Background: Heat stress is known to affect follicular dynamics, oocyte maturation, and fertilization by impairing steroidogenic ability and viability of bovine granulosa cell (bGCs). The present study explored the physiological and molecular response of bGCs to different heat stress intensities in-vitro. We exposed the primary bGCs to heat stress (HS) at 39°C, 40°C and 41°C along with control samples (38°C) for 2 h. To evaluate the impact of heat stress on bGCs, several in vitro cellular parameters including cell apoptosis, intracellular reactive oxygen species (ROS) accumulation and HSP70 kinetics were assessed by flow cytometry, florescence microscopy and western blot, respectively. Furthermore, the ELISA was performed to confirm the 17β-estradiol (E 2 ) and progesterone (P 4 ) levels. In addition, the RNA sequencing (RNA-Seq) method was used to get the molecular based response of bGCs to different heat treatments. Results: Our findings revealed that the HS significantly decreased the cell viability, E 2 and P 4 levels in bGCs, whereas, increased the cellular apoptosis and ROS. Moreover, the RNA-Seq experiments showed that all the treatments (39°C, 40°C and 41°C) significantly regulated many differentially expressed genes (DEGs) i.e. BCL2L1, STAR, CYP11A1, CASP3, SOD2, HSPA13, and MAPK8IP1 and pathways associated with heat stress, apoptosis, steroidogenesis, and oxidative stress. Conclusively, our data demonstrated that the impact of 40°C treatment was comparatively detrimental for cell viability, apoptosis and ROS accumulation. Notably, a similar trend of gene expression was reported by RT-qPCR for RNA-seq data. Conclusions:Our study presented a worthy strategy for the first time to characterize the cellular and transcriptomic adaptation of bGCs to heat stress (39, 40 and 41°C) in-vitro. The results infer that these genes and pathways reported in present study could be useful candidates/indicators for heat stress research in dairy cattle. Moreover, the established model of bGCs to heat stress in the current study provides an appropriate platform to understand the mechanism of how heat-stressed bGCs can affect the quality of oocytes and developing embryo.

The JAK-STAT pathway plays a key role in host immunity. The present study was designed to evaluate the effects of single nucleotide polymorphisms (SNPs) in STAT5A and JAK2 genes on some serum cytokines, mastitis and milk production traits. Two SNPs (SNP1 43046497A/C and SNP2 43047829G/A) in STAT5A, and four SNPs in JAK2 (SNP3 39652267A/G, SNP4 39630048C/T, SNP5 39631044G/A, and SNP6 39631175T/C) were revealed and genotyped in 268 Chinese Holstein cattle. Fixed model was used to analyze the association of SNPs with phenotypes by general linear model procedure of SAS 9.1. SNP1 and SNP4 were significantly associated with IL-6 and IL-17 (P < 0.05), respectively. In JAK2 gene, SNP3 was highly significant (P < 0.01) and SNP5 was significant (P < 0.05) in association with SCC, whereas, the association of SNP6 was found significant (P < 0.05) with both SCC and SCS. Combination genotype analysis revealed that SNPs in JAK2 gene significantly associated with SCC and SCS were associated significantly with the corresponding phenotypes in combinations as well. The GG genotype of SNP3 individually and in any combination genotypes showed lowest SCC. The dominant effect of SNP1, SNP5 and SNP6 was found highly significant (P < 0.01) on the corresponding phenotypes (IL-6, SCC and SCS). As for haplotype analysis, two haplotypes were revealed between the two SNPs of STAT5A gene and four haplotypes amongst four SNPs in JAK2 gene; strong linkage disequilibrium (D' > 0.9) was observed between all these haplotypes. The results imply that the identified SNPs could be powerful markers to select dairy cattle with improved genetic resistance against mastitis.

Bovine brucellosis remains a persistent infection in ruminants in Pakistan. A total of 828 (409 buffaloes and 419 cattle) sera were collected from 11 institutional-owned livestock farms in Punjab, Pakistan. The samples were tested by rose bengal plate agglutination test (RBPT) and indirect enzyme-linked immunosorbent assay (iELISA). The seroprevalence along with 95% confidence interval (CI) was determined. Univariable and multivariable analysis of the epidemiological background data was conducted and odds ratio (OR) was calculated to understand any association between the risk factors and the seroprevalence. An overall seroprevalence of 3.9% (Positive/Tested = 32/828) and 3.3% (27/828) was detected by RBPT and iELISA, respectively. The seroprevalence of 5.6% (CI 3.6–8.3) and 4.7%, (CI 2.8–7.2) and the odds ratio of 2.63 (CI 1.20–5.77) and 2.50 (CI 1.08–5.78) for testing positive by RBPT and iELISA, respectively were significantly higher (p < 0.05) in buffaloes than in cattle. Breed, sex, history of abortion and retention of fetal membranes (RFM) in the animals were not found statistically significantly associated with the infection. RBPT and iELISA based results agreed almost perfect (k = 0.877). In total, Brucella abortus-DNA (9/27) was amplified from seropositive samples by real-time polymerase chain reaction. This study identified for the first time the etiological agents of brucellosis at a molecular level at institutional-owned livestock farms in Pakistan.

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a zoonotic pathogen capable of causing severe respiratory disease in humans. Although dromedary camels are considered as a major reservoir host, the MERS-CoV infection dynamics in camels are not fully understood. Through surveillance in Pakistan, nasal (n = 776) and serum (n = 1050) samples were collected from camels between November 2015 and February 2018. Samples were collected from animal markets, free-roaming herds and abattoirs. An in-house ELISA was developed to detect IgG against MERS-CoV. A total of 794 camels were found seropositive for MERS-CoV. Prevalence increased with the age and the highest seroprevalence was recorded in camels aged [ 10 years (81.37%) followed by those aged 3.1-10 years (78.65%) and B 3 years (58.19%). Higher prevalence was observed in female (78.13%) as compared to male (70.70%). Of the camel nasal swabs, 22 were found to be positive by RT-qPCR though with high Ct values. Moreover, 2,409 human serum samples were also collected from four provinces of Pakistan during 2016-2017. Among the sampled population, 840 humans were camel herders. Although we found a high rate of MERS-CoV antibody positive dromedaries (75.62%) in Pakistan, no neutralizing antibodies were detected in humans with and without contact to camels.

The JAK-STAT pathway plays a key role in mediating immune responses. The genetic effects of single nucleotide polymorphisms (SNPs) in JAK2 and STAT5B were investigated for serum cytokines, mastitis indicators and productions traits in a population of 468 Chinese Holstein cattle. Pooled DNA sequencing revealed one SNP (BTA8:g.39645396A>G) in JAK2 and two SNPs (BTA19:g.43673888A>G and BTA19:g.43660093T>C) in STAT5B. A fixed effect model considering the effects of SNPs, parity, herd, season and year of calving was used by way of the general linear model procedure of sas. Genotype frequencies of these SNPs in the population were in Hardy-Weinberg equilibrium (P > 0.05). A novel SNP (g.39645396A>G) in JAK2 was predicted to change the amino acid from lysine to asparagine and was significantly associated with the somatic cell count (SCC) and somatic cell score (SCS), whereas g.43673888A>G in STAT5B was significantly associated with SCC, SCS and interleukin-4 (IL-4) (P < 0.05). The dominant effect of g.39645396A>G in JAK2 was significant for SCS, and its additive effect was significant for SCC, whereas the dominant effect of g.43673888A>G in STAT5B was significant for SCS and IL-4 (P < 0.05). The combination of g.39645396A>G in JAK2 and g.43673888A>G in STAT5B showed a significant effect on SCC, SCS, IL-4 and TNF-α (P < 0.05). As for mRNA expression analysis, the AA genotype g.39645396A>G and GG genotype g.43673888A>G indicated higher mRNA expression level and were significantly different from other genotypes (P < 0.05). The results imply that JAK2 and STAT5B genes could be useful candidate genes, and the identified polymorphisms might potentially be strong genetic markers for selection of dairy cattle against mastitis development.