Introduction:Chinese herbal medicines, including honeysuckle (Lonicera japonica), exhibit diverse beneficial properties and are increasingly being explored as feed additives in livestock production to improve animal health and product quality. This study investigates the effects of honeysuckle vine powder (HVP) supplementation as an herbal feed additive on growth performance, immune function, and rumengut microbiota in Nanjiang Yellow goats during fattening. Methods:We evaluated four dietary groups: control (basal diet) and three experimental groups supplemented with 1% (EG1), 1.5% (EG2), and 2% HVP (EG3) for 60 days. After sampling, we measured the blood biochemical, immune indicators and carried out 16s RNA sequencing of rumen and gut microbiota for Nanjiang Yellow goat samples. Results:We evaluated four dietary groups: control (basal diet) and three experimental groups supplemented with 1% (EG1), 1.5% (EG2), and 2% HVP (EG3) for 60 days. Results demonstrated that 2% HVP significantly enhanced, average daily gain (ΔADG = 0.05 ± 0.02 /kg, p < 0.05, n = 8), and feed conversion rate (ΔFCR = 5.26 ± 0.69, p < 0.05, n = 8), while improving body length and chest circumference. Serum immunoglobulin (IgG, IgM) levels were elevated, indicating strengthened systemic immunity (p < 0.05). Critically, 16S rRNA analysis revealed HVP-induced rumen and gut microbiota restructuring, characterized by increased Firmicutes abundance. Discussion:These findings validate HVP as a sustainable feed additive that optimizes productivity and immune resilience through microbiota-host crosstalk, supporting its application in ecofriendly livestock farming.

The role of fatty acid transporter 4 (FATP4) in regulating lipid metabolism has been well studied. However, how it affects IMF deposition, especially in goats, remains poorly understood. Here, we cloned the whole coding sequence of the goat FATP4 gene and revealed its closest affinity to sheep by amino acid sequence blast analysis. In addition, we found that the FATP4 reached its highest expression level at day 6 of goat preadipocyte differentiation in vitro. Functionally, in cultured goat intramuscular preadipocytes, siRNA-induced FATP4 knockdown dramatically raised the mRNA expression of lipogenesis-related genes and encouraged lipid deposition. At the same time, FATP4 deficiency inhibited cell proliferation and significantly decreased apoptosis. Unexpectedly, although the overexpression of FATP4 promoted cell proliferation and suppressed apoptosis, it only slightly decreased cellular lipid deposition in goat intramuscular preadipocytes. For RNA-seq (performed on pooled cell samples with three technical replicates), a total of 467 differential genes (DEGs) were identified after silencing of FATP4 in goat preadipocytes, including 47 upregulated genes and 420 downregulated genes. These DEGs were mainly enriched in the signaling pathways of Focal adhesion, HIF-1, and PI3K-Akt by KEGG analysis. To validate these findings, knockdown of FATP4 increased the expression of phosphatidylinositol 3-kinase (PI3k) and vice versa. Convincingly, we rescued the phenotype observed in FATP4 knockout goat preadipocytes by blocking the PI3k-Akt signaling pathway with an AKT inhibitor (LY294002). In summary, in our in vitro model, FATP4 plays a crucial role in directing fatty acids toward cell proliferation (prioritized over cellular lipid deposition) via the PI3K-Akt signaling pathway in goat intramuscular preadipocytes. These findings provide preliminary mechanistic insights into the regulatory network of IMF formation at the cellular level, and offer theoretical clues for future research aimed at enhancing meat quality from the standpoint of IMF deposition.

Enhancing intramuscular fat (IMF) to improve the quality of livestock product has long been a goal in animal breeding. Recent studies have revealed a strong connection between malonylation and lipid metabolism, yet the function of malonylated proteins in ruminants largely unclear. In the present study, we identified the third day of goat intramuscular preadipocyte differentiation as a critical time point for lipid accumulation, with no significant alterations in malonylation levels. We identified 212 and 216 malonylated proteins on day 0 (d0) and day 3 (d3) of differentiation, respectively, enrichment in pathways such as glycolysis/gluconeogenesis, tight junctions, and actin cytoskeleton regulation. Our findings demonstrate the consistent presence of malonylation during preadipocyte differentiation, with minor quantitative variations, and highlight key malonylated proteins closely associated with lipid metabolism, including acetyl-CoA carboxylase (ACACA), translation control tumor protein 1 (TPT1), phosphoglycerate kinase 1 (PGK1), annexin A6 (ANXA6), and annexin A2 (ANXA2). Collectively, our study uncovers critical malonylated proteins during preadipocyte differentiation, establishing a foundation for exploring their roles in intramuscular fat deposition. SIGNIFICANCE: Currently, efforts are being made to improve meat quality by enhancing intramuscular fat (IMF) deposition, thereby promoting the development of the livestock industry. This study addresses a critical gap in our understanding of malonylation, a key post-translational modification, in livestock. By constructing the first malonylation protein modification map in goats and revealing dynamic changes during intramuscular preadipocyte differentiation, this research offers novel insights into the regulatory roles of malonylation in fat deposition. The findings not only advance the field of livestock proteomics but also provide a theoretical foundation for improving meat quality and exploring metabolic regulation in animals.

This study aims to investigate the effects of the vine of Lonicera japonica Thunb (VLT) and marine-derived Bacillus amyloliquefaciens-9 (BA-9) supplementation on the growth performance, antioxidant capacity, and gut microbiota of goat kids. A total of 32 4-week-old kids were randomly assigned into four groups: a control group (CON), a group supplemented with 0.3% BA-9 (BA-9), a group supplemented with 2% VLT (VLT), and a group supplemented with both 0.3% BA-9 and 2% VLT (MIX). The results indicated that VLT supplementation significantly increased both average daily (P < 0.001) and total weight gain (TWG) (P < 0.001), while BA-9 alone had no significant effect (P > 0.05) on the average daily and TWG. Biomarker analysis of oxidative stress revealed that supplementation of VLT or BA-9 alone enhanced antioxidant capacity. The MIX group showing a higher total antioxidant capacity (T-AOC) compared with the CON, VLT, and BA-9 groups (P < 0.05). Plasma albumin (ALB) levels were significantly increased in the both VLT and BA-9 groups. Microbiota analysis revealed significant differences in α-diversity and β-diversity between the MIX and CON groups, with specific genera such as Prevotellaceae_UCG.004 and Rikenellaceae_RC9_gut_group negatively correlated with average daily gain (ADG), while Alistipes was positively correlated with T-AOC. These findings suggest that the combined supplementation of VLT and BA-9 can significantly enhance growth performance and antioxidant capacity in goat kids by modulating the composition of gut microbiota and reducing oxidative stress.

Skeletal muscle is the most widespread tissue in mammals and mediates several functions, and whose development is controlled by a coordinated transcriptional hierarchy that regulates the activities of a range of muscle genes. Skeletal muscle comprises various cells that create communication strategies for exchanging biological information. Nevertheless, the features and developmental programs of several of these cell lines remain unknown. We constructed a complete single-cell landscape of prenatal to postnatal developing bovine skeletal muscle and compared its single-cell transcriptomic characteristics with those of humans and mice. This landscape involved cellular heterogeneity, dynamic gene expression profiles, critical regulons during cell fate decisions, extensive networks of intercellular communication, and a gene regulation network. Overall, our results identify a developmental coordinate of the pluripotency spectrum among bovines, humans, and mice. This finding suggests evolutionary conservation and species-specific differences in the skeletal muscle systems, extending to cell types, gene expression, and regulatory elements. These results offer insights into evolutionary conserved and divergent processes during mammalian skeletal muscle development.

Adipose tissue affects not only the meat quality of domestic animals, but also human health. Adipocyte differentiation is regulated by a series of regulatory genes and cyclins. Four and half-LIM protein (FHL2) is positively correlated with the hypertrophy of adipocytes and can cause symptoms such as obesity and diabetes. In the transcriptome sequencing analysis of intramuscular adipocytes after three days of differentiation, the differentially expressed gene FHL2 was found. To further explore the biological significance of the differentially expressed gene FHL2, which was downregulated in the mature adipocytes. We revealed the function of FHL2 in adipogenesis through the acquisition and loss of function of FHL2. The results showed that the overexpression of FHL2 significantly increased the expression of adipogenic genes (PPARγ, C/EBPβ) and the differentiation of intramuscular and subcutaneous adipocytes. However, silencing FHL2 significantly inhibited adipocyte differentiation. The overexpression of FHL2 increased the number of adipocytes stained with crystal violet and increased the mRNA expression of proliferation marker genes such as CCNE, PCNA, CCND and CDK2. In addition, it significantly increased the rate of EdU positive cells. In terms of apoptosis, overexpression of FHL2 significantly inhibited the expression of P53 and BAX in both intramuscular and subcutaneous adipocytes, which are involved in cell apoptosis. However, overexpression of FHL2 promoted the expression of BCL, but was rescued by the silencing of FHL2. In summary, FHL2 may be a positive regulator of intramuscular and subcutaneous adipocyte differentiation and proliferation, and acts as a negative regulator of intramuscular and subcutaneous adipocyte apoptosis. These findings provide a theoretical basis for the subsequent elucidation of FHL2 in adipocytes.

BackgroundInfectious ecthyma is a severe and highly contagious disease caused by ORF virus (ORFV). The virus is responsible for significant economic losses in the goat industry and threatens humans. Regarding complement component 1, q subcomponent binding protein (C1QBP), we previously showed that C1QBP can interact with ORFV129. However, the role of C1QBP in regulating the apoptosis of goat fetal turbinate cells is unknown.MethodsAfter pcDNA3.1-C1QBP and siRNA were transfected into goat fetal turbinate cells (GFTCs), the expression of C1QBP was detected by western blot and cell cycle and apoptosis using flow cytometry. The expression of cycle-related genes cyclin-dependent protein kinase 2 (CDK2) and cyclin-dependent kinase inhibitors 1A (p21) and apoptosis-related genes cysteinyl aspartate specific proteinase 3 (Caspase 3), cysteinyl aspartate specific proteinase 7 (Caspase 7), p53, poly (ADP-ribose) polymerase 1 (PARP1), B-Cell lymphoma-2 Like-11 (BCL2L11), B-cell lymphoma 2 associated X protein (Bax), and B-cell lymphoma 2 (Bcl-2) were tested by real-time quantitative PCR (RT-qPCR). The effect of MTT on the proliferation of GFTCs was detected. The localization of C1QBP in GFTCs was detected by inverted fluorescence microscope. Finally, transcriptome sequencing was performed and validated by siRNA treatment knockdown of C1QBP, and screening two genes tripartite motif containing 5 (TRIM5) and tumor necrosis factor superfamilymember10 (TNFSF10) with significant changes in expression levels and relevance to cell apoptosis, and to verify their roles in C1QBP-induced cell apoptosis.ResultsKnockdown of C1QBP significantly increased cell viability; cells remained in the G0/G1 phase and reduced apoptosis. Knockdown of C1QBP reduced the mRNA expression of CDK2, p21, Caspase3, Caspase7, Bax, PARP1, BCL2L11, and p53, up regulated the mRNA expression of the Bcl-2. Except for Bcl-2, The opposite effect was observed when C1QBP was overexpressed in GFTCs and the mRNA levels of Bcl-2 had no significant effect. Immunostaining revealed intracellular localization of C1QBP, primarily in the cytoplasm of the GFTCs. Furthermore, gene expression profiling analysis in C1QBP depleted cells compared to the control revealed that a total of 236 differential expression genes (DEGs), including 119 up regulated DEGs and 117 down regulated DEGs, and the expression of TRIM5 and TNFSF10 genes were significantly upregulated. Pathway analysis were predicted to be enriched in Herpes simplex virus 1 infection, Pathways in cancer, PI3K-Akt signaling pathway, Apoptosis, Apoptosis-multiple species and p53 signaling pathway. C1QBP reduced the expression of TRIM5 and TNFSF10 genes. Knockdown of TRIM5 promoted apoptosis in GFTCs, but silencing of TNFSF10 had no change in apoptosis rate. In particular, the apoptosis rate was significantly increased in the TRIM5-siRNA2 or TNFSF10-siRNA2 and C1QBP-siRNA2 group compared to the only C1QBP-siRNA2 group.ConclusionThese findings provide a deeper understanding of the role of C1QBP in apoptosis and could pave the way for further study investigating the role and mechanism of C1QBP protein in mediating the regulation of cell apoptosis by ORFV.

The content and composition of intramuscular fat (IMF) affect the cooked meat palatability such as tenderness and juiciness. Thus, elucidation of lipid deposition and its composition in goat IMF is necessary. Here, Jianzhou big-ear goats with higher IMF content is associated with lower mRNA level of LKB1 gene, compared with those of Chuannan black goats. Functionally, knockdown of LKB1 promoted intramuscular adipocyte lipid accumulation. Next, LC-MS/MS based pseudo target analysis found that 409 lipids existed in goat intramuscular adipocytes, of which polyunsaturated fatty acids accounted for most lipids. Compared with the control, 78 differential lipids were screened in the siLKB1 group, enriched in the triacylglycerols and fatty acids subclasses. The combined analysis between lipidomic and published transcriptomic data showed that siLKB1 enhanced polyunsaturated fatty acids synthesis through upregulation expression of HACD4. Finally, the promotion of lipid accumulation and polyunsaturated fatty acids synthesis in the LKB1 knockdown cells were partly rescued by ablation of HACD4. Collectively, these data provide a genetic target to produce PUFA enriched functional goat meat and expand new insights into improvement of meat quality.

Intramuscular fat (IMF) content is an important factor of goat meat quality, which is related to the proliferation and differentiation of intramuscular preadipocytes. Perilipin 5 (PLIN5) is a lipid droplet-associated protein; however, the specific function and underlying mechanism of PLIN5 in goat IMF deposition are still unclear. In this study, overexpression of PLIN5 significantly enhanced apoptosis and reduced the proliferation of preadipocytes and also promoted cellular lipid deposition via both the upregulation of the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and a significant increase in the expression of lipogenesis-related genes. The inhibition of PLIN5 then confirmed these results. Untargeted lipidomics sequencing identified a total of 34 differentially expressed lipids after PLIN5 overexpression in goat preadipocytes and analysis by KEGG pathway enrichment, which are mainly involved in the PI3K-AKT signaling pathway. The lipid omics findings also show that ceramides and lysophosphatidylinositol were significantly upregulated, e.g., Cer (d35:1), Cer (d18:2/22:1), LPI (18:0), and LPI (16:0), after overexpression of the PLIN5 gene. Higher expression of LPI (16:0) or LPI (18:0) may regulate lipid droplet accumulation by activating PPARγ. Rescue experiments with the PI3K-AKT inhibitor (LY294002) and the PPARγ inhibitor (GW9662) showed that the PI3K-AKT signaling pathway is involved in the regulation of cell proliferation, and PPARγ is involved in the regulation of lipid deposition. In conclusion, our findings demonstrate that PLIN5 regulates lipid reconstitution in goat intramuscular fat via PPARγ and PI3K-AKT signaling pathways. This regulation delivered theoretical support for improving meat quality from the aspect of IMF deposition.

Endothelial lipase (LIPG), a member of the triglyceride lipase family, plays an essential role in human diseases and lipid metabolism. However, its function in goat intramuscular fat (IMF) deposition remains unclear. In this study, we investigated the role of the LIPG gene in IMF deposition by knocking down and overexpressing it in goat intramuscular preadipocytes. We successfully cloned the full-length LIPG gene, which spans 2,131 bp, including a 94 bp 5' untranslated region (5'UTR), a 1,503 bp coding sequence (CDS), and a 534 bp 3' untranslated region (3'UTR). Tissue expression profiles showed that LIPG is expressed in the heart, liver, spleen, Kidney, longest dorsal muscle, and small intestine tissues of goats. LIPG knockdown significantly inhibited both the proliferation of intramuscular preadipocytes and lipid deposition. Moreover, LIPG knockdown markedly decreased mRNA expression of FASN, LPL, CPT1A, CPT1B, FABP3, while increasing the mRNA expression of ATGL, ACOX1, FADS1, and ELOVL6. These findings were further corroborated through LIPG overexpression experiments. Using RNA sequencing (RNA-seq), we identified 1695 differentially expressed genes (DEGs) between the negative control (NC) and LIPG knockdown (Si-LIPG) groups, with KEGG pathway analysis revealing significant enrichment in the PPAR signaling pathway. Additionally, LIPG knockdown significantly upregulated the expression of both mRNA and protein levels of PPARα. The PPARα agonist WY14643 was able to reverse the enhanced lipid deposition induced by LIPG overexpression. In conclusion, our study highlights a key role for LIPG in the regulation of goat intramuscular preadipocyte proliferation and lipid deposition, potentially through the PPARα signaling pathway. These findings provide new insights into the regulatory mechanisms governing IMF deposition and suggest potential strategies for improving goat meat quality.

OBJECTIVE:Cell death-inducing DNA fragmentation factor alpha-like effector B (CIDEB), a family member of Cell death-inducing DFF45-like effectors (CIDEs), is well known as a crucial regulator for lipid metabolic signaling pathways in various metabolic tissues and secretory glands. However, its role in regulating intramuscular fat (IMF) deposition in goat remains unclear. METHODS:The expression vector pcDNA3.1-CIDEB was constructed and transfected into goat intramuscular preadipocytes; the overexpression and interference efficiency and expression of genes related to lipid metabolism were measured by Real-time polymerase chain reaction; the effect of overexpression of CIDEB and interfering with CIDEB on lipid droplet formation was observed by Oil Red O staining and glycerol phosphate oxidase-Trinder enzymatic reaction. Then RNA-Seq was used to investigate the metabolic pathway of CIDEB affecting adipocyte deposition in goat intramuscular preadipocytes. RESULTS:Overexpression of CIDEB significantly promoted the lipid droplets accumulation and the triglyceride deposition, and significantly upregulated the expression of genes related to lipid metabolism. After overexpression of CIDEB in goat intramuscular preadipocytes, 171 differentially expressed genes (DEGs) were found, including 122 up-regulated and 49 down-regulated DEGs, and the top three significantly changed pathways filtered by Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were Cocaine addiction, Amphetamine addiction and Malaria pathways. Conversely, the silencing of CIDEB significantly reduced lipid accumulation in goat intramuscular preadipocytes, meanwhile changing the expression of lipid metabolism genes. For CIDEB silencing, a total of 2140 DEGs were found, including 1252 up-regulated and 888 down-regulated DEGs, and the top three significantly changed pathways filtered by KEGG analysis were Ribosome, Thyroid hormone signaling pathway and Alzheimer disease. CONCLUSION:The expression of CIDEB can significantly promote lipid deposition of intramuscular adipocytes in goats, and these results provide important data to support further clarifying the mechanism of CIDEB gene on the regulation of intramuscular adipogenesis, and the IMF formation in goats.

Intramuscular fat (IMF), or marbling, is a critical indicator of goat meat quality. Non-coding RNAs play a key role in the formation and deposition of IMF in vertebrates by regulating genes involved in its synthesis, degradation, and transport. The competing endogenous RNA (ceRNA) hypothesis identifies circular RNAs (circRNAs) as natural “sponges” for microRNAs (miRNAs). However, the precise mechanisms of circRNAs in goat IMF remain poorly understood. In the current study, we utilized existing sequencing data to construct a ceRNA regulatory network associated with intramuscular adipogenesis and fat deposition in goats. Our goal was to elucidate the post-transcriptional regulatory mechanism of family with sequence similarity 49 member B (FAM49B). Functionally, FAM49B was found to inhibit the differentiation of intramuscular preadipocytes and to directly interact with miR-27a-5p. Mechanistically, dual-luciferase reporter assays and quantitative real-time PCR (qRT-PCR) confirmed the interaction between circ0011446 and miR-27a-5p. Circ0011446 enhanced the expression of FAM49B mRNA and protein through post-transcriptional regulation. As a ceRNA, circ0011446 competitively binds miR-27a-5p, preventing miR-27a-5p from degrading FAM49B. In conclusion, our findings demonstrate that circ0011446 suppresses goat adipogenic differentiation of intramuscular preadipocytes by regulating the expression of the downstream target gene FAM49B through miR-27a-5p sequestration. This study provides a reference for goat meat quality or livestock breeding.

Intramuscular fat (IMF) content determines the quality of goat meat and is regulated by the comprehensive effect of the proliferation and adipogenesis of intramuscular preadipocytes. Our previous RNA-seq data revealed that cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) A was upregulated during the development of intramuscular fat in the longissimus dorsi muscle tissue, implying an important role in lipid homeostasis. However, the mechanism by which CIDEA, a member of the CIDE family, regulates intramuscular fat deposition in goat muscle is unknown, so we explored the function and underlying mechanism of CIDEA in goat intramuscular preadipocytes. To address this, we altered CIDEA in intramuscular preadipocytes and resolved the effect and mechanism of CIDEA in adipogenesis through RT-PCR, Western blot, triglyceride and LD determinations, CCK-8, and RNA-seq. It was found that CIDEA increased lipid droplets (LDs) and triglyceride contents and inhibited cell proliferation. Meanwhile, the lipid metabolism-related genes PPARγ, C/EBPα, SREBP1c, PLIN1, TIP47, ADFP, DGAT1, ACC, FASN, ACSL1, and FABP3 were upregulated, while the lipolysis and β-oxidation genes HSL, ACOX1, and CPT1B, as well as the proliferation marker gene CDK1, were all downregulated upon CIDEA overexpression. Differentially expressed genes in CIDEA dysregulation groups through RNA-seq were selected and were enriched in the apelin and focal adhesion signaling pathways. Specifically, the Western blot and rescue assays found that focal adhesion, but not apelin, was the key signaling pathway in CIDEA regulating lipid deposition in goat intramuscular preadipocytes. In summary, this study reveals that CIDEA promotes lipid deposition in intramuscular preadipocytes through the focal adhesion pathway and inhibits cell proliferation. This work clarifies the functional role and downstream signaling pathway of CIDEA in intramuscular fat deposition and provides theoretical support for improving meat quality by targeting key phenotype-related genes.

Silent Information Regulator 5 (SIRT5) has been established as a crucial regulator of cellular alanylation modification. Furthermore, accumulating evidence suggests that SIRT5 plays a significant regulatory role in key metabolic pathways, including glycolysis, the tricarboxylic acid (TCA) cycle, and fatty acid oxidation, all of which are closely associated with cellular lipid metabolism. Despite these advancements, the specific role of SIRT5 in regulating intramuscular fat (IMF) deposition in goats, as well as the underlying molecular mechanisms, remains largely unexplored. In this study, we cloned the complete coding sequence of the goat SIRT5 gene and, through amino acid sequence alignment, demonstrated its closest phylogenetic relationship with sheep. Additionally, we characterized the higher expression of SIRT5 during the differentiation of goat intramuscular precursor adipocytes. The silencing of SIRT5 by siRNA-mediated knockdown significantly upregulated the expression of lipogenesis-related genes and enhanced lipid deposition in goat intramuscular preadipocytes. Concurrently, SIRT5 deficiency led to the inhibition of cell proliferation and a marked reduction in apoptosis. Interestingly, although overexpression of SIRT5 promoted cell proliferation, it did not significantly alter lipid deposition in goat intramuscular precursor adipocytes. RNA sequencing (RNA-seq) analysis identified a total of 106 differentially expressed genes (DEGs) following SIRT5 silencing in goat preadipocytes, predominantly involved in the Focal adhesion, HIF-1, PI3K-Akt, and MAPK signaling pathways by KEGG pathway enrichment analysis. Notably, we successfully reversed the phenotypic effects observed in SIRT5 knockdown goat precursor adipocytes by inhibiting the PI3K-Akt and MAPK signaling pathways using the AKT inhibitor LY294002 and the p38 MAPK pathway inhibitor PD169316, respectively. In conclusion, our findings demonstrated that SIRT5 may modulate intramuscular fat deposition in goats through PI3k-Akt and MAPK signaling pathways. These results expand the gene regulatory network associated with IMF formation and provide a theoretical foundation for improving meat quality by targeting IMF deposition.

The intestinal microbiota significantly influences the intake, storage, and absorption of nutrients in animals, thereby greatly impacting the growth and development of the animals. Factors such as diet, animal breed, growth stage, and feeding methods may affect variations in the composition of the intestinal microbiota. However, research on the variations in the intestinal microbiota and metabolites of Nanjiang Yellow goats under different feeding methods is still unclear. We measured various serum biochemical indicators and immune biochemical indicators and found that the triglyceride (TC) content in the grazing group (the FMMF) was significantly lower than in the barn-feeding group (the SSMF) ( P <0.05). Serum levels of immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin G (IgM) were higher in the FMMF group. At the phylum level, the most abundant bacteria were Firmicutes, Bacteroidota , and Verrucomicrobiota . At the genus level, the most abundant microbial groups were Christensenellaceae\_R-7\_group, UCG-005 , and Rikenellaceae\_RC9\_gut\_group . Differential metabolite enrichment analysis through KEGG pathways revealed that the most remarkably enriched pathway was “Metabolic pathways,” including Steroid hormone biosynthesis and Arachidonic acid metabolism, among others. Analyzed by multi-omics association, we identified notably different microbial features correlated with immune indicators and metabolites after different feeding methods. We observed a significant negative correlation ( P <0.05) between the concentrations of serum immune factors interleukin-2 (IL-2), interleukin-4 (IL-4), and g\\_\_probable\_genus\_10 . The concentration of IgM in serum showed a highly significant positive correlation ( P <0.01) with the relative abundance of g\_\_Erysipelatoclostridium in the intestine. Interestingly, most differential metabolites were significantly associated with intestinal microbiota. This experiment indicates that different feeding methods may influence the diversity and relative abundance of the intestinal microbiota in Nanjiang Yellow goats. The intestinal microbiota is correlated with immune indicators and metabolism, and regulating the diversity and relative abundance of the intestinal microbiota can be a way to adjust metabolism, thereby promoting the healthy growth of the Nanjiang Yellow goats. ### Competing Interest Statement The authors have declared no competing interest.

The balance of the microbiome, which is sensitive to temperature changes, plays a crucial role in maintaining overall health and reducing the risk of diseases. However, the specific mechanisms by which immunity and microbiota interact to adapt to cold stress have yet to be addressed. In this study, Nanjiang Yellow goats were chosen as a model and sampled during the cold (winter, cold stress) and warm (spring) seasons, respectively. Analyses of serum immune factors, as well as the composition of rumen and fecal microbial communities, were conducted to explore the crosstalk between microbiota and innate immunity under cold stress. Significantly increased levels of IgA (P < 0.01) were observed in the cold season compared to the warm season. Conversely, the levels of IL-2 (P = 0.02) and IL-6 (P < 0.01) diminished under cold stress. However, no significant differences were observed in IgG (P = 0.89), IgM (P = 0.42), and IL-4 (P = 0.56). While there were no significant changes in the diversity of bacterial communities between the warm and cold seasons, positive correlations between serum IgA, IL-2, IL-6 concentrations and several genera were observed. Furthermore, the weighted gene co-expression network analysis indicated that the microbiota enriched in the MEbrown module positively correlated with IgA, while the microbiota enriched in the MEblue module positively correlated with IL-2 and IL-6. The strong correlation between certain probiotics, including Alistipes, Bacteroides, Blautia, and Prevotellaceae_UCG.004, and the concentration of IL-2, and IL-6 suggests their potential role in immunomodulatory properties. This study provides valuable insights into the crosstalk between microbial communities and immune responses under the challenge of cold stress. Further studies on the immunomodulatory properties of these probiotics would contribute to the development of strategies to enhance the stress resistance of animals for improved overall health and survival.

Intramuscular fat refers to the adipose tissue distributed in the muscle. It is an important indicator that affects the quality of goat meat, and can directly affect the tenderness and flavor of goat meat. Our previous study revealed the mRNA that may be crucial for intramuscular fat deposition during goat growth; however, how the microRNAs (miRNAs) are involved in the process is largely unclear. In the present study, a total of 401 known miRNAs and 120 goat novel miRNAs, including 110 differentially expressed (DE) miRNAs, were identified among longissimus dorsi from three growth stages (2, 9, and 24 months) by miRNA sequencing. Combining analysis of the DE mRNAs and DE miRNAs was then performed by miRDB and miRwalk, and miR-145-5p and FOXO1, miR-487b-3p, and PPARG coactivator 1 α (PPARGC1A), miR-345-3p, and solute carrier family 2 member 4 (SLC2A4), etc. were shown to closely associate with lipid metabolism, which was then validated by a correlation analysis. The final DE mRNAs were significantly enriched in fatty acid transmembrane transport, fatty acid homeostasis, apelin signaling pathway, glucagon signaling pathway, insulin signaling pathway, and AMPK signaling pathway by gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis. Besides, miR-145-5p showed a certain effect on goat intramuscular fat metabolism by acting on the possible target gene Forkhead Box O1 (FOXO1). These data provide some theoretical support for improving the quality of goat meat.

Intramuscular adipogenesis plays an important role in muscle development, which determines the quality of goat meat. However, its underlying cellular and molecular mechanisms remain poorly understood. In this study, we provided detailed cellular atlases of goat longissimus dorsi during muscle development at single-nucleus resolution, and identified the subpopulations of fibroblasts/fibro-adipogenic progenitors (FAPs) and muscle satellite cell (MuSC), as well as the differentiation trajectory of FAPs subpopulations. Cellular ligand-receptor interaction analysis revealed enriched BMP and IGF pathways implicated in within-tissue crosstalk centered around FAPs. Through single-nucleus gene regulatory network analysis and in vitro interference verification, we found that TCF7L2 was a critical transcriptional factor (TF) in early adipogenesis in skeletal muscle. Overall, our work reveals the cellular intricacies and diversity of goat longissimus dorsi during muscle development, implementing insights into the critical roles of BMP, IGF pathways and TCF7L2 TF in intramuscular adipogenesis.

Intramuscular fat (IMF) content determines the quality of goat meat, and is strongly associated with the number and volume of adipocytes, which is regulated by the comprehensive effect of proliferation and adipogenesis of intramuscular preadipocytes. Cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) proteins has emerged as lipid droplets (LDs)-related proteins, implying the important roles in lipid homeostasis. However, the mechanism through which CIDEA, one member of CIDE family, regulates intramuscular fat deposition remains unclear. To address this, we dysregulated CIDEA in intramuscular preadipocytes and resolved the effect and mechanism of CIEDA in adipogenesis through RT-PCR, Western blot, triglyceride and LDs determinations, CCK-8 and RNA-seq. It was found that CIDEA increased LDs and triglyceride contents and inhibited cell proliferation. Lipid metabolism-related genes PPARγ, C/EBPα, SREBP1c, PLIN1, TIP47, ADFP, DGAT1, ACC, FASN, ACSL1, FABP3 were upregulated after CIDEA overexpression. Moreover, lipolysis and β oxidation genes HSL, ACOX1, CPT1B and proliferation marker genes CDK1 were upregulated. Differentially expressed genes in RNA-seq results were selected and enriched in the apelin and focal adhesion signaling pathways. Specifically, CIDEA regulated the activation of focal adhesion kinase and AKT signaling proteins, but not p38 signaling. To this end, we did the rescue assay and found that suppressing focal adhesion kinase signaling pathway with PF573228 reversed the lipid droplets and triglyceride contents increase induced by CIDEA overexpressing and further decreased their contents in CIDEA interfering group. In summary, this study reveals that CIDEA promotes lipid deposition in intramuscular preadipocytes through the focal adhesion pathway and inhibiting the cell proliferation. These works clarify the functional role and downstream signaling pathway of CIDEA in intramuscular fat deposition and provide theoretical support for improving meat quality through manipulating phenotype-related key genes.

Meat quality in goats is partly determined by the intramuscular fat (IMF) content, which is associated with the proliferation and differentiation of intramuscular preadipocytes. Emerging studies have suggested that miRNA plays a crucial role in adipocyte proliferation and differentiation. In our recent study, we observed the expression variations in miR-196a in the longissimus dorsi muscle of Jianzhou goats at different ages. However, the specific function and underlying mechanism of miR-196a in IMF deposition are still unclear. This study demonstrated that miR-196a significantly enhanced adipogenesis and apoptosis and reduced the proliferation of preadipocytes. Subsequently, RNA-seq was employed to determine genes regulated by miR-196a, and 677 differentially expressed genes were detected after miR-196a overexpression. The PI3K-Akt pathway was identified as activated in miR-196a regulating intramuscular adipogenesis via Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and further verified via Western blot and rescue assays. Lastly, using RT-qPCR, Western blot, dual-luciferase, and rescue assays, we found that miR-196a promoted adipogenesis and suppressed the proliferation of intramuscular preadipocytes by the downregulation of MAP3K1. In summary, these results suggest that miR-196a regulates IMF deposition by targeting MAP3K1 and activating the PI3K-Akt pathway and provide a theoretical foundation for improving goat meat quality through molecular breeding.