Collagens, as extracellular matrix proteins, support cells for structural integrity and contribute to support mammary basic structure and development. This study aims to perform the genomic identification, evolution, and expression analyses of the collagen gene family in water buffalo (Bubalus bubalis) during lactation. A total of 128 buffalo collagen protein sequences were deduced from the 45 collagen genes identified in silico from buffalo genome, which classified into six groups based on their phylogenetic relationships, conserved motifs, and gene structure analyses. The identified collagen sequences were unequally distributed on 16 chromosomes. The tandem duplicated genes were found within three chromosomes, while only one segmental event occurred between Chr3 and Chr8. Collinearity analysis revealed that a total of 36 collagen gene pairs were orthologous between buffalo and cattle genomes despite having different chromosome numbers. Comparative transcription analyses revealed that a total of 23 orthologous collagen genes were detected in the milk samples at different lactation periods between the two species. Notably, the duplicated gene pair of COL4A1-COL4A2 during lactation had a higher mRNA expression level than that of cattle, while a higher expression level of COL6A1-COL6A2 pair was found in cattle compared with that of buffalo. The present study provides useful information for investigating the potential functions of the collagen family in buffalo during lactation and helps in the functional characterization of collagen genes in additional research.

Identifying key causal genes is critical for unraveling the genetic basis of complex economic traits, yet it remains a formidable challenge. The advent of large-scale sequencing data and computational algorithms, such as transcriptome-wide association studies (TWASs), offers a promising avenue for identifying potential causal genes. In this study, we harnessed the power of TWAS to identify genes potentially responsible for milk production traits, including daily milk yield (MY), fat percentage (FP), and protein percentage (PP), within a cohort of 100 buffaloes. Our approach began by generating the genotype and expression profiles for these 100 buffaloes through whole-genome resequencing and RNA sequencing, respectively. Through comprehensive genome-wide association studies (GWAS), we pinpointed a total of seven and four single nucleotide polymorphisms (SNPs) significantly associated with MY and FP traits, respectively. By using TWAS, we identified 55, 71, and 101 genes as significant signals for MY, FP, and PP traits, respectively. To delve deeper, we conducted protein–protein interaction (PPI) analysis, revealing the categorization of these genes into distinct PPI networks. Interestingly, several TWAS-identified genes within the PPI network played a vital role in milk performance. These findings open new avenues for identifying potentially causal genes underlying important traits, thereby offering invaluable insights for genomics and breeding in buffalo populations.

Acylglycerophosphate acyltransferases (AGPATs) are the rate-limiting enzymes for the de novo pathway of triacylglycerols (TAG) synthesis. Although AGPATs have been extensively explored by evolution, expression and functional studies, little is known on functional characterization of how many members of the AGPAT family are involved in TAG synthesis and their impact on the cell proliferation and apoptosis. Here, 13 AGPAT genes in buffalo were identified, of which 12 AGPAT gene pairs were orthologous between buffalo and cattle. Comparative transcriptomic analysis and real-time quantitative reverse transcription PCR (qRT-PCR) further showed that both AGPAT1 and AGPAT6 were highly expressed in milk samples of buffalo and cattle during lactation. Knockdown of AGPAT1 or AGPAT6 significantly decreased the TAG content of buffalo mammary epithelial cells (BuMECs) and bovine mammary epithelial cells (BoMECs) by regulating lipogenic gene expression (p < 0.05). Knockdown of AGPAT1 or AGPAT6 inhibited proliferation and apoptosis of BuMECs through the expression of marker genes associated with the proliferation and apoptosis (p < 0.05). Our data confirmed that both AGPAT1 and AGPAT6 could regulate TAG synthesis and growth of mammary epithelial cells in buffalo. These findings will have important implications for understanding the role of the AGPAT gene in buffalo milk performance.

Background: While economically vital, buffalo exhibits low reproductive efficiency largely due to embryonic losses during implantation. Successful implantation requires precise embryo–maternal communication and metabolic/immune adaptations in the endometrium. We aimed to identify key serum metabolic signatures and associated peripheral immune responses that characterize the endometrial receptivity window during early pregnancy in water buffalo. Methods: Blood samples from pregnant (Preg, n = 12) and non-pregnant (Non-P, n = 10) buffaloes were collected on days 15, 18, and 21 post-artificial insemination (AI). We measured leukocyte counts and hormone levels and performed untargeted serum metabolomic profiling using LC-MS. Results: Pregnant buffaloes showed significantly reduced total white blood cell count, lymphocyte (LYM%), and neutrophil (NEU%) percentages, indicating immune remodeling at the beginning of pregnancy establishment. Metabolomic analysis identified 131 differentially expressed metabolites (DEMs) associated with pregnancy status at different stages. Enriched pathways included steroid hormone synthesis, retinol metabolism, starch/sucrose metabolism, and phenylalanine biosynthesis. Crucially, alterations in unsaturated fatty acids, retinol, and phenylalanine metabolism, along with monocyte (MON%)/LYM% ratios, were strongly linked to receptivity changes and successful implantation. Conclusions: Endometrial receptivity in buffalo during the embryonic implantation window was associated with changes in immune cells and metabolism in the blood, suggesting that immunometabolism may play an essential role in modulating endometrial receptivity during the implantation window. This study provides potential clues and a metabolic framework for understanding the underlying mechanisms of buffalo embryonic implantation.