Multi-omics strategy reveals potential role of antimicrobial resistance and virulence factor genes responsible for Simmental diarrheic calves caused by Escherichia coli.

Escherichia coli (E. coli) is reported to be an important pathogen associated with calf diarrhea. Antibiotic resistance genes (ARGs) and virulence factor genes (VFGs) pose a considerable threat to both animal and human health. However, little is known about the characterization of ARGs and VFGs presented in the gut microbiota of diarrheic calves caused by E. coli. In this study, we used multi-omics strategy to analyze the ARG and VFG profiles of Simmental calves with diarrhea caused by E. coli K99. We found that gut bacterial composition and their microbiome metabolic functions varied greatly in diarrheic calves compared to healthy calves. In total, 175 ARGs were identified, and diarrheal calves showed a significantly higher diversity and abundance of ARGs than healthy calves. Simmental calves with diarrhea showed higher association of VFGs with pili function, curli assembly, and ferrienterobactin transport of E. coli. Co-occurrence patterns based on Pearson correlation analysis revealed that E. coli had a highly significant (P < 0.0001) correlation coefficient (>0.8) with 16 ARGs and 7 VFGs. Metabolomics analysis showed that differentially expressed metabolites in Simmental calves with diarrhea displayed a high correlation with the aforementioned ARGs and VFGs. Phylotype analysis of E. coli genomes showed that the predominant phylogroup B1 in diarrheic Simmental calves was associated with 10 ARGs and 3 VFGs. These findings provide an overview of the diversity and abundance of the gut microbiota in diarrheic calves caused by E. coli and pave the way for further studies on the mechanisms of antibiotic resistance and virulence in the calves affected with diarrhea.IMPORTANCESimmental is a well-recognized beef cattle breed worldwide. They also suffer significant economic losses due to diarrhea. In this study, fecal metagenomic analysis was applied to characterize the antibiotic resistance gene (ARG) and virulence factor gene (VFG) profiles of diarrheic Simmental calves. We identified key ARGs and VFGs correlated with Escherichia coli isolated from Simmental calves. Additionally, metabolomics analysis showed that differentially expressed metabolites in Simmental calves with diarrhea displayed a high correlation with the aforementioned ARGs and VFGs. Our findings provide an insight into the diversity and abundance of the gut microbiota in diarrheic calves caused by Escherichia coli and pave the way for further studies on the mechanisms of antibiotic resistance and virulence in the diarrheal calves from cattle hosts.

Genome-wide CNV analysis reveals candidate genes associated with milk production traits in water buffalo (Bubalus bubalis).

Buffaloes are vital contributors to the global dairy industry. Understanding the genetic basis of milk production traits in buffalo populations is essential for breeding programs and improving productivity. In this study, we conducted whole-genome resequencing on 387 buffalo genomes from 29 diverse Asian breeds, including 132 river buffaloes, 129 swamp buffaloes, and 126 crossbred buffaloes. We identified 36,548 copy number variant (CNVs) spanning 133.29 Mb of the buffalo genome, resulting in 2,100 copy number variant regions (CNVRs), with 1,993 shared CNVRs being found within the studied buffalo types. Analyzing CNVRs highlighted distinct genetic differentiation between river and swamp buffalo subspecies, verified by evolutionary tree and principal component analyses. Admixture analysis grouped buffaloes into river and swamp categories, with crossbred buffaloes displaying mixed ancestry. To identify candidate genes associated with milk production traits, we employed 3 approaches. First, we used Vst-based population differentiation, revealing 11 genes within CNVRs that exhibited significant divergence between different buffalo breeds, including genes linked to milk production traits. Second, expression quantitative loci (eQTL) analysis revealed differential expression of CNVR-driven genes (DECGs) associated with milk production traits. Notably, known milk production-related genes were among these DECGs, validating their relevance. Last, a genome-wide association study (GWAS) identified 3 CNVRs significantly linked to peak milk yield. Our study provides comprehensive genomic insights into buffalo populations and identifies candidate genes associated with milk production traits. These findings facilitate genetic breeding programs aimed at increasing milk yield and improving quality in this economically important livestock species.

Transcriptome-Wide Association Study Reveals Potentially Candidate Genes Responsible for Milk Production Traits in Buffalo.

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.

Genome-wide identification and evolutionary analysis of the FGF gene family in buffalo.

Fibroblast growth factors (FGFs) are important polypeptide growth factors that play a critical role in many developmental processes, including differentiation, cell proliferation, and migration in mammals. This study employs in silico analyses to characterize the FGF gene family in buffalo, investigating their genome-wide identification, physicochemical properties, and evolutionary patterns. For this purpose, genomic and proteomic sequences of buffalo, cattle, goat, and sheep were retrieved from NCBI database. We identified a total of 22 FGF genes in buffalo. Physicochemical properties observed through ProtParam tool showed notable features of these proteins including in-vitro instability, thermostability, hydrophilicity, and basic nature. Phylogenetic analysis grouped 22 identified genes into nine sub-families based on evolutionary relationships. Additionally, analysis of gene structure, motif patterns, and conserved domains using TBtools revealed the remarkable conservation of this gene family across selected species throughout the course of evolution. Comparative amino acid analysis performed through ClustalW demonstrated significant conservation between buffalo and cattle FGF proteins. Mutational analysis showed three non-synonymous mutations at positions R103 > G, P7 > L, and E98 > Q in FGF4, FGF6, and FGF19, respectively in buffalo. Duplication events revealed only one segmental duplication (FGF10/FGF22) in buffalo and two in cattle (FGF10/FGF22 and FGF13/FGF13-like) with Ka/Ks values <1 indicating purifying selection pressure for these duplications. Comparison of protein structures of buffalo, goat, and sheep exhibited more similarities in respective structures. In conclusion, our study highlights the conservation of the FGF gene family in buffalo during evolution. Furthermore, the identified non-synonymous mutations may have implications for the selection of animals with better performance.Communicated by Ramaswamy H. Sarma.

Metagenomic and metabolomic analyses reveal the role of gut microbiome-associated metabolites in diarrhea calves.

IMPORTANCE: Calf diarrhea is of great concern to the global dairy industry as it results in significant economic losses due to lower conception rates, reduced milk production, and early culling. Although there is evidence of an association between altered gut microbiota and diarrhea, remarkably little is known about the microbial and metabolic mechanisms underlying the link between gut microbiota dysbiosis and the occurrence of calf diarrhea. Here, we used fecal metagenomic and metabolomic analyses to demonstrate that gut microbiota-driven metabolic disorders of purine or arachidonic acid were associated with calf diarrhea. These altered gut microbiotas play vital roles in diarrhea pathogenesis and indicate that gut microbiota-targeted therapies could be useful for both prevention and treatment of diarrhea.

Comparative Genomic Analysis of the Thiolase Family and Functional Characterization of the Acetyl-Coenzyme A Acyltransferase-1 Gene for Milk Biosynthesis and Production of Buffalo and Cattle.

Cattle and buffalo served as the first and second largest dairy animals, respectively, providing 96% milk products worldwide. Understanding the mechanisms underlying milk synthesis is critical to develop the technique to improve milk production. Thiolases, also known as acetyl-coenzyme A acetyltransferases (ACAT), are an enzyme family that plays vital roles in lipid metabolism, including ACAT1, ACAT2, ACAA1, ACAA2, and HADHB. Our present study showed that these five members were orthologous in six livestock species including buffalo and cattle. Transcriptomic data analyses derived from different lactations stages showed that ACAA1 displayed different expression patterns between buffalo and cattle. Immunohistochemistry staining revealed that ACAA1 were dominantly located in the mammary epithelial cells of these two dairy animals. Knockdown of ACAA1 inhibited mammary epithelial cell proliferation and triglyceride and ß-casein secretion by regulating related gene expressions in cattle and buffalo. In contrast, ACAA1 overexpression promoted cell proliferation and triglyceride secretion. Finally, three novel SNPs (g.-681A>T, g.-23117C>T, and g.-24348G>T) were detected and showed significant association with milk production traits of Mediterranean buffaloes. In addition, g.-681A>T mutation located in the promoter region changed transcriptional activity significantly. Our findings suggested that ACAA1 play a key role in regulating buffalo and cattle milk synthesis and provided basic information to further understand the dairy animal lactation physiology.

MAEL gene contributes to bovine testicular development through the m5C-mediated splicing.

Knowledge of RNA molecules regulating testicular development and spermatogenesis in bulls is essential for elite bull selection and an ideal breeding program. Herein, we performed direct RNA sequencing (DRS) to explore the functional characterization of RNA molecules produced in the testicles of 9 healthy Simmental bulls at three testicular development stages (prepuberty, puberty, and postpuberty). We identified 5,043 differentially expressed genes associated with testicular weight. These genes exhibited more alternative splicing at sexual maturity, particularly alternative 3' (A3) and 5' (A5) splice sites usage and exon skipping (SE). The expression of hub genes in testicular developmental stages was also affected by both m6A and m5C RNA modifications. We found m5C-mediated splicing events significantly (p < 0.05) increased MAEL gene expression at the isoform level, likely promoting spermatogenesis. Our findings highlight the complexity of RNA processing and expression as well as the regulation of transcripts involved in testicular development and spermatogenesis.

Comparative analyses of copy number variations between swamp buffaloes and river buffaloes.

As an important source of genomic variation, copy number variation (CNV) contributes to environmental adaptation in worldwide buffaloes. Despite this importance, CNV divergence between swamp buffaloes and river buffaloes has not been studied previously. Here, we report 21 152 CNV regions (CNVRs) in 141 buffaloes of 20 breeds detected through multiple CNV calling strategies. Only 248 CNVRs were shared between river buffalo and swamp buffalo, reflecting great variation of CNVRs between the two subspecies. Population structure analysis based on CNVs successfully separated the two buffalo subspecies. We further assessed CNV divergence by calculating FST for genome-wide CNVs. Totally, we identified 110 significantly divergent CNV segments and 44 putatively selected genes between river buffaloes and swamp buffaloes. In particular, LALBA, a key gene controlling milk production in cattle, presented a highly differentiated CNV in the promoter region, which makes it a strong functional candidate gene for differences between swamp buffaloes and river buffaloes in traits related to milk production. Our study provides useful information of CNVs in buffaloes, which may help explain the genetic differences between the two subspecies.

Whole-genome SNP markers reveal runs of homozygosity in indigenous cattle breeds of Pakistan.

The runs of homozygosity (ROH) were identified in 14 Pakistani cattle breeds (n = 105) by genotyping with the Illumina 50 K SNP BeadChip. These breeds were categorized into Dairy, Dual, and Draft breeds based on their utility and production performance. We identified a total of 10,936 ROHs which mainly consisted of a high number of shorter segments (1-4 Mb). Dairy group exhibited the highest level of inbreeding (FROH: 0.078 ± 0.028) while the lowest (FROH: 0.002 ± 0.008) was observed in Dual group. In 48 genomic regions identified with a high frequency of ROH, 207 genes were detected in the three breed groups. A substantially higher number of ROH islands detected in dairy breeds indicated the impact of the positive selection pressure over the years. Important candidate genes and QTL were detected in the ROH islands associated with economic traits like milk production, reproduction, meat, carcass, and health traits in dairy cattle.

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.

Novel Insight Into the Role of ACSL1 Gene in Milk Production Traits in Buffalo.

Understanding the genetic mechanisms underlying milk production traits contribute to improving the production potential of dairy animals. Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism and was highly expressed in the lactating mammary gland epithelial cells (MGECs). The objectives of the present study were to detect the polymorphisms within ACSL1 in Mediterranean buffalo, the genetic effects of these mutations on milk production traits, and understand the gene regulatory effects on MGECs. A total of twelve SNPs were identified by sequencing, including nine SNPs in the intronic region and three in the exonic region. Association analysis showed that nine SNPs were associated with one or more traits. Two haplotype blocks were identified, and among these haplotypes, the individuals carrying the H2H2 haplotype in block 1 and H5H1 in block 2 were superior to those of other haplotypes in milk production traits. Immunohistological staining of ACSL1 in buffalo mammary gland tissue indicated its expression and localization in MGECs. Knockdown of ACSL1 inhibited cell growth, diminished MGEC lipid synthesis and triglyceride secretion, and downregulated CCND1, PPARγ, and FABP3 expression. The overexpression of ACSL1 promoted cell growth, enhanced the triglyceride secretion, and upregulated CCND1, PPARγ, SREBP1, and FABP3. ACSL1 was also involved in milk protein regulation as indicated by the decreased or increased ß-casein concentration and CSN3 expression in the knockdown or overexpression group, respectively. In summary, our present study depicted that ACSL1 mutations were associated with buffalo milk production performance. This may be related to its positive regulation roles on MGEC growth, milk fat, and milk protein synthesis. The current study showed the potential of the ACSL1 gene as a candidate for milk production traits and provides a new understanding of the physiological mechanisms underlying milk production regulation.

Novel Insight into the Potential Role of Acylglycerophosphate Acyltransferases Family Members on Triacylglycerols Synthesis in Buffalo.

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.

Novel functional mutation of the PDIA3 gene affects milk composition traits in Chinese Holstein cattle.

Protein disulfide isomerase family A member 3 (PDIA3) is a multifunctional protein, and it plays a vital role in modulating various cell biological functions under physiological and pathological conditions. Our previous study on Mediterranean buffalo demonstrated that PDIA3 is a potential candidate gene associated with milk yield based on genome-wide association study analysis. However, the genetic effects of the PDIA3 gene on milk performance in dairy cattle and the corresponding mechanism have not been documented. This study aims to explore the genetic effects of PDIA3 polymorphisms on milk production traits in 362 Chinese Holstein cattle. The results showed that 4 SNPs were identified from the 5' untranslated region of the PDIA3 gene in the studied population, of which 2 SNPs (g.-1713 C>T and g.-934 G>A) were confirmed to be significantly associated with milk protein percentage, whereas g.-434 C>T was significantly associated with milk fat percentage. Notably, linkage disequilibrium analysis indicated that 3 SNPs (g.-1713 C>T, g.-934 G>A, and g.-695 A>C) formed one haplotype block, which was found to be significantly associated with milk protein percentage. The luciferase assay demonstrated that allele C of g.-434 C>T exhibited a higher promotor activity compared with allele T, suggesting that g.-434 C>T might be a potential functional mutation affecting PDIA3 expression. Furthermore, overexpression of the PDIA3 gene was found to induce higher levels of triglyceride and BODIPY fluorescence intensity. In addition, PDIA3 overexpression was also found to positively regulate the synthesis and secretion of α-casein, ß-casein, and κ-casein, whereas knockdown of this gene showed the opposite effects. In summary, our findings revealed significant genetic effects of PDIA3 on milk composition traits, and the identified SNP and the haplotype block might be used as genetic markers for dairy cow selected breeding.

Genome-wide analysis of runs of homozygosity in Italian Mediterranean buffalo.

Runs of homozygosity (ROH) are a powerful tool to explore patterns of genomic inbreeding in animal populations and detect signatures of selection. The present study used ROH analysis to evaluate the genome-wide patterns of homozygosity, inbreeding levels, and distribution of ROH islands using the SNP data sets from 899 Mediterranean buffaloes. A total of 42,433 ROH segments were identified, with an average of 47.20 segments per individual. The ROH comprising mostly shorter segments (1-4 Mb) accounted for approximately 72.29% of all ROH. In contrast, the larger ROH (>8 Mb) class accounted for only 7.97% of all ROH segments. Estimated inbreeding coefficients from ROH (FROH) ranged from 0.0201 to 0.0371. Pearson correlations between FROH and genomic relationship matrix increased with the increase of ROH length. We identified ROH hotspots in 12 genomic regions, located on chromosomes 1, 2, 3, 5, 17, and 19, harboring a total of 122 genes. Protein-protein interaction (PPI) analysis revealed the clustering of these genes into 7 PPI networks. Many genes located in these regions were associated with different production traits. In addition, 5 ROH islands overlapped with cattle quantitative trait loci that were mainly associated with milk traits. These findings revealed the genome-wide autozygosity patterns and inbreeding levels in Mediterranean buffalo. Our study identified many candidate genes related to production traits that could be used to assist in selective breeding for genetic improvement of buffalo.

Evolutionary Analysis of OAT Gene Family in River and Swamp Buffalo: Potential Role of SLCO3A1 Gene in Milk Performance.

The organic anion transporter (OAT) family is the subfamily of the solute carrier (SLC) superfamily, which plays a vital role in regulating essential nutrients in milk. However, little is known about the members' identification, evolutionary basis, and function characteristics of OAT genes associated with milk performance in buffalo. Comparative genomic analyses were performed to identify the potential role of buffalo OAT genes in milk performance in this study. The results showed that a total of 10 and 7 OAT genes were identified in river buffalo and swamp buffalo, respectively. These sequences clustered into three groups based on their phylogenetic relationship and had similar motif patterns and gene structures in the same groups. Moreover, the river-specific expansions and homologous loss of OAT genes occurred in the two buffalo subspecies during the evolutionary process. Notably, the duplicated SLCO3A1 gene specific to river buffalo showed higher expression level in mammary gland tissue than that of swamp buffalo. These findings highlight some promising candidate genes that could be potentially utilized to accelerate the genetic progress in buffalo breeding programs. However, the identified candidate genes require further validation in a larger cohort for use in the genomic selection of buffalo for milk production.

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.

Genetic Features of Reproductive Traits in Bovine and Buffalo: Lessons From Bovine to Buffalo.

Bovine and buffalo are important livestock species that have contributed to human lives for more than 1000 years. Improving fertility is very important to reduce the cost of production. In the current review, we classified reproductive traits into three categories: ovulation, breeding, and calving related traits. We systematically summarized the heritability estimates, molecular markers, and genomic selection (GS) for reproductive traits of bovine and buffalo. This review aimed to compile the heritability and genome-wide association studies (GWASs) related to reproductive traits in both bovine and buffalos and tried to highlight the possible disciplines which should benefit buffalo breeding. The estimates of heritability of reproductive traits ranged were from 0 to 0.57 and there were wide differences between the populations. For some specific traits, such as age of puberty (AOP) and calving difficulty (CD), the majority beef population presents relatively higher heritability than dairy cattle. Compared to bovine, genetic studies for buffalo reproductive traits are limited for age at first calving and calving interval traits. Several quantitative trait loci (QTLs), candidate genes, and SNPs associated with bovine reproductive traits were screened and identified by candidate gene methods and/or GWASs. The IGF1 and LEP pathways in addition to non-coding RNAs are highlighted due to their crucial relevance with reproductive traits. The distribution of QTLs related to various traits showed a great differences. Few GWAS have been performed so far on buffalo age at first calving, calving interval, and days open traits. In addition, we summarized the GS studies on bovine and buffalo reproductive traits and compared the accuracy between different reports. Taken together, GWAS and candidate gene approaches can help to understand the molecular genetic mechanisms of complex traits. Recently, GS has been used extensively and can be performed on multiple traits to improve the accuracy of prediction even for traits with low heritability, and can be combined with multi-omics for further analysis.

Transcriptome-Wide m6A Analysis Provides Novel Insights Into Testicular Development and Spermatogenesis in Xia-Nan Cattle.

Testis is the primary organ of the male reproductive tract in mammals that plays a substantial role in spermatogenesis. Improvement of our knowledge regarding the molecular mechanisms in testicular development and spermatogenesis will be reflected in producing spermatozoa of superior fertility. Evidence showed that N6-Methyladenosine (m6A) plays a dynamic role in post-transcription gene expression regulation and is strongly associated with production traits. However, the role of m6A in bovine testis has not been investigated yet. In this study, we conducted MeRIP-Seq analysis to explore the expression profiles of the m6A and its potential mechanism underlying spermatogenesis in nine bovine testes at three developmental stages (prepuberty, puberty and postpuberty). The experimental animals with triplicate in each stage were chosen based on their semen volume and sperm motility except for the prepuberty bulls and used for testes collection. By applying MeRIP-Seq analysis, a total of 8,774 m6A peaks and 6,206 m6A genes among the studied groups were identified. All the detected peaks were found to be mainly enriched in the coding region and 3'- untranslated regions. The cross-analysis of m6A and mRNA expression exhibited 502 genes with concomitant changes in the mRNA expression and m6A modification. Notably, 30 candidate genes were located in the largest network of protein-protein interactions. Interestingly, four key node genes (PLK4, PTEN, EGR1, and PSME4) were associated with the regulation of mammal testis development and spermatogenesis. This study is the first to present a map of RNA m6A modification in bovine testes at distinct ages, and provides new insights into m6A topology and related molecular mechanisms underlying bovine spermatogenesis, and establishes a basis for further studies on spermatogenesis in mammals.

Knockdown of CYP19A1 in Buffalo Follicular Granulosa Cells Results in Increased Progesterone Secretion and Promotes Cell Proliferation.

Cytochrome P450 aromatase 19A1 (CYP19A1) is a critical enzyme in estrogen synthesis. However, the effect of CYP19A1 on cell growth and hormone secretion of buffalo follicular granulosa cells (BFGCs) is poorly understood. The objective of this study was to assess the role of CYP19A1 in cell proliferation and hormone secretion of BFGCs by knocking down CYP19A1 mRNA expression. The mRNA expression level of CYP19A1 gene was knocked down in BFGCs using the siCYP19A1-296 fragment with the best interference efficiency of 72.63%, as affirmed by real-time quantitative PCR (qPCR) and cell morphology analysis. The CYP19A1 knockdown promoted the proliferation of BFGCs through upregulating the mRNA expression levels of six proliferation-related genes (CCND1, CCNE1, CCNB1, CDK2, CDKN1A, and CDKN1B). Moreover, CYP19A1 knockdown increased (P < 0.05) the concentrations of progesterone secretion (P4) in BFGCs through increasing the mRNA expression levels of three steroidogenic genes (HSD17B1, HSD17B7, and CYP17A1). Our data further found that the FSH could inhibit the mRNA expression level of CYP19A1 in BFGCs, while LH obtains the opposite effect. These findings showed that the CYP19A1 knockdown had a regulatory role in the hormone secretion and cell proliferation in BFGCs.