The mammary gland is the production organ in mammals that is of great importance for milk production and quality. However, characterization of the buffalo mammary gland transcriptome and identification of the valuable candidate genes that affect milk production is limited. Here, we performed the differential expressed genes (DEGs) analysis of mammary gland tissue on day 7, 50, 140, and 280 after calving and conducted gene-based genome-wide association studies (GWAS) of milk yield in 935 Mediterranean buffaloes. We then employed weighted gene co-expression network analysis (WGCNA) to identify specific modules and hub genes related to milk yield based on gene expression profiles and GWAS data. The results of the DEGs analysis showed that a total of 1,420 DEGs were detected across different lactation points. In the gene-based analysis, 976 genes were found to have genome-wide association (P ≤ 0.05) that could be defined as the nominally significant GWAS geneset (NSGG), 9 of which were suggestively associated with milk yield (P < 10-4). Using the WGCNA analysis, 544 and 225 genes associated with milk yield in the turquoise module were identified from DEGs and NSGG datasets, respectively. Several genes (including BNIPL, TUBA1C, C2CD4B, DCP1B, MAP3K5, PDCD11, SRGAP1, GDPD5, BARX2, SCARA3, CTU2, and RPL27A) were identified and considered as the hub genes because they were involved in multiple pathways related to milk production. Our findings provide an insight into the dynamic characterization of the buffalo mammary gland transcriptome, and these potential candidate genes may be valuable for future functional characterization of the buffalo mammary gland.

Fertility is a complex trait that has a major impact on the development of the buffalo industry. Genome-wide association study (GWAS) has increased the ability to detect genes influencing complex traits, and many important genes related to reproductive traits have been identified in ruminants. However, reproductive traits are influenced by many factors. The development of the follicle is one of the most important internal processes affecting fertility. Genes found by GWAS to be associated with follicular development may directly affect fertility. The present study combined GWAS and RNA-seq of follicular granulosa cells to identify important genes which may affect fertility in the buffalo. The 90 K Affymetrix Axiom Buffalo SNP Array was used to identify the SNPs, genomic regions, and genes that were associated with reproductive traits. A total of 40 suggestive loci (related to 28 genes) were identified to be associated with six reproductive traits (first, second and third calving age, calving interval, the number of services per conception and open days). Interestingly, the mRNA expressions of 25 of these genes were also observed in buffalo follicular granulosa cells. The IGFBP7 gene showed high level of expression during whole antral follicle growth. The knockdown of IGFBP7 in buffalo granulosa cells promoted cell apoptosis and hindered cell proliferation, and increased the production of progesterone and estradiol. Furthermore, a notable signal was detected at 2.3–2.7 Mb on the equivalent of bovine chromosome 5 associated with age at second calving, calving interval, and open days. The genes associated with buffalo reproductive traits in this study may have effect on fertility by regulating of follicular growth. These results may have important implications for improving buffalo breeding programs through application of genomic information.

Granulosa cells (GCs) are essential for follicular growth, development, and atresia. The orexin-A (OXA) neuropeptide is widely involved in the regulation of various biological functions. OXA selectively binds to orexin receptor type 1 (OX1R) and mediates all its biological actions via OX1R. This study aimed to explore the expression of OXA and OX1R and their regulatory role in GCs proliferation, cell cycle progression, apoptosis, oocyte maturation, and underlying molecular mechanisms of these processes and elucidate its novel signaling pathway. Western blotting and RT-qPCR showed that OXA and OX1R were expressed during different developmental stages of GCs, and siRNA transfection successfully inhibited the expression of OX1R at the translational and transcriptional levels. Flow cytometry revealed that OX1R knockdown upregulated GCs apoptosis and triggered S-phase arrest in cell cycle progression. RT-qPCR and Western blotting showed significantly reduced expression of Bcl-2 and elevated expression of Bax, caspase-3, TNF-α, and P21 in OX1R-silenced GCs. Furthermore, the CCK-8 assay showed that knockdown of OX1R suppressed GCs proliferation by downregulating the expression of PCNA, a proliferation marker gene, at the translational and transcriptional levels. Western blotting revealed that knockdown of OX1R resulted in a considerable decrease of the phosphorylation level of the AKT and ERK1/2 proteins, indicating that the AKT/ERK1/2 pathway is involved in regulating GCs proliferation and apoptosis. In addition, OX1R silencing enhanced the mRNA expression of GDF9 and suppressed the mRNA expression of BMP15 in mouse GCs. Collectively, these results reveal a novel regulatory role of OXA in the development of GCs and folliculogenesis by regulating proliferation, apoptosis, and cell cycle progression. Therefore, OXA can be a promising therapeutic agent for female infertility.

Linkage disequilibrium (LD) is a useful parameter for guiding the accuracy and power of both genome-wide association studies (GWAS) and genomic selection (GS) among different livestock species. The present study evaluated the extent of LD, persistence of phase and effective population size (Ne) for the purebred (Mediterranean buffalo; n = 411) and crossbred [Mediterranean × Jianghan × Nili-Ravi buffalo, n = 9; Murrah × Nili-Ravi × local (Xilin or Fuzhong) buffalo, n = 36] buffalo populations using the 90K Buffalo SNP genotyping array. The results showed that the average square of correlation coefficient (r²) between adjacent SNP was 0.13 ± 0.19 across all autosomes for purebred and 0.09 ± 0.13 for crossbred, and the most rapid decline in LD was observed over the first 200 kb. Estimated r² ≥ 0.2 extended up to ~50 kb in crossbred and 170 kb in purebred populations, while average r² values ≥0.3 were respectively observed in the ~10 and 60 kb in the crossbred and purebred populations. The largest phase correlation (R_{P, C} = 0.47) was observed at the distance of 100 kb, suggesting that this phase was not actively preserved between the two populations. Estimated Ne for the purebred and crossbred population at the current generation was 387 and 113 individuals, respectively. These findings may provide useful information to guide the GS and GWAS in buffaloes.

Cathepsin B (CTSB), a lysosomal cysteine protease’s high expression and activity, has been reported to cause poor-quality embryos in porcine and bovine. Nevertheless, CTSB functions in mice granulosa cells remain to explore. To discuss the CTSB functional role in follicular dynamics, we studied apoptosis, proliferation, cell cycle progression, and related signaling pathways in primary mouse granulosa cells transfected with small interference RNA specific to CTSB (siCTSB) for 48 h. Further, mRNA and protein expression of cell proliferation regulators (Myc and cyclin D2), apoptosis regulators (caspase 3, caspase 8, TNF-α, and Bcl2), steroidogenesis-related genes (FSHR and CYP11A1), and autophagy markers (LC3-I and ATG5) were investigated. In addition, the effect of CTSB on steroidogenesis and autophagy was also examined. Flow cytometry analysis assay displayed that silencing of CTSB decreased the early and total apoptosis rate by downregulating TNF-α, caspase 8, and caspase 3, and upregulating Bcl2. By regulating Myc and cyclin D2 expression and activating the p-Akt and p-ERK pathways, CTSB knockdown increased GC proliferation and number. A significant decline in estradiol and progesterone concentrations was observed parallel to a significant decrease in autophagy-related markers LC3-I and ATG5 compared to the control group. Herein, we demonstrated that CTSB serves as a proapoptotic agent and plays a critical role in folliculogenesis in female mice by mediating apoptosis, autophagy, proliferation, and steroidogenesis. Hence, CTSB could be a potential prognostic agent for female infertility.

Deoxynivalenol (DON) is one of the most common feed contaminants, and it poses a serious threat to the health of dairy cows. The existing studies of biological toxicity of DON mainly focus on the proliferation, oxidative stress, and inflammation in bovine mammary epithelial cells, while its toxicity on the biosynthesis of milk components has not been well documented. Hence, we investigated the toxic effects and the underlying mechanism of DON on the bovine mammary alveolar cells (MAC-T). Our results showed that exposure to various concentrations of DON significantly inhibited cell proliferation, induced apoptosis, and altered the cell morphology which was manifested by cell distortion and shrinkage. Moreover, the transepithelial electrical resistance (TEER) values of MAC-T cells exposed to DON were gradually decreased in a time- and concentration- dependent manner, but lactate dehydrogenase (LDH) leakage was significantly increased with the maximum increase of 2.4-fold, indicating the cell membrane and tight junctions were damaged by DON. Importantly, DON significantly reduced the synthesis of β-casein and lipid droplets, along with the significantly decreases of phospho-mTOR, phospho-4EBP1, phospho-JAK2, and phospho-STAT5. Gene expression profiles showed that the expressions of several genes related to lipid synthesis and metabolism were changed, including acyl-CoA synthetase short-chain family member 2 (ACSS2), fatty acid binding protein 3 (FABP3), 3-hydroxy-3-methylglutaryl-CoA synthase 1 (HMGCS1), and insulin-induced gene 1 (INSIG1). GO and KEGG enrichment analyses revealed that the differentially expressed genes (DEGs) were significantly enriched in ribosome, glutathione metabolism, and lipid biosynthetic process, which play important roles in the toxicological process induced by DON. Taken together, DON affects the proliferation and functional differentiation of MAC-T cells, which might be related to the cell junction disruption and morphological alteration. Our data provide new insights into functional differentiation and transcriptomic alterations of MAC-T cells after DON exposure, which contributes to a comprehensive understanding of DON-induced toxicity mechanism.