in biosynthesis of glyoxylic acid

Glycolate oxidase was isolated from Medicago falcata Linn. after a screening from 13 kinds of C₃ plant leaves, with higher specific activity than the enzyme from spinach. The M. falcata glycolate oxidase (MFGO) was partially purified and then immobilized onto hydrothermally synthesized magnetic nanoparticles via physical adsorption. The magnetic nanoparticles were characterized with scanning electron microscope (SEM), transmission electron microscopy (TEM) and Fourier transform infrared (FT-IR) spectroscopy. The maximum load of MFGO was 56 mg/g support and the activity recovery was 45%. Immobilization of MFGO onto magnetic nanoparticles enhanced the enzyme stability, and the optimum temperature was significantly increased from 15 °C to 30 °C. The immobilized biocatalyst was successfully used in a batch reactor for repeated oxidization of glycolic acid to synthesize glyoxylic acid, retaining ca. 70% of its initial activity after 4 cycles of reaction at 30 °C for nearly 70 h, and its half-life was calculated to be 117 h.     

Consequences of experimental cortisol manipulations on the thermal biology of the checkered puffer (Sphoeroides testudineus) in laboratory and field environments

Anthropogenic climate change is altering temperature regimes for coastal marine fishes. However, given that temperature changes will not occur in isolation of other stressors, it is necessary to explore the potential consequences of stress on the thermal tolerances and preferences of tropical marine fish in order to understand the thresholds for survival, and predict the associated coastal ecological consequences. In this study, we used exogenous cortisol injections to investigate the effects of a thermal challenge on checkered puffers (Sphoeroides testudineus) as a secondary stressor. There were no significant differences between control and cortisol-treated fish 48 h following cortisol treatment for swimming ability (using a chase to exhaustion protocol), blood glucose concentrations or standard metabolic rate. In the lab, control and cortisol-treated puffers were exposed to ambient (29.1±1.5 °C), ambient +5 °C (heat shock) and ambient −5 °C (cold shock) for 4 h and to evaluate the consequences of abrupt temperature change on puff performance and blood physiology. Following cold shock, control fish exhibited increases in cortisol levels and weak ‘puff’ performance. Conversely, fish dosed with cortisol exhibited consistently high cortisol levels independent of thermal treatment, although there was a trend for an attenuated cortisol response in the cortisol-treated fish to the cold shock treatment. A 20-day complementary field study conducted in the puffer’s natural habitat, a tidal creek in Eleuthera, The Bahamas, revealed that cortisol-injected fish selected significantly cooler temperatures, measured using accumulated thermal units, when compared to controls. These results, and particularly the discrepancies between consequences documented in the laboratory and the ecological trends observed in the field, highlight the need to establish the link between laboratory and field data to successfully develop management policies and conservation initiatives with regards to anthropogenic climate change.     

Heterologous production of a temperature and pH-stable laccase from Bacillus vallismortis fmb-103 in Escherichia coli and its application

To enhance laccase yield, the laccase gene from Bacillus vallismortis fmb-103 was cloned and heterologously expressed in Escherichia coli BL21 (DE3) cells. The auto-induction strategy was applied during fermentation, and the process was controlled, as follows: Cu²⁺ was added when the optical density at 600 nm (OD₆₀₀) was 0.3, the fermentation temperature was adjusted to 16 °C when the OD₆₀₀ was 0.9, and fermentation was stopped after 50 h. The yield of recombinant laccase was up to 3420 U/L, as assayed by 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Recombinant laccase was purified 4.47-fold by heating for 10 min at 70 °C and dialyzing against 50–60% ammonium sulfate, retained more than 50% activity after 10 h at 70 °C, and demonstrated broad pH stability. Malachite green was efficiently degraded by recombinant laccase, especially in combination with mediators. These results provided a basis for the future application of recombinant laccase to malachite green degradation.     

Adult plasticity of cold tolerance in a continental-temperate population of Drosophila suzukii

Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) is a worldwide emerging pest of soft fruits, but its cold tolerance has not been thoroughly explored. We determined the cold tolerance strategy, low temperature thermal limits, and plasticity of cold tolerance in both male and female adult D. suzukii. We reared flies under common conditions (long days, 21 °C; control) and induced plasticity by rapid cold-hardening (RCH, 1 h at 0 °C followed by 1 h recovery), cold acclimation (CA, 5 days at 6 °C) or acclimation under fluctuating temperatures (FA). D. suzukii had supercooling points (SCPs) between −16 and −23 °C, and were chill-susceptible. 80% of control flies were killed after 1 h at −7.2 °C (males) or −7.5 °C (females); CA and FA improved survival of this temperature in both sexes, but RCH did not. 80% of control flies were killed after 70 h (male) or 92 h (female) at 0 °C, and FA shifted this to 112 h (males) and 165 h (females). FA flies entered chill coma (CT_min) at approximately −1.7 °C, which was ca. 0.5 °C colder than control flies; RCH and CA increased the CT_min compared to controls. Control and RCH flies exposed to 0 °C for 8 h took 30–40 min to recover movement, but this was reduced to <10 min in CA and FA. Flies placed outside in a field cage in London, Ontario, were all killed by a transient cold snap in December. We conclude that adult phenotypic plasticity is not sufficient to allow D. suzukii to overwinter in temperate habitats, and suggest that flies could overwinter in association with built structures, or that there may be additional cold tolerance imparted by developmental plasticity.     

Repeatability of a running heat tolerance test

At present there is no standardised heat tolerance test (HTT) procedure adopting a running mode of exercise. Current HTTs may misdiagnose a runner's susceptibility to a hyperthermic state due to differences in exercise intensity. The current study aimed to establish the repeatability of a practical running test to evaluate individual's ability to tolerate exercise heat stress. Sixteen (8M, 8F) participants performed the running HTT (RHTT) (30 min, 9 km h⁻¹, 2% elevation) on two separate occasions in a hot environment (40 °C and 40% relative humidity). There were no differences in peak rectal temperature (RHTT1: 38.82±0.47 °C, RHTT2: 38.86±0.49 °C, Intra-class correlation coefficient (ICC)=0.93, typical error of measure (TEM)=0.13 °C), peak skin temperature (RHTT1: 38.12±0.45, RHTT2: 38.11±0.45 °C, ICC=0.79, TEM=0.30 °C), peak heart rate (RHTT1: 182±15 beats min⁻¹, RHTT2: 183±15 beats min⁻¹, ICC=0.99, TEM=2 beats min⁻¹), nor sweat rate (1721±675 g h⁻¹, 1716±745 g h⁻¹, ICC=0.95, TEM=162 g h⁻¹) between RHTT1 and RHTT2 (p>0.05). Results demonstrate good agreement, strong correlations and small differences between repeated trials, and the TEM values suggest low within-participant variability. The RHTT was effective in differentiating between individuals physiological responses; supporting a heat tolerance continuum. The findings suggest the RHTT is a repeatable measure of physiological strain in the heat and may be used to assess the effectiveness of acute and chronic heat alleviating procedures.     

A reduced core to skin temperature gradient,not a critical core temperature,affects aerobic capacity in the heat

The purpose of this study was to determine the impact of the core to skin temperature gradient during incremental running to volitional fatigue across varying environmental conditions. A secondary aim was to determine if a “critical” core temperature would dictate volitional fatigue during running in the heat. 60 participants (n=49 male, n=11 female; 24±5 yrs, 177±11 cm, 75±13 kg) completed the study. Participants were uniformly stratified into a specific exercise temperature group (18 °C, 26 °C, 34 °C, or 42 °C) based on a 3-mile run performance. Participants were equipped with core and chest skin temperature sensors and a heart rate monitor, entered an environmental chamber (18 °C, 26 °C, 34 °C, or 42 °C), and rested in the seated position for 10 min before performing a walk/run to volitional exhaustion. Initial treadmill speed was 3.2 km h⁻¹ with a 0% grade. Every 3 min, starting with speed, speed and grade increased in an alternating pattern (speed increased by 0.805 km h⁻¹, grade increased by 0.5%). Time to volitional fatigue was longer for the 18 °C and 26 °C group compared to the 42 °C group, (58.1±9.3 and 62.6±6.5 min vs. 51.3±8.3 min, respectively, p<0.05). At the half-way point and finish, the core to skin gradient for the 18 °C and 26 °C groups was larger compared to 42 °C group (halfway: 2.6±0.7 and 2.0±0.6 vs. 1.3±0.5 for the 18 °C, 26 °C and 42 °C groups, respectively; finish: 3.3±0.7 and 3.5±1.1 vs. 2.1±0.9 for the 26 °C, 34 °C, and 42 °C groups, respectively, p<0.05). Sweat rate was lower in the 18 °C group compared to the 26 °C, 34 °C, and 42 °C groups, 3.6±1.3 vs. 7.2±3.0, 7.1±2.0, and 7.6±1.7 g m⁻² min⁻¹, respectively, p<0.05. There were no group differences in core temperature and heart rate response during the exercise trials. The current data demonstrate a 13% and 22% longer run time to exhaustion for the 18 °C and 26 °C group, respectively, compared to the 42 °C group despite no differences in beginning and ending core temperatures or baseline 3-mile run time. This capacity difference appears to result from a magnified core to skin gradient via an environmental temperature advantageous to convective heat loss, and in part from an increased sweat rate.     

Effect of in vitro zinc supplementation on HSPs expression and Interleukin 10 production in heat treated peripheral blood mononuclear cells of transition Sahiwal and Karan Fries cows

The changing climatic scenario with apprehended rise in global temperature is likely to affect the livestock adversely vis-à-vis production and reproduction. This has prompted more focus in addressing the unfavorable effects of thermal stress in livestock system. Presuming that the trace element zinc is indispensible for cellular antioxidant system and immune function, the present study was designed to investigate the effect of zinc treatment on heat stress alleviation and immune modulation in peripheral blood mononuclear cells (PBMC) of indigenous and crossbred transition cows. Twelve cows, six each of Sahiwal and Karan Fries (KF) in their second parity with confirmed pregnancy were selected for the experiment. The blood samples were collected at −21, 0 and +21 days in relation to expected date of calving. The experiment was carried out in vitro after isolating PBMC from whole blood. The 48 h cultured PBMC were subjected to assorted levels of exposures viz. 37 °C, 42 °C to impose heat stress and 42 °C+zinc to alleviate heat stress and modulate immunity. The PBMC viability was 86%, 69% and 78%, respectively. The mRNA expression of heat shock proteins (HSP 40, 70 and 90α) and Interleukin-10 (IL-10) production varied between the two breeds vis-à-vis days and levels of exposure. The mRNA expression of HSP40 and HSP70 was significantly (P<0.05) higher in Karan Fries than the Sahiwal cows. Both the breeds showed maximum expression of HSP on the day of parturition, more so in KF than Sahiwal. There was a significant (P<0.05) difference in the HSP mRNA expression at different levels of exposure. Zinc treatment to heat stressed PBMC caused a significant (P<0.05) down regulation of HSP. For immune status, anti-inflammatory cytokine, IL-10 in the culture supernatant was accessed. The IL-10 was significantly (P<0.05) higher in Karan Fries (168.18±14.09 pg/ml) than the Sahiwal cows (147.24±11.82 pg/ml). The IL-10 concentration was highest on the day of calving. Zinc treatment reduced the IL-10 concentration. From the study, it could be concluded that the zinc supplementation in heat stressed PBMC can ameliorate thermal stress and modulate immune response which can act as a model for reducing heat stress during the periparturient period in tropical livestock.