Differential effect of thermal stress on HSP70 expression, nitric oxide production and cell proliferation among native and crossbred dairy cattle.

In a tropical country like India, thermal stress is one of the major factors which significantly affects the productivity of dairy cattle. The present study was aimed to identify the effect of heat and cold stress on cell viability, mitogen stimulation indices, nitric oxide production and HSP70 expression in Sahiwal and Holstein crossbred (Frieswal) population in India. The results indicated that the Sahiwal breed can better withstand the effect of heat and cold stress significantly (P<0.05) when compared to the crossbred cattle due to the higher survivability of the Peripheral Blood Mononuclear Cells (PBMCs) and Phytohemagglutinin (PHA-P) mitogen based stimulation indices. The study also revealed the significant differences (P<0.05) in the level of nitric oxide (µM) production amongst the pre and post thermal stressed samples of Sahiwal and Frieswal crossbred samples. Further, the expression of HSP70 was significantly (P<0.05) higher in Sahiwal compared to Frieswal immediately after heat/cold shock to 6h of recovery as indirect ELISA analysis showed gradual rise in the Hsp70 protein concentration (ng/ml) immediately after heat and cold stress (0h) and reached the peak at 6h of recovery. Western blot and immune fluorescent assay results were also corroborated with the findings of indirect ELISA. In Sahiwal cattle the mRNA expression of HSP70 and its protein concentration were higher (P<0.05) during peak summer (44°C) and winter (10°C) as compared to Frieswal cattle. This investigation supports the earlier information on the higher adaptability of indigenous cattle breeds to hot and humid conditions compared to the crossbreds of temperate cattle breeds.

Bhanuprakash V ，Singh U ，Sengar G ，Sajjanar B ，Bhusan B ，Raja TV ，Alex R ，Kumar S ，Singh R ，Ashish Kumar ，Alyethodi RR ，Kumar S ，Deb R ... -  《JOURNAL OF THERMAL BIOLOGY》

Effect of heat stress on the expression profile of Hsp90 among Sahiwal (Bos indicus) and Frieswal (Bos indicus × Bos taurus) breed of cattle: a comparative study.

We evaluated the effect of thermal challenge on the expression profile of heat shock protein 90 (Hsp90) among Sahiwal (Bos indicus) and Frieswal (Bos indicus × Bos taurus) breeds of cattle. The present investigation was focused on the comparative studies on Hsp90 expression among Frieswal and Sahiwal under in vitro and environmental heat stress. Measured immediately after the in vitro heat shock to the peripheral blood mononuclear cells (PBMCs), the relative expression of Hsp90 mRNA was significantly (P<0.05) higher in Sahiwal compared to those in Frieswal. In later intervals of time, the differences in the expression levels between the two breeds become negligible coming down towards the basal level. A similar pattern was observed in the protein concentration showing significantly (P<0.05) higher levels in Sahiwal compared to those in Frieswal. The second sets of experiments were undertaken during summer months (March to May) when temperature peaked from 37 to 45 °C. During these months, Frieswal cows consistently recorded higher rectal temperatures than the Sahiwal breed. Further during this peak summer stress, Sahiwal showed significantly higher levels of mRNA transcripts as well as protein concentration compared to the Frieswal breed. Our findings also interestingly showed that, the cell viability of PBMC are significantly higher among the Sahiwal than Frieswal. Taken together, the experiments of both induced in vitro and environmental stress conditions indicate that, Sahiwal may express higher levels of Hsp90 then Frieswal to regulate their body temperature and increase cell survivality under heat stressed conditions.

Deb R ，Sajjanar B ，Singh U ，Kumar S ，Singh R ，Sengar G ，Sharma A ... -  《-》

Expression pattern of bta-mir-2898 miRNA and their correlation with heat shock proteins during summer heat stress among native vs crossbred cattle.

Earlier studies identified the role of bta-mir-2898 in bovine. Our earlier study identified that, bta-mir-2898 can be over expressed in crossbred cattle during heat stress. Nevertheless the differential expression of bta-mir-2898 among native vs crossbred cattle during summer stress along with it's correlation with different heat shock proteins (HSPs) is not yet studied. In the present context, we studied the differential expression of bta-mir-2898 among Frieswal (Bos indicus x Bos taurus) and Sahiwal (Bos indicus) breeds of cattle during a range of environmental air temperatures and further investigated the correlation of bta-mir-2898 with different HSPs (HSP70, HSP90, HSP60. HSF, HSPB8 and HSP27). It was observed that, at peak air temperature the relative miRNA expression level (p < 0.05) of bta-mir-2898 was 3.4 ± 0.41 and 0.79 ± 0.22 among Frieswal and Sahiwal, respectively. We also observed significant levels (p < 0.05) of mRNA abundance of HSP70, HSP90, HSPB8 and HSP27 among the breeds. In all the cases Sahiwal found to exhibited higher level of HSPs in comparison to Frieswal. Studies revealed that the expression profile of bta-mir-2898 was negatively correlated with the expression of all the HSPs during thermal stress in post anti-mir2898 treated PBMC invitro cultured model originated from both Frieswal and Sahiwal cattle breeds. However, significantly (p < 0.05) higher negative correlations were observed between bta-mir-2898 and HSP70, HSP60 and HSPB8. Present findings highlighted the preliminary role of overexpressed bta-mir-2898 in cattle during thermal stress and its impact on different heat shock proteins.

Deb R ，Sengar GS 《JOURNAL OF THERMAL BIOLOGY》

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 48h 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±14.09pg/ml) than the Sahiwal cows (147.24±11.82pg/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.

Sheikh AA ，Aggarwal A ，Aarif O 《JOURNAL OF THERMAL BIOLOGY》

Comparative CpG methylation kinetic patterns of cis-regulatory regions of heat stress-related genes in Sahiwal and Frieswal cattle upon persistent heat stress.

The kinetic patterns of CpG methylation of the cis-regulatory region of heat stress-related genes on exposed to heat stress (at 42 °C) between the Sahiwal and Frieswal cattle was compared in the present study. Using an in vitro whole blood culture model, cells were continuously exposed to heat stress (at 42 °C) for 6 h. Methylation levels of five genes, viz., GPX1, HSP70, HSP90, c-FOS, and JUN were estimated by SyberGreen-based quantitative methylation-specific PCR (qMSP) assay. CpG methylation kinetics at different time points of heat stress (0.5, 1, 2, 4, 6 h) were analyzed using mixed ANOVA. The initial methylation level, estimated at 37 °C, of HSP70 was significantly high in the Sahiwal breed. A significant (p<0.001) time-dependent hypomethylation of an antioxidant gene (GPX1) CpG islands was detected at the acute phase of the stress. Heat shock protein gene (HSP70) showed a similar CpG methylation kinetics where the hypomethylation was prominent from 1 h and persisted up to 4 h. The heat stress responses of both Sahiwal and Frieswal cattle were identical as there was no distinctiveness in the methylation kinetics of CpG islands of studied genes. The acclimatization of Frieswal cattle-a breed developed in India over the years to the tropical climatic conditions, maybe one of the reasons for this similarity. Thus, the present study results could pave a path to understand the molecular mechanism of heat stress and adaptation of indigenous and crossbred cattle populations to the changing scenario in tropical climate conditions.

Verma N ，Alyethodi RR ，Kathuria A ，Alex R ，Hussain S ，Singh U ，Tyagi S ，Sirohi AS ，Kumar S ，Sengar GS ，Raja TV ，Prakash B ... -  《-》