Buffalo milk is nutritionally rich but vulnerable to adulteration, posing challenges to food safety. This study analyzes buffalo milk, soybean milk, Holstein milk, and five common additives-ammonium chloride, urea, sodium bicarbonate, sodium citrate, and sucrose-using Raman spectroscopy. Six spectral preprocessing methods were systematically evaluated to enhance model performance. For qualitative detection, PLS-DA models combined with Multiplicative Scatter Correction (MSC) preprocessing achieved excellent classification accuracy (up to 100 %) for pure buffalo milk, water, and soybean milk adulteration. For quantitative analysis, both PLS and MSC-CNN regression models were developed. The MSC-CNN model achieved high predictive performance for sodium bicarbonate (R-2 = 0.97) and sodium citrate (R-2 = 0.93), with RMSEP <5 % of full scale. Detection limits were as low as 17.4 mg/kg for sodium bicarbonate and 20.9 mg/kg for sodium citrate, meeting practical sensitivity requirements. Compared with existing PLS-based methods, our approach improved predictive accuracy and expanded low-concentration detection. The proposed Raman-deep learning strategy offers a rapid, accurate, and non-destructive solution for milk adulteration monitoring and quality control.

Spermatogonial stem cells (SSCs) serve as the foundation of spermatogenesis and play a critical role in livestock fertility and genetic improvement. However, maintaining these cells in long-term culture remains challenging, especially in pigs, where preserving both proliferative capacity and the undifferentiated state has proven difficult. To overcome this limitation, we investigated SB216763 (SB), a selective GSK-3β inhibitor reported to enhance proliferation in other stem cell types, for its potential effects on porcine spermatogonial cells (pSCs). In a 9-day concentration screening (0, 1.25, 2.5, and 5 μM SB), SB treatment significantly enhanced the proliferation of pSCs. It upregulated the proliferation-related gene PCNA and the antiapoptotic gene BCL2, while downregulating key apoptosis-related genes, including BAX and Caspase-3. It also promoted the expression of DAZL, UCHL1, NANOS2, and OCT4. Furthermore, over a 55-day period, treatment with 2.5 μM SB consistently supported higher proliferation rates, a larger proportion of DAZL-positive cells, and elevated expression of undifferentiation-associated markers (NANOS2, OCT4) compared with untreated controls at each passage. Overall, our results demonstrate that SB216763 not only promotes the proliferation of pSCs but also helps maintain their germline identity, offering a feasible strategy to improve the long-term in vitro culture of porcine spermatogonial cells.

The content of unsaturated fatty acids (UFAs) is significantly associated with the flavor, taste, and nutritional value of beef. Adipose tissue (AT) develops in multiple depots, and the UFA content of AT varies among different depots. To elucidate the regulatory role of long non-coding RNAs (lncRNAs) in UFA synthesis across different AT depots in buffaloes, we conducted RNA sequencing (RNA-seq) on AT samples from six distinct depots. A total of 8926 lncRNAs were identified and 1363 of them were novel. The numbers of lncRNAs identified in different AT depots were similar. Through weighted gene co-expression network analysis (WGCNA), a module including 207 lncRNAs with a high correlation with UFA content was revealed. Functional enrichment analysis showed that these lncRNAs were significantly enriched in fat deposition and fatty acid metabolism. Notably, two lncRNAs (MSTRG.11229 and MSTRG.16994) were further identified. Both lncRNAs exhibited predominant expression in sternum subcutaneous AT (SSAT) and were upregulated during adipogenic differentiation in SSAT-derived adipocytes. What is more, the expressions of the two lncRNAs presented a high correlation to adipogenesis and UFA synthesis genes as well as UFA content. Collectively, this study provides a comprehensive atlas of lncRNA profiles across six AT depots and identifies two lncRNAs with high correlation with UFA content in buffaloes. These findings offer valuable insights and lncRNAs for the regulation of UFA synthesis in buffaloes.

Intramuscular fat (IMF) and unsaturated fatty acid (UFA) contents are critical factors influencing meat quality in livestock. Circular RNAs (circRNAs) have recently emerged as key regulators in lipid metabolism. However, their roles in IMF deposition and UFA synthesis remain largely unexplored in buffalo. Here, high-throughput sequencing of six adipose depots combined with WGCNA identified a novel circRNA (circFAM129B) that was highly correlated with UFA content in buffaloes. The authenticity of circFAM129B was confirmed by sequencing and RNase R treatment. Overexpression of circFAM129B upregulated the expression of adipogenesis and UFA synthesis genes, enhancing lipid accumulation and UFA content, while inhibiting cell proliferation. Mechanistically, circFAM129B sponges bta-let-7d, alleviating its inhibitory effect on SCD. These findings unveil a novel circRNA-mediated regulatory axis modulating adipogenesis and UFA synthesis, offering potential molecular targets for enhancing meat quality and nutritional value in buffaloes.

BACKGROUND:The intramuscular fat content and the level of unsaturated fatty acids influence meat quality and nutritional value. microRNAs can participate in lipid metabolic processes. Understanding how novel miRNAs regulate lipogenesis and fatty acid metabolism is key to enhancing buffalo meat quality and nutritional value. RESULTS:In this study, miRNA sequencing was conducted on 36 adipose tissues from six anatomical sites in river buffaloes (Murrah). A total of 1,682 miRNAs were identified in 36 adipose tissues, of which 987 were novel and 695 were known miRNAs. Weighted gene co-expression networks analysis (WGCNA) revealed that bta-novel-miR-25336 was highly expressed in sternum subcutaneous adipose tissue (SSAT) and had a high correlation with unsaturated fatty acids (UFA). Functional studies showed that bta-novel-miR-25336 was positively correlated with the proliferation and adipogenic differentiation of both buffalo preadipocytes and intramuscular adipocytes. Additionally, we used bioinformatics analysis and a 3' untranslated region (3'UTR) luciferase reporter assay to validate acyl-CoA dehydrogenase short chain (ACADS) as a target gene of bta-novel-miR-25336. CONCLUSIONS:In summary, bta-novel-miR-25336 may regulate biological processes associated with cell proliferation, lipid differentiation and unsaturated fatty acid synthesis through ACADS. This study provides a theoretical basis for further elucidating the regulation of lipid metabolism by novel miRNAs in farm animals.

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.

As a major energy source in the metabolism of early embryos, lactate also participates in regulating zygotic gene expression and subsequently contributes to preimplantation embryonic development. Nevertheless, the influence of lactate on embryonic development in bovine cloned embryos remains elusive. For this reason, this research explored the differences in metabolic pathways between in vitro fertilization (IVF) embryos and somatic cell nuclear transfer (SCNT) embryos during zygotic genome activation (ZGA) using Smart-seq and observed changes in SCNT embryo development in response to lactate supplementation. Weighted gene coexpression network analysis (WGCNA) revealed that LDHA functions as a hub gene that significantly influences the gene expression profile of 8-cell SCNT embryos. Compared with those of IVF embryos, SCNT embryos showed lower LDHA levels and lactate contents. Lactate supplementation was found to increase the developmental potential and blastocyst quality of SCNT embryos. Furthermore, the addition of lactate significantly increased the immunofluorescence intensity of both Pan Kla and H3K18la as well as the expression levels of the zygotic genes ZSCAN5B and SUPT4H1 in SCNT embryos. These results indicate that lactate deficiency leading to the downregulation of histone lactylation may be an important factor affecting the in vitro development of SCNT embryos.

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.

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.

An efficient promoter with specific transcriptional activity plays significant roles in the regulation of expression of exogenous genes. The efficient promoter specific to skeletal muscles can achieve high expression of exogenous genes in skeletal muscles. This is of great significance for the targeted improvement of livestock meat quality by combining gene editing and traditional breeding techniques. To identify efficient promoters specific to the skeletal muscles of buffalo, in the present study, a total of 14 genes, CACNG1, LRRC30, CACNG6, MYOG, VGLL2, MYOD1, KCNA7, DUPD1, PRR32, LBX1, IGFN1, ACTN3, PITX3, and MURC, were firstly screened as skeletal-muscle-specific expressed genes based on high-throughput sequencing data. Among them, only two genes - namely, VGLL2 and CACNG1 - were identified to be specifically and efficiently expressed in the skeletal muscles of buffalo by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Then, the transcriptional activity of different truncated fragments of the upstream putative promoter region of VGLL2 and CACNG1 were evaluated by the dual luciferase reporter gene detection system in mouse C2C12 cells and buffalo skeletal muscle cells. As a result, both core promoters of VGLL2 and CACNG1 were identified to have specifically and efficiently transcriptional activity in skeletal muscle tissue while the transcriptional activity of the core promoters of VGLL2 was more efficient. These results provide significant information for the targeted improvement of meat quality in buffaloes and other livestock animals.

The intramuscular fat content and the unsaturated fatty acid (UFA) composition are both critical indicators of buffalo meat quality. While microRNAs regulate fatty acid metabolism, their specific roles in buffaloes remain unclear. Our previous WGCNA identified bta-miR-30f as a hub miRNA positively correlated with UFA levels. In the present study, bta-miR-30f was found to be highly expressed in sternum subcutaneous adipose tissue and mature adipocytes. Functional studies indicated that bta-miR-30f increased lipid accumulation via enhanced adipogenesis and UFA levels, upregulating key genes including PPARG, C/EBPα, SCD, and FADS1/2. It also promoted cell proliferation. Mechanistically, the dual luciferase assay confirmed that bta-miR-30f interacted with RAD23B, whose knockdown similarly increased lipid deposition and UFA content in buffalo intramuscular preadipocytes. Thus, this study demonstrated that bta-miR-30f enhances adipogenesis differentiation and UFA accumulation by targeting RAD23B in buffalo intramuscular preadipocytes, which provides significant information for the genetic improvement of meat quality in buffaloes.

In vitro maturation (IVM) of oocytes is pivotal for successful embryo production. Cumulus cells (CCs) contribute to oocyte maturation through the secretion of hormones and nutrients, with proper autophagic activity being crucial for this process. However, the role of autophagy in CCs remains underexplored. Siraitia grosvenorii extract Mogroside III (MIII), known for its antioxidant properties, has yet to be extensively studied for its impact on bovine oocyte IVM and its potential regulatory effects on autophagy. This study assessed the influence of MIII on autophagic activity in CCs and its subsequent effects on oocyte developmental potential. The results demonstrated that MIII enhanced bovine oocyte IVM, promoted CC expansion, and supported embryonic development. Transcriptomic analysis indicated that MIII upregulated the expression of autophagy-related genes. In vitro experiments on CCs revealed that MIII increased LC3B protein levels, reduced SQSTM1 accumulation, and upregulated the gene expression of LC3, Beclin1, and ATG5. In co-culture systems, autophagy inhibition in CCs impaired oocyte IVM and embryonic development, but MIII alleviated these effects, restoring oocyte developmental capacity compromised by 3-MA-induced autophagy inhibition. Mechanistically, MIII facilitated the degradation of WT1 by upregulating LC3B, influencing CC differentiation, enhancing FSHR synthesis, and increasing estrogen and progesterone secretion. In conclusion, MIII enhances oocyte developmental potential by modulating autophagy in CCs.

Background/Objectives: Buffalo populations exhibit distinct genetic variations influenced by domestication history, geographic distribution, and selection pressures. This study investigates the genetic structure and differentiation of 11 buffalo populations, focusing on five loci related to milk protein (CSN1S1 and CSN3) and fat metabolism (LPL, DGAT1 and SCD). The aim is to assess genetic variation between river, swamp, and wild-type buffaloes and identify key loci contributing to population differentiation. Methods: Genetic diversity was analyzed through allele frequency distribution, the Hardy−Weinberg equilibrium testing, and observed (Ho) and expected heterozygosity (He) calculations. Population structure was assessed using principal component analysis (PCA), FST statistics, and phylogenetic clustering (k-means and UPGMA tree). The silhouette score (SS) and the Davies−Bouldin index (DBI) were applied to determine optimal population clustering. Results: Significant genetic differentiation was observed between river and swamp buffaloes (p < 0.001). DGAT1 and CSN3 emerged as key markers distinguishing buffalo types. The Italian Mediterranean buffalo exhibited the highest genetic diversity (Ho = 0.464; He = 0.454), while the Indonesian, Chinese, and Vietnamese populations showed low heterozygosity, likely due to selection pressures and geographic isolation. The global FST (0.2143; p = 0.001) confirmed moderate differentiation, with closely related populations (e.g., Nepal and Pakistan) exhibiting minimal genetic divergence, while distant populations (e.g., Egypt and Indonesia) showed marked differences, and the Romanian population showed a unique genetic position. Conclusions: These findings contribute to a deeper understanding of buffalo genetic diversity and provide a valuable basis for exploiting the potential of this species in the light of future breeding and conservation strategies specific for each buffalo type.

The Wnt/β-catenin signaling pathway is integral to follicular development, yet its precise role in buffalo folliculogenesis remains poorly defined. This study aimed to elucidate its involvement in follicular selection and underlying regulatory mechanisms. Buffalo follicles were classified as dominant or atretic based on the follicular fluid estradiol-to-progesterone (E2: P4) ratio. Quantitative PCR revealed significantly elevated expression of Wnt/β-catenin pathway components in the granulosa cells of dominant follicles. Functional experiments demonstrated that pharmacological inhibition of Wnt/β-catenin signaling markedly suppressed both FSH-induced estradiol (E2) production and oocyte maturation. Conversely, Wnt/β-catenin pathway activation enhanced oocyte maturation, cleavage, and blastocyst formation rates. Further investigation identified AKT/GSK3β/β-catenin cascade as a key mediator of Wnt's positive regulatory role in FSH-supported steroidogenesis. These findings clarify the pivotal role of Wnt/β-catenin signaling in buffalo follicular selection and provide a foundational strategy for enhancing the efficiency of in vitro embryo production (IVP) in buffalo breeding programs.

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.

This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform in vitro maturation, in vitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the in vitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.

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.

Abstract Background Brilliant cresyl blue (BCB) staining can stain oocytes and differentiated oocytes will lead to different developmental outcomes. This technique has been studied in multiple species, but it is still unclear whether buffalo oocytes can be used for developmental potential prediction through BCB staining methods. This study used the BCB staining method to group buffalo oocytes (BCB + and BCB-) and perform in vitro maturation, in vitro fertilization, and embryo culture. By statistical analysis, the effect of BCB staining on predicting the developmental potential of buffalo oocytes will be explored. At the same time, molecular biology techniques will be used to detect gap junction protein and oxidative stress-related indicators to explore the molecular mechanism of BCB staining predicting oocyte cell developmental potential. Methods The oocytes were divided into BCB + and BCB- groups using BCB staining technique. And then mainly uses in vitro maturation, in vitro fertilization and embryo culture techniques of buffalo oocytes to analyze their developmental potential, and uses immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels, and qPCR to detect the expression levels of the antioxidant related genes SOD2 and GPX1. Results Our results showed that the in vitro maturation rate, embryo cleavage rate, and blastocyst rate of buffalo oocytes in the BCB + group were significantly higher than those in the BCB- group and the control group (P < 0.05). The expression level of CX43 protein in the BCB + group was higher than that in the BCB- group both before and after maturation (P < 0.05). The intensity of ROS in the BCB + group was significantly lower than that in the BCB- group (P < 0.05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB + group were significantly higher than those in the BCB- group (P < 0.05). Conclusions BCB staining can effectively predict the developmental potential of buffalo oocytes. The results of BCB staining are positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the ROS level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes may be closely related to antioxidant activity.