Chunyan Yang

Buffalo milk plays a vital role in the dairy industry, with milk yield regulated by both transcriptomic and epigenetic mechanisms. While previous studies have primarily focused on differences among individuals or breeds, the epigenetic basis underlying milk yield variation in genetically identical animals remains poorly understood. In this study, we employed a cloned buffalo model and integrated whole-genome bisulfite sequencing (WGBS) with RNA sequencing (RNA-seq) to investigate how DNA methylation and transcriptional regulation contribute to milk yield variation. Results tentatively revealed that low-yielding buffalo exhibited globally reduced DNA methylation in mammary tissues, with distinct distribution patterns across genomic features and regulatory regions. Differentially methylated genes were enriched in PI3K-Akt, HIF-1, and immune-related pathways, whereas hypomethylated genes were associated with calcium signaling, cAMP pathways, and metabolic processes. Transcriptome analysis showed that high-yielding buffalo upregulated genes involved in lipid metabolism and cell proliferation, while low-yielding buffalo displayed enrichment in immune stress and amino acid metabolism. Integrative analysis identified 126 hypo-upregulated genes and highlighted hub regulators such as KLF6, NR4A1, ESR1, KCNQ1. Collectively, this study outlines a preliminary multi-omics regulatory landscape of milk yield variation in cloned buffalo, suggests the interplay between DNA methylation and transcription, provides preliminary insights into the potential interplay between DNA methylation and transcription, and suggests potential connections that merit further investigation.

Hypoxia benefits the proliferation and maintenance of animal spermatogonial cells; however, the underlying mechanism remains incompletely understood. This study aims to investigate the role and mechanism of the hypoxia–glycolysis–histone lactylation axis in the proliferation of buffalo spermatogonial cells (bSCs). bSCs were cultured under different oxygen concentrations to observe changes in cell proliferation. RNA-seq was used to analyze gene expression and signaling pathways. Changes in lactylation were monitored, and CUT&Tag-seq was utilized to determine the regulatory effects of lactylation on gene expression. The glycolytic pathway was regulated to validate the results of the bioinformatic analysis. Oxygen concentrations between 2.5% and 10% support the proliferation of bSCs, with 5% having the most pronounced effect. An amount of 5% oxygen significantly increased the proliferation and pluripotency of bSCs while also promoting glycolysis and lactylation. Inhibition of glycolysis eliminated the proliferative effects of hypoxia. By analyzing genes associated with the key lactylation site H3K18la using CUT&Tag technology, we found that it is closely linked to genes involved in the regulation of proliferation. After inhibition of HK-2 expression, cell proliferation, H3K18la expression, and the expression of these target genes were all suppressed. Hypoxia promotes the proliferation of bSCs via activation of glycolysis, leading to an increase in H3K18la and altered expression of its target genes.

Background: The reproductive performance of water buffalo significantly impacts the economic aspects of production. Traditional breeding methods are constrained by low heritability and numerous influencing factors, making it difficult to effectively improve reproductive efficiency. Genome-wide association studies (GWAS) offer new possibilities for exploring reproductive traits in water buffalo, opening up new avenues for efficient breeding. Methods: Using whole-genome resequencing, we identified quantitative trait loci (QTLs) associated with four suggestive reproductive traits: calving interval (CI), calf birth weight (CBW), dam birth weight (BW), and age at first calving (FCA). The study focused on identifying genetic variants that influence these reproductive traits. Results: Our research identified 52 suggestive regulatory loci associated with reproductive traits in water buffalo. Based on a 50 kb interval, we annotated these loci to 58 candidate genes. These loci involve genes such as AGBL4, GRM1, NCKAP5, and NRXN1, which are primarily enriched in pathways including the FOXO signaling pathway, calcium ion pathways, estrogen signaling pathway, and phospholipase D signaling pathway. These pathways directly or indirectly regulate the reproductive efficiency of water buffalo. Conclusions: This study has revealed suggestive regulatory genes (AGBL4, GRM1, NCKAP5, NRXN1) associated with reproductive traits in water buffalo. This not only enhances our understanding of the molecular mechanisms underlying complex traits but also points towards strategies for improving the reproductive capacity of water buffalo. These findings provide a solid foundation for future breeding programs aimed at enhancing water buffalo productivity.

Background: Water buffaloes represent a crucial genetic resource for the global dairy industry, yet enhancements in their production performance remain relatively constrained. The advent of advanced sequencing technologies, coupled with genome-wide association studies (GWASs), has significantly boosted the potential for breeding superior-quality water buffalo. Methods: An integrated genomic analysis was performed on sequencing data from 100 water buffaloes, utilizing the high-quality UOA_WB_1 genome assembly as a reference. This study particularly emphasized reproduction-related traits, with a focus on age at first calving (AFC). Results: Our analysis revealed two significant single-nucleotide polymorphisms (SNPs). Based on these genetic markers, the GRM1 gene was identified as a candidate gene. This gene shows substantial involvement in various reproduction-associated pathways, including the FOXO signaling pathway, calcium signaling pathway, and estrogen signaling pathway. Conclusions: The identification of GRM1 as a candidate gene provides a robust theoretical basis for molecular breeding strategies aimed at enhancing fertility in water buffaloes. These findings offer critical scientific support for optimizing breeding programs, thereby improving overall production efficiency.

Background: Buffaloes are considered an indispensable genetic resource for dairy production. However, improvements in lactation performance have been relatively limited. Advances in sequencing technology, combined with genome-wide association studies, have facilitated the breeding of high-quality buffalo. Methods: We conducted an integrated analysis of genomic sequencing data from 120 water buffalo, the high-quality water buffalo genome assembly designated as UOA_WB_1, and milk production traits, including 305-day milk yield (MY), peak milk yield (PM), total protein yield (PY), protein percentage (PP), fat percentage (FP), and total milk fat yield (FY). Results: The results identified 56 significant SNPs, and based on these markers, 54 candidate genes were selected. These candidate genes were significantly enriched in lactation-related pathways, such as the cAMP signaling pathway (ABCC4), TGF-β signaling pathway (LEFTY2), Wnt signaling pathway (CAMK2D), and metabolic pathways (DGAT1). Conclusions: These candidate genes (e.g., ABCC4, LEFTY2, CAMK2D, DGAT1) provide a substantial theoretical foundation for molecular breeding to enhance milk production in buffaloes.

Buffalo is a globally important livestock species, but its reproductive performance is relatively low than cattles. At present, dominant follicle development specific process and mechanistic role of follicular growth related genes in water buffaloes are not well understood. Therefore, we comprehensively performed transcriptomics of granulosa cells and oocytes from different-sized follicles in water buffalo to identify key candidate genes that influence follicle development and diameter, and further explored the potential regulatory mechanisms of granulosa cells and oocytes in the process of water buffalo follicle development. In this study, we found918 granulosa cell transcripts and 1401 oocyte transcripts were correlated in follicles of different diameters, and the expression differences were significant. Subsequent enrichment analysis of the co-expressed differentially expressed transcripts identified several genes targeted by long non-coding RNAs (lncRNAs) and associated with follicular development. Notably, the upregulation of BUB1 regulated by MSTRG.41325.4 and interactive action of SMAD2 and SMAD7 might have key regulatory role in follicular development. Additionally, we also detected key differentially expressed genes that potentially influence follicular hormone metabolism and growth, like ID2, CHRD, TGIF2 and MAD2L1, and constructed an interaction network between lncRNA transcripts and mRNAs. In summary, this study preliminarily revealed the differences in gene expression patterns among buffalo follicles of different sizes and their potential molecular regulatory mechanisms. It provides a new perspective for exploring the mechanism of buffalo follicular dominance and improving buffalo reproductive performance.

Atresia is a process in ovarian follicles that is regulated by hormone-induced apoptosis. During atresia, granulosa cell (GC) apoptosis is a key mechanism orchestrated through diverse signaling pathways. Cocaine- and amphetamine-regulated transcript (CART) signaling within ovarian GCs has been demonstrated to play a key role in the regulation of follicular atresia in cattle, pigs, and sheep. The present work aimed to investigate the potential local regulatory role of CART in GC apoptosis-induced follicular atresia in buffalo, focusing on the modulation of the AKT/GSK3β/β-catenin signaling pathways, which are the intracellular signaling pathways involved in cell viability. Our findings revealed increased expression of CARTPT and BAX and decreased levels of AKT, β-catenin, and CYP19A1 genes in atretic follicles compared to healthy follicles. Subsequently, CART treatment in the presence of FSH inhibited the FSH-induced increase in GC viability by reducing estradiol production and increasing apoptosis. This change was accompanied by an increase in the gene expression levels of both CARTPT and BAX. At the protein level, treatment with CART in the presence of FSH negatively affected the activity of AKT, β-catenin, and LEF1, while the activity of GSK3β was enhanced. In conclusion, our study shows how CART negatively influences buffalo GC viability, underlying the modulation of the AKT/GSK3β/β-catenin pathway and promoting apoptosis—a key factor in follicular atresia.

Background: Atresia, a prevailing phenomenon within the majority of ovarian follicles, is a process regulated by hormone-induced apoptosis and influenced by changes in both endocrine and paracrine factors. Amongthese alterations, granulosa cell (GC) apoptosis is a key mechanism orchestrated through diverse signaling pathways. Cocaine- and amphetamine-regulated transcript (CART) signaling within ovarian GCs has been demonstrated to play a key role in the regulation of follicular atresia in cattle, pigs and sheep. In the present study, we aimed to investigate the potential local regulatory role of CART in the process of GCapoptosis-induced follicular atresia in buffalo, focusing on the modulation of the AKT/GSK3β/β-catenin pathways, the intracellular signaling pathways involved in cell viability. To address our objective, we firstinvestigated the association between CART gene (CARTPT) expression levels and follicular atresia by conducting gene expression analyses for CARTPT and related genes in both healthy and atretic follicles. Next, we investigated the influence of CART supplementation, with or without FSH, on GCestradiol production and apoptosis, exploring its potential regulatory role in the AKT/GSK3β/β-catenin pathways. Results: Our findings revealed increased expression of the CARTPT and BAX genes in atretic follicles, accompanied by decreased levels of AKT, β-catenin, and CYP19A1, compared to those in healthy follicles. Subsequently, CART treatment in the presence of FSH potently inhibited the FSH-induced rise in GC viability by reducing estradiol production and increasing apoptosis. This change was accompanied by an increase in the gene expression levels of both CARTPT and BAX, while the expression levels of AKT and CYP19A1 weresignificantly decreased. Moreover, at the protein level, treatment with CART in the presence of FSH negatively affects the activity of AKT, β-catenin and LEF1, while the activity of GSK3β was enhanced. Conclusion: In conclusion, our study illuminates how CART negatively influences buffalo GC viability by affecting estradiol production and promoting apoptosis—a key factor in follicular atresia. This regulatory mechanism involves modulation of the AKT/GSK3β/β-catenin pathway, providing valuable insights into the intricate mechanisms governing ovarian follicle development and granulosa cell function. These findings have implications for reproductive biology not only for buffalo but also for different species.

This work aimed to investigate the effect of fucoidan (FPS) on urate transporters induced by uric acid (UA). The results showed that UA stimulated the expression of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) in HK-2 cells, and FPS could reverse the effect. Moreover, UA could activate NF-κB, JNK and PI3K/Akt pathways, but both pathway inhibitors and FPS inhibited the UA-induced activation of these three pathways. These data suggested that FPS effectively inhibited the expression induction of reabsorption transporters URAT1 and GLUT9 by UA, through repressing the activation of NF-κB, JNK and PI3K/Akt signal pathways in HK-2 cells. The in vitro research findings support the in vivo results that FPS reduces serum uric acid content in hyperuricemia mice and rats through inhibiting the expression of URAT1 and GLUT9 in renal tubular epithelial cells. This study provides a theoretical basis for the application of FPS in the treatment of hyperuricemia.