Identification of novel molecular markers of mastitis caused by Staphylococcus aureus using gene expression profiling in two consecutive generations of Chinese Holstein dairy cattle

Background: Mastitis in dairy cows caused by Staphylococcus aureus is a major problem hindering economic growth in dairy farms worldwide. It is difficult to prevent or eliminate due to its asymptomatic nature and long persistence of infection. Although transcriptomic responses of bovine mammary gland cells to pathogens that cause mastitis have been studied, the common responses of peripheral blood leukocytes to S. aureus infection across two consecutive generations of dairy cattle have not been investigated.Methods: In the current study, RNA-Seq was used to profile the transcriptomes of peripheral blood leukocytes sampled from S. aureus-infected mothers and their S. aureus-infected daughters, and also healthy non-infected mothers and their healthy daughters. Differential gene expression was evaluated as follows: 1) S. aureus-infected cows versus healthy non-infected cows(S vs. H, which include all the mothers and daughters), 2) S. aureus-infected mothers versus healthy non-infected mothers(SM vs. HM), and 3) S. aureus-infected daughters versus healthy noninfected daughters(SMD vs. HMD).Results: Analysis of all identified expressed genes in the four groups(SM, SMD, HM, and HMD) showed that EPOR,IL9, IFNL3, CCL26, IL26 were exclusively expressed in both the HM and HMD groups, and that they were significantly(P < 0.05) enriched for the cytokine-cytokine receptor interaction pathway. A total of 17, 13 and 10 differentially expressed genes(DEGs)(FDR Padj. < 0.1 and |FC| > 1.2) were detected in the three comparisons, respectively. DEGs with P < 0.05 and |FC| > 2 were used for functional enrichment analyses. For the S vs. H comparison, DEGs detected included CCL20, IL13 and MMP3, which are associated with the IL-17 signaling pathway. In the SM vs. HM and SMD vs. HMD comparisons, five(BLA-DQB, C1 R, C2, FCGR1 A, and KRT10) and six(BLA-DQB, C3 AR1, CFI, FCAR, FCGR3 A, and LOC10498484) genes, respectively, were involved in the S. aureus infection pathway.Conclusions: Our study provides insights into the transcriptomic responses of bovine peripheral blood leukocytes across two generations of cattle naturally infected with S. aureus. The genes highlighted in this study could serve as expression biomarkers for mastitis and may also contain sequence variation that can be used for genetic improvement of dairy cattle for resilience to mastitis.

Weighted single-step GWAS and RNA sequencing reveals key candidate genes associated with physiological indicators of heat stress in Holstein cattle

Background: The study of molecular processes regulating heat stress response in dairy cattle is paramount for developing mitigation strategies to improve heat tolerance and animal welfare. Therefore, we aimed to identify quantitative trait loci(QTL) regions associated with three physiological indicators of heat stress response in Holstein cattle, including rectal temperature(RT), respiration rate score(RS), and drooling score(DS). We estimated genetic parameters for all three traits. Subsequently, a weighted single-step genome-wide association study(WssGWAS) was performed based on 3200 genotypes, 151,486 phenotypic records, and 38,101 animals in the pedigree file. The candidate genes located within the identified QTL regions were further investigated through RNA sequencing(RNA-seq) analyses of blood samples for four cows collected in April(non-heat stress group) and four cows collected in July(heat stress group).Results: The heritability estimates for RT, RS, and DS were 0.06, 0.04, and 0.03, respectively. Fourteen, 19, and 20 genomic regions explained 2.94%, 3.74%, and 4.01% of the total additive genetic variance of RT, RS, and DS, respectively. Most of these genomic regions are located in the Bos taurus autosome(BTA) BTA3, BTA6, BTA8, BTA12, BTA14, BTA21, and BTA24. No genomic regions overlapped between the three indicators of heat stress, indicating the polygenic nature of heat tolerance and the complementary mechanisms involved in heat stress response. For the RNA-seq analyses, 2627 genes were significantly upregulated and 369 downregulated in the heat stress group in comparison to the control group. When integrating the WssGWAS, RNA-seq results, and existing literature, the key candidate genes associated with physiological indicators of heat stress in Holstein cattle are: PMAIP1, SBK1, TMEM33, GATB, CHORDC1, RTN4IP1, and BTBD7.Conclusions: Physiological indicators of heat stress are heritable and can be improved through direct selection. Fifty-three QTL regions associated with heat stress indicators confirm the polygenic nature and complex genetic determinism of heat tolerance in dairy cattle. The identified candidate genes will contribute for optimizing genomic evaluation models by assigning higher weights to genetic markers located in these regions as well as to the design of SNP panels containing polymorphisms located within these candidate genes.

Evaluation of heat stress effects on cellular and transcriptional adaptation of bovine granulosa cells

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℃,40℃ and 41℃ along with control samples(38℃)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(E2)and progesterone(P4)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,E2 and P4 levels in bGCs,whereas,increased the cellular apoptosis and ROS.Moreover,the RNA-Seq experiments showed that all the treatments(39℃,40℃ and 41℃)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.