Novel Insight into the Role of Squalene Epoxidase (SQLE) Gene in Determining Milk Production Traits in Buffalo.

Understanding the genetic mechanisms underlying milk production traits contribute to improving the production potential of dairy animals. Squalene epoxidase (SQLE) is one of the rate-limiting enzymes for cholesterol biosynthesis and was highly expressed in the buffalo mammary. The objectives of the present study were to detect the polymorphisms within SQLE in buffalo, the genetic effects of these mutations on milk production traits, and to understand the gene regulatory effects on buffalo mammary epithelial cells (BuMECs). A total of five SNPs were identified by sequencing, g.18858G > A loci were significantly associated with fat yield, and g.22834C > T loci were significantly associated with peak milk yield, milk yield, fat yield, and protein yield. Notably, linkage disequilibrium analysis indicated that 2 SNPs (g.18858G > A and g.22834C > T) formed one haplotype block, which was found to be significantly associated with milk fat yield, fat percentage, and protein yield. Furthermore, expression of SQLE was measured in different tissues of buffalo and was found to be higher in the mammary. Knockdown of SQLE gene expression significantly affected the growth of BuMECs, including proliferation, cell cycle, and apoptosis, and significantly downregulated the expression of related genes MYC, PCNA, and P21. In addition, knockdown of the SQLE gene significantly reduces triglyceride concentrations and the signal intensity of oil red O staining. In addition, silencing of SQLE was also found to regulate the synthesis and secretion of ß-casein and κ-casein negatively. Furthermore, SQLE knockdown is accompanied by the downregulation of critical genes (RPS6KB1, JAK2, eIF4E, and SREBP1) related to milk fat and protein synthesis. The current study showed the potential of the SQLE gene as a candidate for buffalo milk production traits. It provides a new understanding of the physiological mechanisms underlying buffalo milk production regulation.

Evolutionary analysis of buffalo sterol regulatory element-binding factor (SREBF) family genes and their affection on milk traits.

The sterol regulatory element-binding factor (SREBF) genes are a vital group of proteins binding to the sterol regulatory element 1 (SRE-1) regulating the synthesis of fatty acid. Two potential candidate genes (SREBF1 and SREBF2) have been identified as affecting milk traits. This study aims to identify the SREBF family of genes and find candidate markers or SREBF genes influencing lactation production in buffalo. A genome-wide study was performed and identified seven SREBF genes randomly distributed on 7 chromosomes and 24 protein isoforms in buffalos. The SREBF family of genes were also characterized in cattle, goat, sheep and horse, and using these all-protein sequences, a phylogenetic tree was built. The SREBF family genes were homologous between each other in the five livestock. Eight single nucleotide polymorphisms (SNPs) within or near the SREBF genes in the buffalo genome were identified and at least one milk production trait was associated with three of the SNP. The expression of SREBF genes at different lactation stages in buffalo and cattle from published data were compared and the SREBF genes retained a high expression throughout lactation with the trend being the same for buffalo and cattle. These results provide valuable information for clarifying the evolutionary relationship of the SREBF family genes and determining the role of SREBF genes in the regulation of milk production in buffalo.

Effects of capsicum oleoresin supplementation on rumen fermentation and microbial abundance under different temperature and dietary conditions in vitro.

This study aimed to determine the effect of capsicum oleoresin (CAP) on rumen fermentation and microbial abundance under different temperature and dietary conditions in vitro. The experimental design was arranged in a 2 × 2 × 3 factorial format together with two temperatures (normal: 39°C; hyperthermal: 42°C), two forage/concentrate ratios (30:70; 70:30), and two CAP concentrations in the incubation fluid at 20 and 200 mg/L with a control group. Regarding the fermentation characteristics, high temperature reduced short-chain fatty acids (SCFA) production except for molar percentages of butyrate while increasing acetate-to-propionate ratio and ammonia concentration. The diets increased total SCFA, propionate, and ammonia concentrations while decreasing acetate percentage and acetate-to-propionate ratio. CAP reduced acetate percentage and acetate-to-propionate ratio. Under hyperthermal condition, CAP could reduce acetate percentage and increase acetate-to-propionate ratio, lessening the negative effect of high heat on SCFA. Hyperthermal condition and diet altered the relative abundance of microbial abundance in cellulose-degrading bacteria. CAP showed little effect on the microbial abundance which only increased Butyrivibrio fibrisolvens. Thus, CAP could improve rumen fermentation under different conditions, with plasticity in response to the ramp of different temperature and dietary conditions, although hardly affecting rumen microbial abundance.

Evaluation of type traits in relation to production, and their importance in early selection for milk performance in dairy buffaloes.

Type traits (TTs) can contribute to breeding animals with good economic traits such as production, longevity, fertility, and profitability. Dairy buffaloes are the second largest source of milk supply in the world, and their TTs should be taken into consideration in future dairy buffalo breeding programmes. However, the relationship between TTs and milk production traits in buffalo remains largely unknown. The study aimed to establish an early selection method for buffaloes with desirable milk performance by TTs. Using 1 908 records from 678 buffaloes, the relationship between TTs and milk production traits was analysed and the optimal growth curves of TTs related to milk production traits were constructed. We examined the correlations between 45 TTs (33 body structural, 12 udder and teat morphological traits) and three milk production traits (milk yield (MY), milk fat percentage (MF), and milk protein percentage (MP)). The results showed that the highest correlation was found between MY and udder circumference (r = 0.438), teat length (r = -0.380) or heart girth (r = -0.341). The teat distance and teat circumference exhibited a significant negative correlation with MF and MP. Rump length was the only trait that had a significant positive correlation with milk production traits, suggesting that milk performance could be comprehensively improved by including rump length in the selection procedure. Notably, we found that high milk production traits was obtained from the buffaloes with short teats (<6 cm), small heart girth (<200 cm), large udder circumference (>104 cm), long rump (>39 cm), and small distance between teats. Moreover, an early selection method for buffaloes with excellent milk performance was developed based on the non-linear models. Brody model exhibited the best fitting effect for heart girth and rump length, while the Logistic model displayed the best fitting effect for teat length. Our findings provide theoretical basis for the early selection of buffaloes with desirable milk performance.

Preliminary Study on Gene Regulation and its Pathways in Chinese Holstein Cows with Clinical Mastitis Caused by Staphylococcus Aureus.

Introduction: Clinical mastitis (CM) is one of the most common diseases of dairy cows globally, has a complex aetiology and recurs easily. Staphylococcus aureus is a frequently isolated pathogen responsible for bovine mastitis and remains difficult to eradicate. Material and Methods: To characterise the transcriptional profiles of dairy cows infected by S. aureus, we performed an RNA-seq analysis of peripheral blood leukocytes in lactating Chinese Holstein dairy cows with CM and did the same with healthy cows' samples as controls. Results: A total of 4,286 genes were detected in the CM cases infected with S. aureus which were differentially expressed compared to the controls, 3,085 of which were upregulated, the remainder being downregulated. Notably, we observed that some differentially expressed genes (DEGs) had strong protein-protein interaction. Of these, six downregulated DEGs (AKR1C4, PTGS2, HNMT, EPHX2, CMBL, and IDH1) were involved in the metabolic pathway, while eight upregulated DEGs (VWF, GP9, MYLK, GP6, F2RL3, ITGB3, GP5, and PRKG1) were associated with the platelet activation pathway. Conclusion: The transcriptome dataset of CM cases would be a valuable resource for clinical guidance on anti-inflammatory medication and for deeper understanding of the biological processes of CM response to S. aureus infection, and it would enable us to identify specific genes for diagnostic markers and possibly for targeted therapy.

Evolutionary and Association Analysis of Buffalo FABP Family Genes Reveal Their Potential Role in Milk Performance.

The fatty acid-binding protein (FABP) family gene encode a group of proteins that affect long-chain fatty acid (LCFAs) trafficking and play a crucial function in the regulation of milk fat synthesis. Nevertheless, little is known regarding the identification of members, theevolutionary background, and functional characteristics of FABP genes in buffalo. In this study, in silico analysis was performed to identify the members of FABPs in buffalo. The results revealed that a total of 17 FABP genes were identified. Based on their phylogenetic relationships, these sequences clustered into five groups with similar motif patterns and gene structures. According to positive selection analyses, all duplicated gene pairs containing FABPs in buffalo had Ka/Ks (nonsynonymous/synonymous) ratios that were less than 1, suggesting that they were under purifying selection. Association analysis showed that one SNP in LOC102401361 was found significantly associated with buffalo milk yield. The expression levels of several FABPs in buffalo mammary epithelial cells were regulated by palmitic and stearic acid treatment. The findings of this study provide valuable information for further research on the role of FABPs in regulating buffalo milk synthesis.

Ginsenoside Rb1 Mitigates Escherichia coli Lipopolysaccharide-Induced Endometritis through TLR4-Mediated NF-κB Pathway.

Endometritis is the inflammatory response of the endometrial lining of the uterus and is associated with low conception rates, early embryonic mortality, and prolonged inter-calving intervals, and thus poses huge economic losses to the dairy industry worldwide. Ginsenoside Rb1 (GnRb1) is a natural compound obtained from the roots of Panax ginseng, having several pharmacological and biological properties. However, the anti-inflammatory properties of GnRb1 in lipopolysaccharide (LPS)-challenged endometritis through the TLR4-mediated NF-κB signaling pathway has not yet been researched. This study was planned to evaluate the mechanisms of how GnRb1 rescues LPS-induced endometritis. In the present research, histopathological findings revealed that GnRb1 ameliorated LPS-triggered uterine injury. The ELISA and RT-qPCR assay findings indicated that GnRb1 suppressed the expression level of pro-inflammatory molecules (TNF-α, IL-1ß and IL-6) and boosted the level of anti-inflammatory (IL-10) cytokine. Furthermore, the molecular study suggested that GnRb1 attenuated TLR4-mediated NF-κB signaling. The results demonstrated the therapeutic efficacy of GnRb1 in the mouse model of LPS-triggered endometritis via the inhibition of the TLR4-associated NF-κB pathway. Taken together, this study provides a baseline for the protective effect of GnRb1 to treat endometritis in both humans and animals.

Cathepsin B Regulates Mice Granulosa Cells' Apoptosis and Proliferation In Vitro.

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.

Association analysis between FASN genotype and milk traits in Mediterranean buffalo and its expression among different buffalo tissues.

Fatty acid synthase (FASN) is a multifunctional protein that catalyzes the synthesis of long-chain saturated fatty acid. In this study, we identified the single nucleotide polymorphisms (SNPs), and their association with milk traits in Mediterranean buffalo, and the expression of FASN gene in different tissues was measured. Nine SNPs (g.-1640G > A, g.-1099C > T, g.1095C > A, g.3221G > A, g.4762G > A, g.5299G > A, g.7164G > A, g.7272 T > C, and g.8927 T > C) were identified by DNA pooled sequencing and then genotyped. Seven identified SNPs except g.3221G > A and g.8927 T > C were found significantly associated with both fat and protein percentage, and also the g.7164G > A and g.8927 T > C had significant association with peak milk yield and protein percentage, respectively. One haplotype block was successfully constructed by linkage disequilibrium (LD) analysis and it showed a significant association with both fat percentage and protein percentage. Expression of FASN gene was found in almost all the buffalo tissues including mammary gland, heart, liver, spleen, lung, kidney, uterus, and ovary, and to be highest in lung and mammary gland. Our findings suggest that polymorphisms in the buffalo FASN gene are associated with milk production traits and can be used as a candidate gene for milk traits and marker-assisted selection in buffalo breeding program.

Identifying Hub Genes for Heat Tolerance in Water Buffalo (Bubalus bubalis) Using Transcriptome Data.

Heat stress has a detrimental effect on the physiological and production performance of buffaloes. Elucidating the underlying mechanisms of heat stress is challenging, therefore identifying candidate genes is urgent and necessary. We evaluated the response of buffaloes (n = 30) to heat stress using the physiological parameters, ELISA indexes, and hematological parameters. We then performed mRNA and microRNA (miRNA) expression profiles analysis between heat tolerant (HT, n = 4) and non-heat tolerant (NHT, n = 4) buffaloes, as well as the specific modules, significant genes, and miRNAs related to the heat tolerance identified using the weighted gene co-expression network analysis (WGCNA). The results indicated that the buffaloes in HT had a significantly lower rectal temperature (RT) and respiratory rate (RR) and displayed a higher plasma heat shock protein (HSP70 and HSP90) and cortisol (COR) levels than those of NHT buffaloes. Differentially expressed analysis revealed a total of 753 differentially expressed genes (DEGs) and 16 differentially expressed miRNAs (DEmiRNAs) were identified between HT and NHT. Using the WGCNA analysis, these DEGs assigned into 5 modules, 4 of which were significantly correlation with the heat stress indexes. Interestingly, 158 DEGs associated with heat tolerance in the turquoise module were identified, 35 of which were found within the protein-protein interaction network. Several hub genes (IL18RAP, IL6R, CCR1, PPBP, IL1B, and IL1R1) were identified that significantly enriched in the Cytokine-cytokine receptor interaction. The findings may help further elucidate the underlying mechanisms of heat tolerance in buffaloes.

Genome-Wide SNP Data Revealed the Extent of Linkage Disequilibrium, Persistence of Phase and Effective Population Size in Purebred and Crossbred Buffalo Populations.

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 (r 2) 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 r 2 ≥ 0.2 extended up to ~50 kb in crossbred and 170 kb in purebred populations, while average r 2 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.

Anti-inflammatory potential of ß-cryptoxanthin against LPS-induced inflammation in mouse Sertoli cells.

ß-cryptoxanthin (CX), a major carotenoid pigment, can inhibit inflammatory gene expression in mice with nonalcoholic steatohepatitis. In the present study, we examined the anti-inflammatory effects of CX on lipopolysaccharide (LPS)-induced inflammation in mouse primary Sertoli cells and the possible molecular mechanisms behind its effects. The results showed that CX significantly inhibited LPS-induced decreases in cell viability and in the percentage of apoptotic cells. Moreover, CX inhibited the LPS-induced up-regulation of tumor necrosis factor α (TNF-α), interleukin-10 (IL-10), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) in Sertoli cells. In addition, CX significantly limited the LPS-induced down-regulation of AR, HSF2, CREB, FSHR, INHBB and ABP in Sertoli cells. Western blot analysis showed that CX significantly suppressed NF-κB (p65) activation as well as MAPK phosphorylation. All the results suggested that CX suppressed inflammation, possibly associated with the NF-κB activation and MAPK of phosphorylation. Thus, CX may possess therapeutic potential against inflammation-related diseases.