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.

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.

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) 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.

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.

[目的]以羊口疮病毒129(ORFV129)蛋白为研究对象,在构建山羊鼻甲骨原代细胞cDNA文库的基础上,通过酵母双杂交筛选与其相互作用的蛋白.[方法]采用Smart?技术构建山羊鼻甲骨原代细胞cDNA文库.构建诱饵质粒pGBKT7-129,并将其转化至Y2HGold酵母感受态细胞,验证质粒pGBKT7-129是否具有自激活性.对转化了质粒pGBKT7-129的菌液进行生长曲线测定,验证该质粒是否对酵母细胞有毒性作用.以ORFV129为诱饵蛋白,利用酵母双杂交系统筛选出与ORFV129相互作用的宿主胞内蛋白并对阳性菌落进行PCR和测序鉴定.利用DAVID 6.7的GO数据库对胞内宿主蛋白进行功能分类和通路分析,并依据Cytoscape v 3.8.0软件绘制ORFV129与胞内蛋白相互作用的网络图.以山羊脾脏为组织样本,采用RT-PCR技术克隆山羊补体C1q结合蛋白(complement C1q binding protein,C1QBP)基因CDS区序列,并采用在线软件进行生物信息学分析.将C1QBP基因CDS区序列连接至pcDNA3.1(+)构建真核表达载体pcDNA3.1(+)-C1QBP,并将其转染至山羊鼻甲骨原代细胞进行亚细胞定位分析.[结果]试验成功构建山羊鼻甲骨原代细胞cDNA文库,文库容量约为6.0×106 CFU/mL.成功构建诱饵质粒pGBKT7-129,重组质粒pGBKT7-129无自激活能力且对酵母细胞无毒性.利用酵母双杂交筛选出14个与ORFV129进行互作的胞内蛋白并对阳性克隆进行PCR、测序验证.试验成功克隆山羊C1QBP基因CDS区,长度为837 bp,编码279个氨基酸.系统进化树显示,山羊和绵羊亲缘关系最近.C1QBP蛋白为不稳定的亲水性蛋白,无信号肽结构和跨膜结构域,主要包括3种磷酸化位点,分别是18个丝氨酸、4个苏氨酸和3个酪氨酸位点.C1QBP蛋白二级结构由α-螺旋(33.81％)、无规则卷曲(46.40％)、延伸链(16.91％)和β-转角(2.88％)组成,三级结构与二级结构一致.间接免疫荧光试验表明,C1QBP蛋白散在分布于细胞质中.[结论]筛选出了与ORFV129蛋白相互作用且在天然免疫应答中发挥作用的宿主胞内蛋白C1QBP,通过间接免疫荧光试验验证C1QBP定位于细胞质中,推测ORFV129与能C1QBP相互作用诱导炎症,为进一步验证ORFV129蛋白介导ORFV抑制机体免疫应答的过程奠定基础.

The aim of the present study was to clone goat BIRC5, and to reveal the role of BIRC5 gene in regulating the cycle and apoptosis of goat testis cells through the overexpression or interference of BIRC5. These data will be beneficial for exploring the role of BIRC5 gene in goat. The BIRC5 gene sequence was cloned by RT-PCR from spleen tissue of 3 three-day-old healthy Jianzhou male goats with 3 kg of body weight, and sequenced for further bioinformatics analysis using online softwares. The clone recovery product was ligated to a eukaryotic expression vector to construct pcDNA3.1（+）-BIRC5. Effective siRNA was designed and screened based on goat BIRC5 gene sequence. After pcDNA3.1（+）-BIRC5 and siRNA were respectively transfected into goat testicular cells, the expression of BIRC5 protein was detected by Western blot, and cell cycle and apoptosis were detected by flow cytometry. Meanwhile, the expression of apoptosis-related genes Bax, Caspase3, Caspase7, Bcl-2, p53, BCL2L11 and PARP1 were detected by RT-qPCR. A length of 506 bp BIRC5 gene sequence was cloned successfully, including 28 bp of 5′UTR, 429 bp of CDS region, and 49 bp of 3′UTR,encoding 142 amino acids. The phylogenetic tree showed that BIRC5 had the closest relative to Bos taurus. The overexpression of BIRC5 gene inhibited the apoptosis of cells, and the cells were arrested in G2/M+S phase; it also down-regulated the expression of Caspase7, p53, BCL2L11, Bcl-2 and Bax gene mRNA, but did not significantly change the mRNA expression of Caspase3 and PARP1. siRNA interference of BIRC5 gene promoted the apoptosis of cells, and the cells were arrested in G0/G1 phase; it also up-regulated the expression of Caspase3, Caspase7 and PARP1 gene mRNA, and down-regulated the expression of p53 and Bcl-2 gene mRNA, but did not significantly change the mRNA expression of Bax and BCL2L11. Goat BIRC5 gene could inhibit the apoptosis of testis cells and promote the arrest of goat testicular cells in G2/M+S phase. These data may lay a foundation for further study of BIRC5 gene function.

Intramuscular fat (IMF) deposition is one of the most important factors affecting meat quality and is closely associated with the expression of carnitine palmitoyl transferase 1A (CPT1A) which facilitates the transfer of long-chain fatty acids (LCFAs) into the mitochondria. However, the role of how CPT1A regulates the IMF formation remains unclear. Herein, we established the temporal expression profile of CPT1A during the differentiation of goat intramuscular precursor adipocytes. Functionally, the knockdown of CPT1A by siRNA treatment significantly increased the mRNA expression of adipogenic genes and promoted lipid deposition in goat intramuscular precursor adipocytes. Meanwhile, a CPT1A deficiency inhibited cell proliferation and promoted cell apoptosis significantly. CPT1A was then supported by the overexpression of CPT1A which significantly suppressed the cellular triglyceride deposition and promoted cell proliferation although the cell apoptosis also was increased. For RNA sequencing, a total of 167 differential expression genes (DEGs), including 125 upregulated DEGs and 42 downregulated DEGs, were observed after the RNA silencing of CPT1A compared to the control, and were predicted to enrich in the focal adhesion pathway, cell cycle, apoptosis and the MAPK signaling pathway by KEGG analysis. Specifically, blocking the MAPK signaling pathway by a specific inhibitor (PD169316) rescued the promotion of cell proliferation in CPT1A overexpression adipocytes. In conclusion, the expression variation of CPT1A may reconstruct the lipid distribution between cellular triglyceride deposition and cell proliferation in goat intramuscular precursor adipocyte. Furthermore, we demonstrate that CPT1A promotes the proliferation of goat adipocytes through the MAPK signaling pathway. This work widened the genetic regulator networks of IMF formation and delivered theoretical support for improving meat quality from the aspect of IMF deposition.

Objective: Contagious ecthyma is a severe and highly contagious disease caused by an orf virus (ORFV). The virus is responsible for substantial economic losses in the goat industry and threatens humans. We previously determined the role of ORFV129 protein, one of the five ankyrin-repeat proteins coded by the orf genome, in suppressing the transcription of pro-inflammatory cytokines IL-6, IL-1 & beta; and IFN-& gamma;. In the present study, we identified 14 cellular proteins (complement C1q binding protein [C1QBP], MCM7, EIF5A, PKM, SLC6A, TSPAN6, ATP6AP2, GPS1, MMADHC, HSPB6, SLC35B1, MTF1, P3H4, and IL15RA) that interact with ORFV129 using a yeast two-hybrid system in goat turbinate bone cells (GFTCs). The interaction between ORFV129 and (C1QBP), an immune-related protein, was confirmed using immunofluorescence co-localization and coimmunoprecipitation assays. C1QBP overexpression inhibited ORFV replication, whereas the knockdown of C1QBP promoted ORFV replication in GFTCs. Furthermore, ORFV or ORFV129 increased C1QBP expression in GFTCs, indicated that ORFV129-C1QBP interaction might contribute to the ORFV-induced host immune process. In addition, our research showed that ORFV increased the expression of ORFV129, cytokine IL-6, IL-1 & beta; and IFN-& gamma;. C1QBP overexpression induced IFN-& gamma; production and reduced IL-6 and IL-1 & beta; production. Conversely, C1QBP knockdown induced IL-1 & beta; production and reduced IFN-& gamma; and IL-1 & beta; production. Moreover, augmentation of ORFV129 expression enhanced the inhibition of the secretion of cytokines IL-6, IL-1 & beta;, and IFN-& gamma; induced by the altered expression of C1QBP. These findings suggest different downstream pathways might be involved in regulating different cytokines induced by ORFV129 expression in GFTCs.

【Background】Fatty acid transporter 1 (FATP1) can promote the uptake of fatty acids in mammals. This process is very important to maintain the balance of lipid metabolism, and also has an important impact on the meat quality of livestock.【Objective】The aim of this study was to obtain the CDS sequence of goat FATP1 gene, to detect the expression of FATP1 gene in different tissues of goats, and to explore its effect on lipid metabolism of goat intramuscular adipocytes, so as to provide a reference for further revealing the mechanism of FATP1 gene in goat lipid metabolism, which can provide a theoretical basis for genetic and breeding improvement of goats.【Method】The CDS of goat FATP1 gene was cloned by real-time fluorescence quantitative PCR (RT-PCR), its biological characteristics, such as hydrophobicity, transmembrane region and signal peptide, were analyzed by online tools, and its amino acid sequence phylogenetic tree was constructed. The expression level of FATP1 gene in different goat tissues was detected by RT-qPCR and its tissue expression pattern was constructed. The constructed eukaryotic expression vector and screened siRNA were used to overexpress and interfere with FATP1 in goat intramuscular adipocytes, the effects of FATP1 gene overexpression and interference on lipid deposition in goat intramuscular adipocytes were detected by oil red O staining and triglyceride determination, and the effects of FATP1 gene overexpression and interference on the expression of genes related to lipid metabolism were further explored by RT-qPCR.【Result】The CDS of FATP1 gene was 1 941 bp, encoding 646 amino acids residues. It was predicted that its molecular formula was C3196H5026N884O898S25, and the protein was a basic hydrophobic stable protein. Phylogenetic tree analysis of amino acid sequence showed that goat FATP1 was closely related to sheep. RT-qPCR showed that the expression of FATP1 gene was the highest in goat small intestine. Oil red O staining and triglyceride determination showed that the number of lipid droplets and triglyceride content in goat intramuscular adipocytes increased after overexpression of FATP1, but the opposite results were obtained after interference with FATP1. After overexpression of FATP1 in goat adipocytes, the expression levels of fatty acid synthesis, transport and other related genes AGPAT6(P<0.01), PLIN1(P<0.01), DGAT2(P<0.01), FADS2(P<0.01), FADS1(P<0.01), ACSL1(P<0.01) and ELOVL3 (P<0.05) increased significantly, while the expression level of lipolysis related genes ACOX1 (P<0.01) decreased significantly. After interfering FATP1, the expression of fatty acid transport, elongation and other related genes SCD5 (P<0.01), FABP3 (P<0.01) and ELOVL3 (P<0.05) decreased significantly, and the expression of lipolysis related genes ACOX1 (P<0.01) and CPT1B (P<0.05) increased significantly.【Conclusion】FATP1 might significantly promote the lipid deposition of goat intramuscular precursor adipocytes by promoting the expression of genes related to cell lipid production and reducing the expression of genes related to lipolysis, which provided an experimental reference for further revealing the role and molecular mechanism of FATP1 gene in regulating lipid metabolism.

旨在对山羊脂肪酸转运蛋白2(fatty acid transport protein 2,FATP2)基因进行克隆及生物信息学分析,检测FATP2基因在山羊不同组织中的表达差异,并进一步揭示干扰FATP2基因对山羊肌内前体脂肪细胞脂质代谢的影响.本试验以10月龄健康简州大耳羊(n=12)为试验动物.采用RT-PCR法扩增并克隆山羊FATP2基因,进行序列对比、系统发育树构建及生物信息学分析;利用实时荧光定量PCR检测FATP2基因在山羊不同组织中及在山羊肌内前体脂肪细胞不同分化时期的相对表达水平,合成SI-RNA干扰序列并转染至山羊肌内前体脂肪细胞;使用RT-qPCR检测FATP2干扰效率及脂质代谢相关基因的表达情况;通过Bodipy染色法观察干扰FATP2对脂滴形成的影响,利用GPO-Trinder酶学反应检测甘油三酯含量.结果显示,获得山羊FATP2基因序列全长2 335 bp,CDS区1 863 bp,5'UTR 188 bp,3'UTR 284 bp;共编码621个氨基酸,山羊FATP2基因在肝脏表达量最高;RT-qPCR检测结果显示,FATP2表达在细胞中被显著干扰;Bodipy染色结果显示,干扰FATP2基因后脂滴显著减少;甘油三酯测定结果显示,干扰FATP2基因山羊肌内前体脂肪细胞甘油三酯含量显著降低.本研究成功获得山羊FATP2基因CDS区序列,干扰FATP2后山羊肌内前体脂肪细胞脂质沉积显著减少并显著影响脂代谢相关基因的表达,这些结果为进一步阐明FATP2对调控山羊肌内脂肪形成的作用机制提供了重要数据.

旨在通过探究大豆油添加日粮对育肥山羊肠道菌群结构的影响,筛选获得大豆油最适添加水平,从而为简州大耳羊集约化高效养殖提供参考.随机选取24只6月龄健康、体重相近、体况相似的公羔作为试验对象.于直肠末端采集山羊粪便进行16S rRNA测序分析.通过比较各样本间的reads数比例得到细菌的相对丰度,Alpha多样性和Beta多样性计算结果显示,饲喂大豆油添加日粮对群落物种多样性没有显著影响(P＞0.05).物种注释结果表明,2％大豆油添加组中变形菌门的相对丰度显著高于其他组(P＜0.05).而与消化吸收相关的差异菌属中,Paludibacteraceae的相对丰度在2％大豆油添加组中显著高于3％和对照组大豆油添加组(P＜0.05).在日粮中添加大豆油不会破坏山羊肠道菌群多样性,而添加2％大豆油全混合日粮饲喂肉用山羊可以调节特定菌群丰度,一定程度上提高了山羊的消化吸收能力.这些结果为大豆油在肉羊集约化养殖中的安全推广使用提供了数据参考.

旨在克隆山羊PSMD9基因序列并对其进行生物信息学分析,检测PSMD9在山羊各组织中的表达情况和肌内前体脂肪细胞不同分化时期的表达水平,并进一步揭示过表达和干扰PSMD9基因对山羊肌内前体脂肪细胞脂质沉积的影响.本研究以1周岁健康简州大耳羊公羊(n=12)为试验对象,采用T-A克隆技术获得山羊PSMD9基因序列,进行生物信息学分析,并通过双酶切方法构建PSMD9过表达载体.利用脂质体转染在山羊肌内脂肪细胞中过表达PSMD9,小干扰RNA(small interfering RNA,siRNA)干扰PSMD9表达.通过油红O染色法观察PSMD9对脂滴形成的影响,GPO-Trinder酶学反应检测甘油三酯含量;同时利用实时荧光定量PCR(RT-qPCR)检测PSMD9在山羊不同组织中的表达水平和肌内前体脂肪细胞不同分化时期的表达水平.结果,获得山羊PSMD9基因核苷酸序列763 bp,其中5'UTR 67 bp,CDS区564 bp,3'UTR 132 bp,编码187个氨基酸残基;山羊PSMD9基因在肝脏中的表达量最高,在脾脏中表达量最低,在诱导分化前4 d其表达水平随着山羊前体脂肪细胞分化而逐渐降低,随后上升,并在第8天表达量最高;过表达PSMD9基因,肌内前体脂肪细胞的脂滴明显增加,甘油三酯含量显著提高,同时可显著上调DGAT2、FASN和ACC的相对表达量,DGAT1、PPARγ和SCD1表达量无显著变化;干扰PSMD9基因肌内前体脂肪细胞脂滴减少,甘油三酯含量降低,同时可显著下调DGAT1、PPARγ、FASN和ACC表达量,DGAT2和SCD1无显著变化.本试验成功获得山羊PSMD9基因CDS区序列,其在肝脏组织中表达量最高,山羊PSMD9基因的表达随着肌内前体脂肪细胞的分化而逐渐降低,在第4天表达量最低,之后表达量逐渐升高,PSMD9的表达能够显著促进山羊肌内前体脂肪细胞脂质沉积,这些结果为进一步阐明PSMD9基因对调控山羊肌内脂肪形成的作用机制提供了重要数据支撑.