Testis is the primary organ of the male reproductive tract in mammals that plays a substantial role in spermatogenesis. Improvement of our knowledge regarding the molecular mechanisms in testicular development and spermatogenesis will be reflected in producing spermatozoa of superior fertility. Evidence showed that N6-Methyladenosine (m6A) plays a dynamic role in post-transcription gene expression regulation and is strongly associated with production traits. However, the role of m6A in bovine testis has not been investigated yet. In this study, we conducted MeRIP-Seq analysis to explore the expression profiles of the m6A and its potential mechanism underlying spermatogenesis in nine bovine testes at three developmental stages (prepuberty, puberty and postpuberty). The experimental animals with triplicate in each stage were chosen based on their semen volume and sperm motility except for the prepuberty bulls and used for testes collection. By applying MeRIP-Seq analysis, a total of 8,774 m6A peaks and 6,206 m6A genes among the studied groups were identified. All the detected peaks were found to be mainly enriched in the coding region and 3′- untranslated regions. The cross-analysis of m6A and mRNA expression exhibited 502 genes with concomitant changes in the mRNA expression and m6A modification. Notably, 30 candidate genes were located in the largest network of protein-protein interactions. Interestingly, four key node genes (PLK4, PTEN, EGR1, and PSME4) were associated with the regulation of mammal testis development and spermatogenesis. This study is the first to present a map of RNA m6A modification in bovine testes at distinct ages, and provides new insights into m6A topology and related molecular mechanisms underlying bovine spermatogenesis, and establishes a basis for further studies on spermatogenesis in mammals.

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Background: Heat stress is a key issue of growing concern for livestock industry worldwide due to its negative effects not only on milk production, fertility, health, welfare, and economic returns of dairy cows, but also on the microbial communities in the rumen. However, the underlying relationship between rumen microbiome and its associated metabolism with heat tolerance in cow have not been extensively described yet. Therefore, the main objective of this study was to investigate differential heat resistance in Holstein cows using rumen microbiome and metabolome analyses.Methods: We performed both principal component analysis and membership function analysis to select 7 heat-tolerant (HT) and 7 heat-sensitive (HS) cows. The ruminal fluid samples of two groups were collected at two hours post feeding on 7th day of heat stress period, for analyses including rumen fermentation parameters, rumen microbiome and nontargeted metabolomics.Results: Under heat stress conditions, the HT cows had a significantly higher propionic acid content than the HS cows; whereas measures of the respiratory rate (RR), rectal temperature (RT), acetic,butyric acid and acetic acid to propionic acid ratio (A:P) in the HT cows were lower compared with the HS cows. Omics sequencing revealed that the relative abundance of Rikenellaceae_RC9_gut_group, Succiniclasticum, Ruminococcaceae_NK4A214_group and Christensenellaceae_R-7_group were significantly higher in the HT than HS cows; whereas Prevotella_1, Ruminococcaceae_UCG-014, and Shuttleworthia were significantly higher in the HS cows compared to HT cows. Substances mainly involved in carbohydrate metabolism, including glycerol, mannitol, and maltose, showed significantly higher content in the HT cows compared to that in the HS cows. Simultaneously, RR was significantly correlated with both differential microorganisms and distinct metabolites, suggesting three metabolites could be potential biomarkers for determining heat resistance that require further research.Conclusion: Overall, distinct changes in the rumen microbiome and metabolomics in the HT cows may be associated with better adaptability to heat stress. These findings suggest their use as diagnostic tools of heat tolerance in dairy cattle breeding schemes.