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.

Bos taurus is known for its tolerance of coarse grains, adaptability, high temperature, humidity, and disease resistance. Primarily, cattle are raised for their meat and milk, and pinpointing genes associated with traits relevant to meat production can enhance their overall productivity. The aim of this study was to identify the genome, analyze the evolution, and explore the function of the Pax gene family in B. taurus to provide a new molecular target for breeding in meat-quality-trait cattle. In this study, 44 Pax genes were identified from the genome database of five species using bioinformatics technology, indicating that the genetic relationships of bovids were similar. The Pax3 and Pax7 protein sequences of the five animals were highly consistent. In general, the Pax gene of the buffalo corresponds to the domestic cattle. In summary, there are differences in affinity between the Pax family genes of buffalo and domestic cattle in the Pax1/9, Pax2/5/8, Pax3/7, and Pax4/6 subfamilies. We believe that Pax1/9 has an effect on the growth traits of buffalo and domestic cattle. The Pax3/7 gene is conserved in the evolution of buffalo and domestic animals and may be a key gene regulating the growth of B. taurus. The Pax2/5/8 subfamily affects coat color, reproductive performance, and milk production performance in cattle. The Pax4/6 subfamily had an effect on the milk fat percentage of B. taurus. The results provide a theoretical basis for understanding the evolutionary, structural, and functional characteristics of the Pax family members of B. taurus and for molecular genetics and the breeding of meat-production B. taurus species.

BACKGROUND: Swamp-type buffaloes with varying degrees of white spotting are found exclusively in Tana Toraja, South Sulawesi, Indonesia, where spotted buffalo bulls are highly valued in accordance with the Torajan customs. The white spotting depigmentation is caused by the absence of melanocytes. However, the genetic variants that cause this phenotype have not been fully characterized. The objective of this study was to identify the genomic regions and variants responsible for this unique coat-color pattern.
RESULTS: Genome-wide association study (GWAS) and selection signature analysis identified MITF as a key gene based on the whole-genome sequencing data of 28 solid and 39 spotted buffaloes, while KIT was also found to be involved in the development of this phenotype by a candidate gene approach. Alternative candidate mutations included, in addition to the previously reported nonsense mutation c.649 C > T (p.Arg217*) and splice donor mutation c.1179 + 2T > A in MITF, a nonsense mutation c.2028T > A (p.Tyr676*) in KIT. All these three mutations were located in the genomic regions that were highly conserved exclusively in Indonesian swamp buffaloes and they accounted largely (95%) for the manifestation of white spotting. Last but not the least, ADAMTS20 and TWIST2 may also contribute to the diversification of this coat-color pattern.
CONCLUSIONS: The alternative mutations identified in this study affect, at least partially and independently, the development of melanocytes. The presence and persistence of such mutations may be explained by significant financial and social value of spotted buffaloes used in historical Rambu Solo ceremony in Tana Toraja, Indonesia. Several de novo spontaneous mutations have therefore been favored by traditional breeding for the spotted buffaloes.

Peroxisome proliferator-activated receptor γ (PPARG) has various splicing variants and plays essential roles in the regulation of adipocyte differentiation and lipogenesis. However, little is known about the expression pattern and effect of the PPARG on milk fat synthesis in the buffalo mammary gland. In this study, we found that only PPARG-X17 and PPARG-X21 of the splicing variant were expressed in the buffalo mammary gland. Amino acid sequence characterization showed that the proteins encoded by PPARG-X17 and PPARG-X21 are endonuclear non-secreted hydrophilic proteins. Protein domain prediction found that only the PPARG-X21-encoded protein had PPAR ligand-binding domains (NR_LBD_PPAR), which may lead to functional differences between the two splices. RNA interference (RNAi) and the overexpression of PPARG-X17 and PPARG-X21 in buffalo mammary epithelial cells (BMECs) were performed. Results showed that the expression of fatty acid synthesis-related genes (ACACA, CD36, ACSL1, GPAT, AGPAT6, DGAT1) was significantly modified (p < 0.05) by the RNAi and overexpression of PPARG-X17 and PPARG-X21. All kinds of FAs detected in this study were significantly decreased (p < 0.05) after RNAi of PPARG-X17 or PPARG-X21. Overexpression of PPARG-X17 or PPARG-X21 significantly decreased (p < 0.05) the SFA content, while significantly increased (p < 0.05) the UFA, especially the MUFA in the BMECs. In conclusion, there are two PPARG splicing variants expressed in the BMECs that can regulate FA synthesis by altering the expression of diverse fatty acid synthesis-related genes. This study revealed the expression characteristics and functions of the PPARG gene in buffalo mammary glands and provided a reference for further understanding of fat synthesis in buffalo milk.

The gold standard of milk is human milk, not cow milk. The present study expects to explored the comprehensive nutritional value of different kinds of milk and the differences between them through multi-omics analysis and found functional components that are more similar to human milk. This study employed untargeted LC-MS/MS metabolomics, untargeted LC-MS/MS lipidomics, and 4D label-free proteomics analysis techniques. The findings revealed substantial disparities in metabolites, lipids, and proteins among the five types of milk. Notably, pig milk exhibited a remarkable abundance of N-acetylneuraminic acid (Neu5Ac) and specific polar lipids. Yak milk stood out with significantly elevated levels of creatine and lipoprotein lipase (LPL) compared to other species. Buffalo milk boasted the highest concentrations of L-isoleucine, echinocystic acid, and alkaline phosphatase, tissue-nonspecific isozyme (ALPL). The concentrations of iminostilbene and osteopontin (OPN) were higher in cow milk.

Mastitis is one of the most serious diseases that threatens the health of dairy animals. The somatic cell count (SCC) in milk is widely used to monitor mastitis. This study aimed to reveal the diversity of microorganisms in buffalo milk with high somatic cell count (SCC ≥ 3 × 105 cells/mL, n = 30) and low somatic cell count (SCC ≤ 5 × 104 cells/mL, n = 10), and identify the dominant bacteria that cause mastitis in a local buffalo farm. We also investigated the potential method to treat bacterial mastitis. The V3-V4 region of 16 S rDNA was sequenced. Results showed that, compared to the milk with low SCC, the high SCC samples showed lower microbial diversity, but a high abundance of bacteria and operational taxonomic units (OTUs). By in vitro isolation and culture, Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae were found to be the leading pathogens, which is consistent with the 16 S rDNA sequencing data. We further isolated 3 of the main pathogens and established a pathogen detection method based on ELISA. In addition, the antibacterial effects of 10 antimicrobials and 15 Chinese herbal extracts were also investigated. Results showed that the microbial has developed tolerance to several of the antimicrobials. While the water extracts of Chinese herbal medicine such as Galla Chinensis, Coptis chinensis Franch, Terminalia chebula Retz, and Sanguisorba officinalis L can effectively inhibit the growth of main pathogens. This study provides novel insight into the microbial diversity in buffalo milk and a reference for the prevention, diagnosis, and treatment of mastitis.

Water buffalo Hunnivirus (BufHuV) belongs to the family Picornaviridae and is a newly discovered member of the Hunnivirus A genus. It causes intestinal diseases in cattle, mainly lead to subclinical infections, thereby seriously threatening the health of cattle herds. In addition, it can also bring about various clinical disease syndromes which results in severe economic losses to the cattle industry. To date, there have been no reports worldwide on the study of Hunnivirus virus infecting host cells and causing innate immune responses. In this study, we found that interferon treatment effectively blocked BufHuV replication and infection with the virus weakened the host antiviral responses. Inhibiting the transcription of IFN-β and ISGs induced by either Sendai virus (SeV) or poly(I:C) in MDBK and HCT-8 cells, were dependent on the IRF3 or NF-κB signaling pathways, and this inhibited the activation of IFN-β promoter by TBK1 and its upstream molecules, RIGI and MDA5. By constructing and screening five BufHuV proteins, we found that VP2, 2 C, 3 C and 3D inhibited the activation of IFN-β promoter induced by SeV. Subsequently, we showed that VP2 inhibited the activation of IRF3 induced by SeV or poly (I:C), and it inhibited IRF3 activation by inhibiting its phosphorylation and nuclear translocation. In addition, we confirmed that VP2 inhibited the activation of IFNβ induced by signaling molecules, MDA5 and TBKI. In summary, these findings provide new insights into the pathogenesis of Hunnivirus and its mechanisms involved in evading host immune responses.

Whether adulteration exists is a difficult problem in the identification of traditional Chinese medicine(TCM). Bubali Cornu is mainly available in the medicinal material market in the form of buffalo horn silk or buffalo horn powder but lacks obvious identification characteristics, so there is a risk of adulteration. However, the method of identification of adulteration in Bubali Cornu is lacking at present. In order to ensure authenticity and identify adulteration of TCM Bubali Cornu, control the quality of TCM Bubali Cornu, and ensure the authenticity of clinical use, the DNA fingerprints of 43 batches of samples from pharmaceutical companies and medicinal material markets were identified, and the amplification primers of fluorescent DNA fingerprints of Bubali Cornu and Bovis Grunniens Cornu were screened. The DNA fingerprints of Bubali Cornu were obtained by fluorescent capillary typing. The identification effect of fluorescent capillary typing on different adulteration ratios was also tested. Two pairs of fluorescent STR typing primers, namely 16Sa and CRc, which can distinguish Bubali Cornu and Bovis Grunniens Cornu, were screened out, and a DNA fingerprint identification method was established. The 16Sa migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 223.4-223.9 bp and 225.5-226.1 bp. The CRc migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 518.8-524.8 bp and 535.9-542.5 bp. The peak height of the migration peak could be used for preliminary quantification of the adulterants with an adulteration ratio below 50%, and the quantitative results were similar to the adulteration ratio. In this study, a simple and quick universal DNA fingerprint method was established for the identification of Bubali Cornu and its adulterants, which could realize the identification of TCM Bubali Cornu and the semi-quantitative identification of the adulterants.

Due to the highly stable structure of keratin, the extraction and dissolution steps of animal medicines rich in keratin are complex, which seriously restricts the detection efficiency and flux. Therefore, this study simplified the pre-treatment steps of horn samples and optimized the detection methods of characteristic peptides to improve the efficiency of identifying the specificity of horn-derived animal medicines. For detection of the characteristic peptides in horn-derived animal medicines treated with/without iodoace-tamide(IAA), the ion pair conditions of the characteristic peptides were optimized, and the retention time, intensity and other data of the specific peptides were compared between the samples treated with/without IAA. Two pre-treatment methods, direct enzymatic hydrolysis and total protein extraction followed by enzymatic hydrolysis, were used to prepare horn-derived animal medicine samples. The effects of different methods on the detection of specific peptides in the samples of Saiga antelope horn, water buffalo horn, goat horn, and yak horn were compared regarding the retention time of specific peptides and ion intensity. The results indicated that after direct enzymatic hydrolysis, the specific peptides in the samples without IAA treatment can be detected. Compared with the characteristic peptides in the samples treated with IAA, their retention time shifted back and the mass spectrometry response slightly decreased. The specific peptides of the samples without IAA treatment had good specificity and did not affect the specificity identification of horn-derived animal medicines. Overall, the process of direct enzymatic hydrolysis can be used to treat horn samples, omitting the steps of protein extraction and dithiothreitol and IAA treatment, significantly improving the pre-treatment efficiency without affecting the specificity identification of horn-derived animal medicines. This study provides ideas for quality research and standard improvement of horn-derived animal medicines.

Dairy buffaloes are typically fed a high-forage, low-quality diet with high fiber. These conditions result in an inherent energy and protein inefficiency. In order to make full and rational use of feed resources and improve the production level and breeding efficiency of dairy buffaloes, the effects of various roughages on nutrient digestibility, ruminal fermentation parameters, and microorganisms in dairy buffaloes were studied in this experiment. Three ternary hybrid buffaloes, with an average body weight of 365 ± 22.1 kg, were selected and fitted with permanent rumen fistulas. They were fed six different diets, each consisting of 1 kg concentrate supplement and one of six types of roughage, including alfalfa hay (A diet), oat hay (O diet), whole corn silage (W diet), king grass (K diet), sugarcane shoot silage (S diet), and rice straw hay (R diet) according to an incomplete Latin square design of 3 × 6, respectively. The pre-feeding period of each period was 12 d. From day 13 to 15 was the official experimental period. During the prefeeding period, free feed intake for each roughage was determined, and during the experiment, the roughage was fed at 90% of the voluntary feed intake. Digestion and metabolism tests were carried out using the total manure collection method to determine the feed intake and fecal output of each buffalo, and to collect feed and fecal samples for chemical analysis. On day 15, rumen fluid samples were collected two hours after morning feeding to determine rumen fermentation parameters and bacterial 16 S rRNA high-throughput sequencing was performed. The results showed that DM and OM digestibility were greatest for the W diet and lowest for the S diet. The rumen pH of the O diet was significantly greater than that of the W diet. The concentration of rumen fluid NH3-N (mg/dL) increased with increased CP content. The concentration of total volatile fatty acids (mmol/L) in the rumen decreased with increased NDF content but increased with increased NFC content. The relative abundances of Bacteroidetes, Firmicutes, and Spirochaetes were 57.03-74.84%, 14.29-21.86%, and 0.44-1.43% in the different quality roughage groups. Bacteroidetes were mainly Prevotellaceae1 and Rikenellaceae RC_gut_group with relative abundances of 30.17-45.75% and 3.23-7.82%. The relative abundance of Patescibacteria and Spirochaetes decreased with increasing roughage quality. These results provide a theoretical and practical basis for evaluating the nutritional value of dairy buffalo feed, utilizing feed resources, matching rations, feeding scientifically, and protecting animal health.