Jianfang Wang

Single-cell omics technologies have transformed the study of cellular heterogeneity, enabling high-resolution analysis of tissue development and complex traits. In sheep and goats, these approaches have been applied to skin, hair follicles, reproductive organs, metabolic tissues, and adipose tissue, revealing cell type-specific regulatory programs underlying traits such as wool quality, fertility, growth, and fat deposition. However, most studies rely on single-cell RNA sequencing (scRNA-seq) and are limited by incomplete genome annotation, insufficient coverage of production traits, and weak integration with population genetics, restricting their application in molecular breeding. This review summarizes advances in single-cell omics in sheep and goats, focusing on tissue development and trait formation. We further discuss emerging strategies that integrate single-cell multi-omics, spatial transcriptomics, and population genetics to resolve regulatory mechanisms in a cell type-specific and spatially informed context. Finally, we discuss CRISPR/Cas9-based validation to link genotype and phenotype, accelerating gene discovery and precision breeding in small ruminants.

Adipogenesis, a complex process, is primarily determined by the number of preadipocytes and their adipogenic capacity. Preadipocyte proliferation and apoptosis regulate this cellular reserve, which is essential for adipogenic differentiation. CircRNAs are significant regulators of various cellular processes, yet their roles in preadipocyte proliferation and apoptosis remain underexplored. In this study, gain- and loss-of-function experiments show that circGLIS3 promotes proliferation and restrains the apoptosis of bovine intramuscular preadipocytes. Mechanistically, circGLIS3 serves as a molecular sponge for miR-21-3p, thereby derepressing PDPK1 and activating the Akt signaling pathway, as validated by dual-luciferase reporter, RNA immunoprecipitation, and rescue assays. Notably, circGLIS3 exhibits high sequence conservation between the Bos taurus and Mus musculus. In vivo overexpression of circGLIS3 in mouse skeletal muscle increased PDGFRα- and Pref-1-positive preadipocyte populations. This study elucidates the molecular mechanism by which the circGLIS3/miR-21-3p/PDPK1/Akt axis regulates bovine preadipocyte proliferation and apoptosis, thereby providing significant theoretical insights into bovine IMF deposition.

MicroRNAs (miRNAs) have a critical role in controlling myogenesis and the quality of meat in beef cattle. Numerous miRNA types have been identified to have regulatory roles in the development of bovine skeletal muscle, including Bta-miR-130, miR-148a-3p, miR-224, Bta-miR-182, Bta-miR-183, and Bta-miR-338, as well as miR-214, miR-26a, miR-206, miR-885, and miR-196a. Exploring the intricate regulation system of microRNAs in the development of muscles and quality traits can offer valuable insights for enhancing beef production and meat quality. Investigations into the effects of microRNAs on myogenesis and meat characteristics could result in novel breeding strategies and genetic enhancement initiatives. Additionally, microRNAs could function as biomarkers for assessing meat quality in beef cattle, providing opportunities to modulate muscle growth, composition, and overall characteristics within livestock species ultimately yielding high-quality meats aligned with consumer preferences. This paper delves into role of microRNAs in myogenesis and their functions in beef cattle meat quality while focusing on their potential as targets for genetic improvement to increase production potential and quality. Recognizing the impact of microRNA on shaping meat quality has the potential to revolutionize global competitiveness within the Beef industry. Recent genomics advancements, including molecular markers and genetic variations, can boost animal productivity, meeting global demands for high-quality meat products. This review paper will establishes a foundation for future research on understanding regulatory networks linked to Myogenesis and function of micro RNAs in meat quality, contributing to both scholarly knowledge advancement and practical applications within animal agriculture.

MicroRNAs (miRNAs) and epigenetic processes in bovine adipocytes can provide valuable new insights into controlling adipogenesis in livestock. It modulates gene expression at the posttranscriptional stage by affecting mRNA translation and stability. It plays a significant role in the regulation of a wide range of physiological and cellular functions, such as immune system processes and cellular and intramuscular fat development. Transcriptional regulators such as PPARγ and C/EBP proteins are key mediators of the critical processes governing progenitor cell differentiation into adipocytes. The differentiation of these progenitor cells into adipocytes, as well as the subsequent lipid deposition within the adipocytes, is influenced by a variety of factors. These include genetics; epigenetics; nutritional; hormonal regulation; weaning and slaughtering; aging and weight; and stress levels. Epigenetic modifications such as chromatin reorganization, histone alterations, and DNA methylation subsequently affect the activation of genes related to adipogenesis and the progression of adipocyte differentiation. By investigating how fat deposition is epigenetically regulated in beef cattle, scientists aim to unravel molecular mechanisms, identify key regulatory genes and pathways, and develop targeted strategies for modifying fat deposition to enhance desirable traits such as marbling and meat tenderness. This review paper delves into miRNAs and epigenetic factors and their role in regulating bovine adipocytes and it will identify links to transcription factors that influence diverse aspects of meat quality, encompassing flavor, juiciness, tenderness, firmness, texture, and visual appearance. Cutting-edge developments in genomics, including the identification of molecular markers and genetic variations, hold promise for enhancing animal productivity and meeting the global demand for premium-quality meat. This review establishes a foundation for future research on understanding regulatory networks linked to miRNAs and epigenetic changes, contributing to both scholarly knowledge advancement and practical applications within animal agriculture.

Objective: Our study aimed to investigate the gender and age-related variations in rumen fermentation, serum metabolites, and microbiota in Qinchuan cattle.Methods: A total of 38 Qinchuan beef cattle were selected and maintained on a uniform diet for three months. Rumen fluid and blood samples were collected to determine rumen fermentation, serum metabolites, and microbial 16S rRNA sequencing.Results: The results revealed that the concentration of rumen butyrate in female Qinchuan cattle was significantly higher than in males (p<0.05). Isobutyrate, butyrate, and isovalerate exhibited significant age-related differences. Females exhibited lower serum glucose (GLU) and higher triglycerides (TG), nonesterifiedfatty acid (NEFA) levels compared to males (p<0.05). Serum albumin (ALB) and urea (UA) levels increased with age (p<0.05). Furthermore, the alpha diversity of rumen bacteria improved with age (p<0.05), with no gender differences observed. Males had higher relative abundances of Bacteroidota, Verrucomicrobiota, and Cyanobacteria, while females had higher Firmicutes and Desulfobacterota (p<0.05). The cellulose-degrading genus Ruminococcus and propionateproducing genus Succiniclasticum were more abundant in females, whereas the antiinflammatory genus Lachnospiraceae_NK4A136_group and the hemicellulose-degrading genus Prevotella were more abundant in males (p<0.05). Age-related differences in bacteria were found in Pseudobutyrivibrio and several members of the Lachnospiraceae. Functional prediction indicated that “Amino acid metabolism” and “Lipid metabolism” were mainly enriched in females, whereas “Carbohydrate metabolism” and “Glycan biosynthesis and metabolism” were enriched in males (p<0.05). RDA analysis highlighted butyrate as a key factor influencing the rumen bacterial community. NK4A214_group and Ruminococcus were positively correlated with butyrate, while Prevotella and Pseudobutyrivibrio were negatively correlated with butyrate (p<0.05).Conclusion: We observed a significant improvement in the diversity and stability of rumen microbiota as age increased. Ruminococcus, NK4A214_group, and Prevotella were likely contributors to variations in energy utilization and fat deposition between male and female Qinchuan cattle.

Leptin can indirectly regulate fatty‐acid metabolism and synthesis in muscle in vivo and directly in incubated muscle ex vivo. In addition, non‐synonymous mutations in the bovine leptin gene ( LEP ) are associated with carcass intramuscular fat (IMF) content. However, the effects of LEP on lipid synthesis of adipocytes have not been clearly studied at the cellular level. Therefore, this study focused on bovine primary intramuscular preadipocytes to investigate the effects of LEP on the proliferation and differentiation of intramuscular preadipocytes, as well as its regulatory mechanism in lipid synthesis. The results showed that both the LEP and leptin receptor gene ( LEPR ) were highly expressed in IMF tissues, and their mRNA expression levels were positively correlated at different developmental stages of intramuscular preadipocytes. The overexpression of LEP inhibited the proliferation and differentiation of intramuscular preadipocytes, while interference with LEP had the opposite effect. Additionally, LEP significantly promoted the phosphorylation level of AMPKα by promoting the protein expression of CAMKK2. Meanwhile, rescue experiments showed that the increasing effect of AMPK inhibitors on the number of intramuscular preadipocytes was significantly weakened by the overexpression of LEP . Furthermore, the overexpression of LEP could weaken the promoting effect of AMPK inhibitor on triglyceride content and droplet accumulation, and prevent the upregulation of adipogenic protein expression (SREBF1, FABP4, FASN, and ACCα) caused by AMPK inhibitor. Taken together, LEP acted on the AMPK signaling pathway by regulating the protein expression of CAMKK2, thereby downregulating the expression of proliferation‐related and adipogenic‐related genes and proteins, ultimately reducing intramuscular adipogenesis.

As cellular transcription factors and DNA replicators, nuclear factor I (NFI) family members play an important role in mammalian development. However, there is still a lack of research on the muscle regeneration of NFI family members in cattle. In this study, the analysis of NFI family factors was conducted on their characterization, phylogenetics, and functional domains. We found that NFI family members were relatively conserved among different species, but there was heterogeneity in amino acid sequences, DNA coding sequences, and functional domain among members. Furthermore, among NFI family factors, we observed that NFIC exhibited highly expression in bovine muscle tissues, particularly influencing the expression of proliferation marker genes in myoblasts. To investigate the influence of NFIC on myoblast proliferation, we knocked down NFIC (si-NFIC) and found that the proliferation of myoblasts was significantly promoted. In terms of regulation mechanism, we identified that si-NFIC could counteract the inhibitory effect of the cell cycle inhibitor RO-3306. Interestingly, CENPF, as the downstream target gene of NFIC, could affect the expression of CDK1, CCNB1, and actively regulate the cell cycle pathway and cell proliferation. In addition, when CENPF was knocked down, the phosphorylation of p53 and the expression of Bax were increased, but the expression of Bcl2 was inhibited. Our findings mainly highlight the mechanism by which NFIC acts on the CENPF/CDK1 axis to regulate the proliferation of bovine myoblasts.

Tropomyosin 3 (TPM3) plays a significant role as a regulatory protein in muscle contraction, affecting the growth and development of skeletal muscles. Despite its importance, limited research has been conducted to investigate the influence of TPM3 on bovine skeletal muscle development. Therefore, this study revealed the role of TPM3 in bovine myoblast growth and development. This research involved conducting a thorough examination of the Qinchuan cattle TPM3 gene using bioinformatics tools to examine its sequence and structural characteristics. Furthermore, TPM3 expression was evaluated in various bovine tissues and cells using quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that the coding region of TPM3 spans 855 bp, with the 161st base being the T base, encoding a protein with 284 amino acids and 19 phosphorylation sites. This protein demonstrated high conservation across species while displaying a predominant α-helix secondary structure despite being an unstable acidic protein. Notably, a noticeable increase in TPM3 expression was observed in the longissimus dorsi muscle and myocardium of calves and adult cattle. Expression patterns varied during different stages of myoblast differentiation. Functional studies that involved interference with TPM3 in Qinchuan cattle myoblasts revealed a very significantly decrease in S-phase cell numbers and EdU-positive staining (P < 0.01), and disrupted myotube morphology. Moreover, interference with TPM3 resulted in significantly (P < 0.05) or highly significantly (P < 0.01) decreased mRNA and protein levels of key proliferation and differentiation markers, indicating its role in the modulation of myoblast behavior. These findings suggest that TPM3 plays an essential role in bovine skeletal muscle growth by influencing myoblast proliferation and differentiation. This study provides a foundation for further exploration into the mechanisms underlying TPM3-mediated regulation of bovine muscle development and provides valuable insights that could guide future research directions as well as potential applications for livestock breeding and addressing muscle-related disorders.

The intramuscular fat (or marbling fat) content is an essential economic trait of beef cattle and improves the flavor and palatability of meat. Several studies have highlighted the correlation between long non-coding RNAs (lncRNAs) and intramuscular fat development; however, the precise molecular mechanism remains unknown. Previously, through a high-throughput sequencing analysis, we found a lncRNA and named it a long non-coding RNA BNIP3 (lncBNIP3). The 5′ RACE and 3′ RACE explored 1945 bp total length of lncBNIP3, including 1621 bp of 5′RACE, and 464 bp of 3′RACE. The nucleoplasmic separation and FISH results explored the nuclear localization of lncBNIP3. Moreover, the tissue expression of lncBNIP3 was higher in the longissimus dorsi muscle, followed by intramuscular fat. Furthermore, down-regulation of lncBNIP3 increased the 5-Ethynyl-2′- deoxyuridine (EdU)-EdU-positive cells. The flow cytometry results showed that the number of cells in the S phase was significantly higher in preadipocytes transfected with si-lncBNIP3 than in the control group (si-NC). Similarly, CCK8 results showed that the number of cells after transfection of si-lncBNIP3 was significantly higher than in the control group. In addition, the mRNA expressions of proliferative marker genes CyclinB1 (CCNB1) and Proliferating Cell Nuclear Antigen (PCNA) in the si-lncBNIP3 group were significantly higher than in the control group. The Western Blot (WB) results also showed that the protein expression level of PCNA transfection of si-lncBNIP3 was significantly higher than in the control group. Similarly, the enrichment of lncBNIP3 significantly decreased the EdU-positive cells in the bovine preadipocytes. The results of flow cytometry and CCK8 assay also showed that overexpression of lncBNIP3 inhibited the proliferation of bovine preadipocytes. In addition, the overexpression of lncBNIP3 significantly inhibited the mRNA expressions of CCNB1 and PCNA. The WB results showed that the overexpression of lncBNIP3 significantly inhibited the expression of the CCNB1 protein level. To further explore the mechanism of lncBNIP3 on the proliferation of intramuscular preadipocytes, RNA-seq was performed after interference with si-lncBNIP3, and 660 differentially expressed genes (DEGs) were found, including 417 up-regulated DEGs and 243 down-regulated DEGs. The KEGG pathway analysis showed that the cell cycle was the most significant pathway for the functional enrichment of DEGs, followed by the DNA replication pathway. The RT-qPCR quantified the expression of twenty DEGs in the cell cycle. Therefore, we speculated that lncBNIP3 regulated intramuscular preadipocyte proliferation through the cell cycle and DNA replication pathways. To further confirm this hypothesis, the cell cycle inhibitor Ara-C was used to inhibit DNA replication of the S phase in intramuscular preadipocytes. Herein, Ara-C and si-lncBNIP3 were simultaneously added to the preadipocytes, and the CCK8, flow cytometry, and EdU assays were performed. The results showed that the si-lncBNIP3 could rescue the inhibitory effect of Ara-C in the bovine preadipocyte proliferation. In addition, lncBNIP3 could bind to the promoter of cell division control protein 6 (CDC6), and down-regulation of lncBNIP3 promoted the transcription activity and the expression of CDC6. Therefore, the inhibitory effect of lncBNIP3 on cell proliferation might be understood through the cell cycle pathway and CDC6 expression. This study provided a valuable lncRNA with functional roles in intramuscular fat accumulation and revealed new strategies for improving beef quality.