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Simple Summary: This study aimed to reveal the single nucleotide polymorphisms (SNPs) of the bovine Stearoyl-CoA desaturase-1 (SCD1) to explore the correlation between genotypes and carcass, meat quality, adipogenic traits, fatty acid composition, and milk production traits in cattle. Four SNPs, g.21272246 A>G, g.21272306 T>C, g.21272422 C>T, and g.21272529 A>G, were found by Sanger sequencing; further statistical analysis showed that these four SNPs of the SCD1 gene were significantly associated with carcass traits and meat quality, including carcass weight, carcass fat coverage rate, rib eye area, marbling score, adipogenic traits, and fatty acid composition. Additionally, a modest effect on milk production traits, such as average milk yield and milk fat content, was observed in cows. Further haplotype analysis indicated that the combinations of H2H3 and H2H2 of SNPs had a higher value than others. Our results indicate that these four SNPs are potentially effective markers and could be used in marker-assisted breeding to improve meat and milk quality simultaneously in the future. Stearoyl-CoA desaturase-1 (SCD1) is a key enzyme in the biosynthesis of monounsaturated fatty acids and is considered a candidate gene for improving milk and meat quality traits. Sanger sequencing was employed to investigate the genetic polymorphism of the fifth exon and intron of bovine SCD1, revealing four SNPs, g.21272246 A>G, g.21272306 T>C, g.21272422 C>T, and g.21272529 A>G. Further variance analysis and multiple comparisons were conducted to examine the relationship between variation sites and economic traits in Chinese Simmental cattle, as well as milk production traits in Holstein cows. The findings revealed these four loci exhibited significant associations with carcass traits (carcass weight, carcass length, backfat thickness, and waist meat thickness), meat quality (pH value, rib eye area, and marbling score), adipogenic traits (fat score and carcass fat coverage rate), and fatty acid composition (linoleic acid and α-linolenic acid). Furthermore, these loci were additionally found to be significantly associated with average milk yield and milk fat content in cows. In addition, a haplotype analysis of combinations of SNPs showed that H2H3 has a significant association with adipogenic traits and H2H2 was associated with higher levels of linoleic acid and α-linolenic acid than the other combinations. These results suggest that the four SNPs are expected to be prospective genetic markers for the above economic traits. In addition, the function of SNPs in exon 5 of SCD1 on gene expression and protein structure needs to be explored in the future. [ABSTRACT FROM AUTHOR]

Delta-like non-canonical Notch ligand 1 (DLK1), which inhibits the differentiation of precursor adipocytes, is a recognized marker gene for precursor adipocytes. Lipids play a crucial role in energy storage and metabolism as a vital determinant of beef quality. In this study, we investigated the mechanism of the DLK1 gene in lipid metabolism by constructing adipose tissue-specific knockout mice. We examined some phenotypic traits, including body weight, liver coefficient, fat index, the content of triglyceride (TG) and cholesterol (CHOL) in abdominal white adipose tissue (WAT) and blood. Subsequently, the fatty acid content and genes related to lipid metabolism expression were detected in DLK1−/− and wild-type mice via GC-MS/MS analysis and quantitative real-time PCR (qRT-PCR), respectively. The results illustrated that DLK1−/− mice exhibited significant abdominal fat deposition compared to wild-type mice. HE staining and immunohistochemistry (IHC) results showed that the white adipocytes of DLK1−/− mice were larger, and the protein expression level of DLK1−/− was significantly lower. Regarding the blood biochemical parameters of female mice, DLK1−/− mice had a strikingly higher triglyceride content (p < 0.001). The fatty acid content in DLK1−/− mice was generally reduced. There was a significant reduction in the expression levels of the majority of genes that play a crucial role in lipid metabolism. This study reveals the molecular regulatory mechanism of fat metabolism in mice and provides a molecular basis and reference for the future application of the DLK1 gene in the breeding of beef cattle with an excellent meat quality traits. It also provides a molecular basis for unravelling the complex and subtle relationship between adipose tissue and health. [ABSTRACT FROM AUTHOR]

Breast milk is widely considered to be the most natural, safe, and complete food for infants. However, current breastfeeding rates fall short of the recommendations established by the World Health Organization. Despite this, there are few studies that have focused on the promotion of human lactation through nutrient supplementation. Therefore, the aim of this study was to investigate the effect of methionine on milk synthesis in human mammary epithelial cells (MCF-10A cells) and to explore the underlying mechanisms. To achieve this, MCF-10A cells were cultured with varying concentrations of methionine, ranging from 0 to 1.2 mM. Our results indicated that 0.6 mM of methionine significantly promoted the synthesis of milk protein. An RNA-seq analysis revealed that methionine acted through the PI3K pathway. This finding was validated through real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. In addition, PI3K inhibition assays confirmed that methionine upregulated the expression of both mTOR and p-mTOR through activation of PI3K. Taken together, these findings suggest that methionine positively regulates milk protein synthesis in MCF-10A cells through the PI3K-mTOR signaling pathway. [ABSTRACT FROM AUTHOR]

Transcriptome sequencing showed that syndecan-3 (SDC3) was differentially expressed in high-fat and low-fat mammary epithelial cells of Chinese Holstein cows. Previous studies found that SDC3 plays an important role in inflammatory diseases and virus infection. However, those studies did not confirm whether or not the functional gene SDC3, which plays an important role in regulating milk fat metabolism, has an effect on susceptibility to breast tissue diseases. Therefore, we studied the effects of SDC3 on milk lipid metabolism and inflammation in bovine mammary epithelial cells (BMECs) and further explored the common regulatory pathway of SDC3 in both. The overexpression of SDC3 increased the contents of triglycerides and cholesterol, reduced the content of non-esterified fatty acids, inhibited the expression of inflammatory factors (IL-6, IL-1β, TNF-α and COX-2), and reduced the production of ROS in BMECs. However, silenced SDC3 had the opposite effect. Further exploring the mechanisms of SDC3, we found that SDC3 upregulated the expression of peroxisome proliferator-activated receptor gamma (PPARG) through the AMPK/SIRT1 signal pathway to promote milk fat synthesis. It also regulated the activation of the NF-κB pathway through the AMPK/SIRT1 signal pathway, reducing the expression of inflammatory factors and ROS production, thus inhibiting the inflammatory response of BMECs. Nuclear factor kappa B subunit 1 (NF-κB p50) was an important target of SDC3 in this process. To sum up, our results showed that SDC3 coregulated milk fat metabolism and inflammation through the AMPK/SIRT1 signaling pathway. This study laid a foundation for the comprehensive evaluation of breeding value based on multi-effect functional genes in dairy cow molecular breeding. [ABSTRACT FROM AUTHOR]

Meat quality has a close relationship with fat and connective tissue; therefore, screening and identifying functional genes related to lipid metabolism is essential for the production of high-grade beef. The transcriptomes of the Longissimus dorsi muscle in Wagyu and Chinese Red Steppe cattle, breeds with significant differences in meat quality and intramuscular fat deposition, were analyzed using RNA-seq to screen for candidate genes associated with beef quality traits. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the 388 differentially expressed genes (DEGs) were involved in biological processes such as short-chain fatty acid metabolism, regulation of fatty acid transport and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. In addition, crystallin alpha B (CRYAB), ankyrin repeat domain 2 (ANKRD2), aldehyde dehydrogenase 9 family member A1 (ALDH9A1) and enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (EHHADH) were investigated for their effects on intracellular triglyceride and fatty acid content and their regulatory effects on genes in lipogenesis and fatty acid metabolism pathways. This study generated a dataset from transcriptome profiling of two cattle breeds, with differing capacities for fat-deposition in the muscle, and revealed molecular evidence that CRYAB, ANKRD2, ALDH9A1 and EHHADH are related to fat metabolism in bovine fetal fibroblasts (BFFs). The results provide potential functional genes for maker-assisted selection and molecular breeding to improve meat quality traits in beef cattle. [ABSTRACT FROM AUTHOR]

Carnitine palmitoyltransferase 1B (CPT1B) is a candidate gene that regulates livestock animal lipid metabolism and encodes the rate-limiting enzyme in fatty acid β-oxidation. To explore the effect of this gene on lipid metabolism in cattle, this study examined CPT1B gene polymorphism in Chinese Simmental cattle and the effect of CPT1B on lipid metabolism. The results showed that the triglyceride content increased significantly with increasing CPT1B gene expression in bovine fetal fibroblasts (BFFs) (p < 0.05), while CPT1B knockout led to decreased CPT1B expression and a downward trend in triglyceride levels. Correlation analysis showed a significant association between the g.119896238 G > C locus and Chinese Simmental cattle backfat thickness (p < 0.05). Backfat thickness was significantly greater in individuals with the GC genotype (0.93 ± 0.67 cm) than in those with the CC genotype (0.84 ± 0.60 cm). The g.119889302 T > C locus was significantly correlated with arachidonic acid content in Chinese Simmental cattle (p < 0.05). The arachidonic acid content in the longissimus muscle was significantly higher in CC genotype beef cattle (0.054 g/100 g) than in those with the other two genotypes (0.046 g/100 g, 0.049 g/100 g). These molecular markers can be effectively used for marker-assisted selection in cattle breeding. [ABSTRACT FROM AUTHOR]

3-β-hydroxysteroid-Δ-24-reductase (DHCR24) gene regulates the abdominal fat of broilers and it is hypothesized that it can also play a role in the fat metabolism of cattle. A mutation in the 3' untranslated region of the DHCR24 gene was identified that led to the differential expression of DHCR24 in cattle. In this study, we detected mutation at g.32435 from G > A in 3'UTR of DHCR24 gene in a Chinese Simmental steer population. In mutated individuals at g. 32435 G > A of DHCR24 the kidney fat weight of GG was higher than that of GA and AA individuals. Steers of the GG and AA genotype having high backfat thickness (p < 0.05) and a higher carcass fat coverage rate (p < 0.05). Furthermore, GG genotype individuals had higher marble grade than GA genotype individuals (p < 0.05). It is predicted through bioinformatics analysis that bta-miR-12059 can bind to 3'UTR of the DHCR24 gene. Our results indicate that single nucleotide polymorphisms might be used as a molecular marker for marker-assisted selection in beef cattle breeding. [ABSTRACT FROM AUTHOR]

Simple Summary: In order to study the effect of chitosan oligosaccharides on milk fat synthesis of bovine mammary epithelial cells (BMECs), we did a series of related experiments. The results showed that chitosan oligosaccharide (COS) could inhibit the fatty acid synthesis and promote milk fat decomposition and oxidation through AMPK/SREBP1/SCD1, AMPK/HSL and AMPK/PPARα signaling pathways to reduce the milk fat content in bovine mammary epithelial cells. We elucidated the important role of COS in BMECs lipid metabolism. COS may be the potential small-molecule component in milk cow molecular breeding to regulate milk fat synthesis and metabolism. These findings will help us to further understand the mechanism of COS on milk fat metabolism. Chitosan oligosaccharide (COS) is a variety of oligosaccharides, and it is also the only abundant basic amino oligosaccharide in natural polysaccharides. Chitosan oligosaccharide is a low molecular weight product of chitosan after enzymatic degradation. It has many biological effects, such as lipid-lowering, antioxidant and immune regulation. Previous studies have shown that chitosan oligosaccharide has a certain effect on fat synthesis, but the effect of chitosan oligosaccharide on milk fat synthesis of bovine mammary epithelial cells (BMECs) has not been studied. Therefore, this study aimed to investigate chitosan oligosaccharide's effect on milk fat synthesis in bovine mammary epithelial cells and explore the underlying mechanism. We treated bovine mammary epithelial cells with different concentrations of chitosan oligosaccharide (0, 100, 150, 200, 400 and 800 μg/mL) for 24 h, 36 h and 48 h respectively. To assess the effect of chitosan oligosaccharide on bovine mammary epithelial cells and determine the concentration and time for chitosan oligosaccharide treatment on cells, several in vitro cellular experiments, including on cell viability, cycle and proliferation were carried out. The results highlighted that chitosan oligosaccharide (100, 150 μg/mL) significantly promoted cell viability, cycle and proliferation, increased intracellular cholesterol content, and reduced intracellular triglyceride and non-esterified fatty acids content. Under the stimulation of chitosan oligosaccharide, the expression of genes downstream of Phosphorylated AMP-activated protein kinase (P-AMPK) and AMP-activated protein kinase (AMPK) signaling pathway changed, increasing the expression of peroxisome proliferator-activated receptor alpha (PPARα) and hormone-sensitive lipase (HSL), but the expression of sterol regulatory element-binding protein 1c (SREBP1) and its downstream target gene stearoyl-CoA desaturase (SCD1) decreased. In conclusion, these results suggest that chitosan oligosaccharide may inhibit milk fat synthesis in bovine mammary epithelial cells by activating the AMP-activated protein kinase signaling pathway, promoting the oxidative decomposition of fatty acids and inhibiting fatty acid synthesis. [ABSTRACT FROM AUTHOR]

Mitochondrial glycerol-3-phosphate acyltransferase (GPAM) catalyses the initial and rate-regulated first-stage pathway of glycerol lipid synthesis and helps to allocate acyl-CoA (acyl-coenzyme A) to triglyceride (TG) synthesis and away from degradation pathways in animal lipometabolism-related pathways. In this study, RNA interference (RNAi) and GPAM gene overexpression were used to examine the correlation between the expression of GPAM and adipogenesis in bovine mammary epithelial cells (bMECs). Additionally, three novel polymorphisms were identified within the bovine key functional domain of GPAM with Sanger sequencing. The relationship between variants of the GPAM gene and milk quality traits of Chinese Holstein cows was then analysed using statistical methods. The results showed that knockdown of the GPAM gene significantly reduced the synthesis of triglycerides in the bMECs (p < 0.05), whereas the overexpression of the GPAM gene significantly increased the synthesis of TG (p < 0.05). In Chinese Holstein dairy cattle, the polymorphic locus of the GPAM gene E20-3386G > A was significantly correlated with fat, protein and somatic cell count (p < 0.05); I18-652A > G was significantly correlated with fat, total fat content, protein, dry matter and somatic cell count (p < 0.05); and I18-726A > G was significantly correlated with protein, milk yield, dry matter and somatic cell count (p < 0.05). Specifically, individuals with the AA genotype of the I18-652A > G and E20-3386G > A polymorphic loci had a higher milk fat percentage (p < 0.05). In summary, GPAM plays a pivotal role in the intracellular regulation of triglyceride, and its mutations could work as a competent molecular marker for selective breeding in dairy cattle. [ABSTRACT FROM AUTHOR]

microRNA is a class of single-stranded RNA molecules of about 22–24 nucleotides in length, which regulate a variety of biological processes, including lipid metabolism and triglyceride synthesis at transcriptional and translational levels by degrading target mRNAs or interfering with the protein production. In this study, the effect of miR-2382-5p on triglyceride levels was examined in bovine mammary epithelial cells (BMECs), and the results showed that miR-2382-5p could decrease the content of triglyceride. Furthermore, miR-2382-5p regulated the expression of lipoprotein lipase (LPL), peroxisome proliferator-activated receptor gamma co-activator 1beta (PPARGC1B), hormone-sensitive lipase (HSL), and peroxisome proliferator-activated receptor gamma (PPARγ), which are known to increase triglyceride decomposition in lipid metabolism. Luciferase reporter assay and quantitative real-time PCR (qPCR) validated that miR-2382-5p downregulated the mRNA expression of target gene N-myc downstream-regulated gene 2 (NDRG2) by specifically recognizing and binding to its 3′-untranslated region (UTR). Meanwhile, overexpression of NDRG2 led to increased triglyceride and cholesterol production in BMECs. In summary, this study suggested that miR-2382-5p regulated lipid metabolism by targeting NDRG2, which might be a potential target for molecular manipulation of milk fat composition to produce healthy milk. This study also provided basic data for further understanding lipid metabolism in dairy cattle. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) can regulate a variety of biological functions such as fat and cholesterol synthesis, translocation, and utilization by regulating degradation or by inhibiting target mRNA translation. In this study, the target gene of miR-21-3p was analyzed through bioinformatics, which predicted Elovl5. Furthermore, miR-21-3p was verified for regulation of triglyceride expression. The results showed that luciferase activity was significantly lower in cells cotransfected with miR-21-3p and Elovl5 reporter vector, pmiR-RB-REPORT-Elovl5-WT, than in cells with miR-21-3p and reporter control vectors, pmiR-RB-REPORT-Elovl5-mut and pmiR-RB-REPORT-Elovl5-si, indicating that target sites exist in the 3′UTR of Elovl5. Further analysis using qPCR and Western blotting showed that the expression of miR-21-3p negatively correlated with the levels of Elovl5 mRNA and protein, suggesting that miR-21-3p might play an important role in regulating Elovl5 gene expression. Finally, transfection with bta-miR-21-3p mimics, bta-miR-21-3p inhibitor, or miRNA-ShNC in mammary epithelial cells suggested that bta-miR-21-3p promotes triglyceride production, which might be attributed to the expression of the target gene Elovl5. [ABSTRACT FROM AUTHOR]

Acyl-CoA synthetase long-chain family member 5 (ACSL5) is a member of the acyl coenzyme A (CoA) long-chain synthase families (ACSLs), and it plays a key role in fatty acid metabolism. In this study, we proved an association between the ACSL5 gene and triglyceride metabolism at the cellular level in cattle. pBI-CMV3- ACSL5 and pGPU6/GFP/Neo- ACSL5 plasmids were constructed and transfected into bovine preadipocytes by electroporation. The expression level of ACSL5 was detected by real-time quantitative PCR and western blot. The triglyceride content was detected by a triglyceride kit. The results indicated that the expression level of ACSL5 mRNA and protein in the pBI-CMV3- ACSL5 -transfected group was significantly increased compared with those in the control group. Furthermore, the pGPU6/GFP/Neo- ACSL5 -transfected group was significantly decreased compared with those in the control group. A cell triglyceride test showed that overexpression or silencing of the ACSL5 gene could affect synthesis of cellular triglycerides. This study investigated the mechanism of ACSL on bovine fat deposition, and also provides a new candidate gene for meat quality traits in beef cattle. [ABSTRACT FROM AUTHOR]

Unlike specific expression in the skin of wild mice, the agouti signaling protein (ASIP) is expressed widely in the tissue of cattle, including adipose and muscle tissue. Hence, it has been suggested that ASIP plays a role in bovine fat metabolism. An inserted L1-BT element was recently identified upstream of the ASIP locus which led to an ectopic expression of ASIP mRNA in cattle. In this study, we detected the indel of the L1-BT element at g. -14643 nt and three SNPs in introns of the ASIP gene (g. -568 A > G, g. -554 A > T, and g. 4805A > T) in a Chinese Simmental steer population. The association analysis between variants of ASIP and economic traits showed that the homozygous genotype of L1-BT element insertion, AA genotype of g. -568 A > G, and AT genotype of g. 4805A > T were significantly correlated with carcass and fat-related traits, such as live weight and back fat thickness. Moreover, three haplotypes (H1: AT; H2: AA; H3: GT) were identified by linkage disequilibrium analysis and formed six combined genotypes. Results indicated that Chinese Simmental steers with an H1H2 combined genotype had a higher measured value of fat-deposition-related traits (p<0.05), including thickness of back fat and percentage of carcass fat coverage, but a lower content of linoleic acid and α -linolenic acid (p<0.05). Individuals of an H3H3 combination had a lower marbling score, perirenal fat weight, and carcass weight (p<0.05). This suggests that these three SNPs and two combined haplotypes might be molecular markers for beef cattle breeding selection. [ABSTRACT FROM AUTHOR]

Short/branched chain acyl-CoA dehydrogenase (ACADSB) is a member of the acyl-CoA dehydrogenase family of enzymes that catalyze the dehydrogenation of acyl-CoA derivatives in the metabolism of fatty acids. Our previous transcriptome analysis in dairy cattle showed that ACADSB was differentially expressed and was associated with milk fat metabolism. The aim of this study was to elucidate the background of this differential expression and to evaluate the role of ACADSB as a candidate for fat metabolism in dairy cattle. After analysis of ACADSB mRNA abundance by qRT-PCR and Western blot, overexpression and RNA interference (RNAi) vectors of ACADSB gene were constructed and then transfected into bovine mammary epithelial cells (bMECs) to examine the effects of ACADSB on milk fat synthesis. The results showed that the ACADSB was differentially expressed in mammary tissue of low and high milk fat dairy cattle. Overexpression of ACADSB gene could significantly increase the level of intracellular triglyceride (TG), while ACADSB gene knockdown could significantly reduce the TG synthesis in bMECs. This study suggested that the ACADSB was important in TG synthesis in bMECs, and it could be a candidate gene to regulate the metabolism of milk fat in dairy cattle. [ABSTRACT FROM AUTHOR]

Hormone-sensitive lipase (HSL) is an intracellular neutral lipase capable of hydrolysing a variety of esters and is considered to be a candidate gene affecting fat deposition traits. Gene expression profiles of HSL were analysed in various adipose tissues of cattle, and the effect of HSL on lipid metabolism genes was analysed by a PCR array. Novel polymorphisms were identified within the HSL regulatory domain by sequencing, and the relationship between HSL variants and fat deposition traits was analysed. HSL mRNA was highly expressed in the subcutaneous and visceral fat of cattle. CPT1B/CPT1C and other lipocatabolic genes were upregulated, and lipogenesis-related genes ( FASN , LPL and ACOT12 ) were downregulated by HSL overexpression in BFFs. Five novel variants in the HSL functional domain were significantly associated with fat deposition traits, including FCR, LBT, MFW and fatty acid composition. HSL plays a pivotal role in the regulation of lipolysis and fatty acid biosynthesis in beef cattle. [ABSTRACT FROM AUTHOR]

DNA methylation is an important epigenetic mechanism involved in expression of genes in many biological processes including muscle growth and development. Its effects on economically important traits are evinced from reported significant differences in meat quality traits between Japanese black (Wagyu) and Chinese Red Steppes cattle, thus presenting a unique model for analyzing the effects of DNA methylation on these traits. In the present study, we performed whole genome DNA methylation analysis in the two breeds by whole genome bisulfite sequencing (WGBS). Overall, 23150 differentially methylated regions (DMRs) were identified which were located in 8596 genes enriched in 9922 GO terms, of which 1046 GO terms were significantly enriched (p<0.05) including lipid translocation (GO: 0034204) and lipid transport (GO: 0015914). KEGG analysis showed that the DMR related genes were distributed among 276 pathways. Correlation analysis found that 331 DMRs were negatively correlated with the expression levels of differentially expressed genes (DEGs) with 21 DMRs located in promoter regions. Our results identified novel candidate DMRs and DEGs correlated with meat quality traits, which will be valuable for future genomic and epigenomic studies of muscle development and for marker assisted selection of meat quality traits. [ABSTRACT FROM AUTHOR]

Bama Xiang pig (BM) and Tibetan mini-pig (TM) are used as experimental animals in China; however, the dwarf molecular mechanisms of these Chinese local pig breeds are unknown. IGFBP-3 affects animal growth, carcass and meat quality. The aim of this study was to identify the polymorphisms in the promoter of theIGFBP-3and analyse their effect on theIGFBP-3mRNA expression level in liver and muscle tissues. High-density single-nucleotide polymorphisms (SNPs) (31) and InDels (5) were detected in the promoter of theIGFBP-3in the BM, TM and Junmu No. 1 White (JM, control) pig breeds from 114 individuals by re-sequencing. A perfect Linkage disequilibrium consisted of 13 SNPs was observed in the promoter region and 2 main haplotypes were identified, of which theh1genotype (GCA-ATGTACATAT) was more prevalent in JM breed than in TM or BM breeds (P < .0001),h2(ATGTGCACG--CGC) was the dominant haplotype in TM and BM breeds (P < .0001). Expression analysis showed that haplotype of the promoter region is highly associated with theIGFBP-3mRNA expression level in liver and muscle tissues of pigs. TheIGFBP-3mRNA expression level was determined higher in the liver and muscle tissues of pigs withh2genotype as compared to that in pigs withh1genotype (P < .05). The results suggest that the SNPs and haplotypes in the promoter of theIGFBP-3gene may serve as useful molecular markers for the body growth traits and the breeding in swine. [ABSTRACT FROM AUTHOR]

microRNAs are involved in various biological processes by regulating the degradation or repressing the translation of target genes. In this study, the target genes of miR-29b were analyzed and predicted by bioinformatics. And lipoprotein lipase ( LPL) and thymine DNA glycosylase ( TDG) were selected for further validation by dual luciferase reporter assay. In addition, we investigated the effects of miR-29b on triglyceride synthesis and mammary epithelial cell (MEC) apoptosis. The result showed that luciferase activity was significantly lower in cells that miR-29b cotransfected with LPL and TDG gene reporter vectors ( pmiR-RB-REPORT-LPL-WT, pmiR-RB-REPORT-TDG-WT) than in cells of miR-29b cotransfected with gene reporter vectors ( pmiR-RB-REPORT-LPL-mut and pmiR-RB-REPORT-LPL-si; pmiR-RB-REPORT-TDG-mut and pmiR-RB-REPORT-TDG-si) ( p < 0.05), indicating that target sites existed in 3′UTR of LPL and TDG. Furthermore, the expressions of miR-29b were negatively correlated with levels of mRNA and protein of LPL and TDG gene using quantitative real-time polymerase chain reaction and western blot analysis, suggesting that miR-29b might play an important role in regulation of LPL and TDG gene expression. Finally, miR-29b promoted triglyceride production and suppressed apoptosis in MECs, which might be attributed to the expressions of target genes LPL and TDG. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor binding protein 6 (IGFBP6) is a key factor in regulating the effects of insulin-like growth factor 2 (IGF2) on the animal growth and development, but the mechanism is far from known. In this study, the 5'-terminal sequence of theIGFBP6gene (from −920 to −1 bp) which may regulate gene expression was sequenced in the Bama mini-pigs, the Tibetan mini-pigs, the Landrace pigs, the Large White pigs and the Northeast wild boars to screen for the single nucleotide polymorphisms (SNPs) and analyze the relations with the body size traits of swine through a chi-square test analysis. The genotype frequencies of the SNPs in the 5'-terminal sequence have shown that c.-726C > T, c.-722T > C, c.-535C > A, c.-488T > C, c.-403A > G, and c.-378T > C may be related to the dwarf traits of the Bama mini-pig and the Tibetan mini-pigs (P < .05). A haplotype analysis of the 5'-terminal sequence of theIGFBP6gene in the Landrace pigs, the Large White pigs and the Northeast wild boars found the two SNPs at −403 nt and −378 nt were in linkage and formed three kinds of haplotypes; AT was the dominant haplotype and the haplotype block was not formed in the Bama mini-pigs and Tibetan mini-pigs. Above all, the SNPs and haplotypes of the 5'-terminal sequence of theIGFBP6gene may be involved in the regulation of swine body size. [ABSTRACT FROM AUTHOR]

ABSTRACT Growth-hormone-releasing peptide 6 (GHRP-6) plays an important role in animal growth. However, there have been few studies focusing on the effect of GHRP-6 on animal growth through controlled release systems. We synthesized the poly(lactic-co-glycolic acid) (PLGA)-poly(ethylene glycol) (PEG)-PLGA copolymer to investigate its controlled released effect on GHRP-6 in vitro and to study the effect of a GHRP-6-copolymer hydrogel on the growth of rex rabbits. The copolymer was synthesized with ring-opening copolymerization and characterized by 1H-NMR. The interaction between GHRP-6 and the copolymer was characterized by Fourier transform infrared spectroscopy and X-ray diffraction. The body weight, serum level of insulin-like growth factor 1 (IGF-1), and hair coat quality were studied in rex rabbits. The results show that hydrogen bonds formed between the NH group in GHRP-6 and the CO group in the copolymer. The release mechanism of GHRP-6 was a combination of a diffusion-controlled mechanism and an erosion-controlled mechanism in the copolymer. The serum level of IGF-1, hair coat quality, and body weight were all significantly higher in the GHRP-6-copolymer hydrogel group than in the other groups. These results indicate that the copolymer effectively controlled the release of GHRP-6. In addition, the GHRP-6-copolymer hydrogel increased the synthesis of IGF-1 for a prolonged period and, thereby, increased the rex rabbits' growth and hair coat quality. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40185. [ABSTRACT FROM AUTHOR]

Simple Summary: MicroRNAs are small molecules that can regulate the relative abundance of their target genes by binding to the 3′ untranslated region of the target genes at the post-transcriptional level to affect various biological processes, such as biosynthesis, fat metabolism and proliferation, apoptosis, and cell differentiation. Fertility is one of the most important economic traits in livestock production. Bulls require the continuous production of high-quality spermatozoa in abundance. The quality of semen is an exceptionally important factor affecting the fertilization rate of the dairy cow and is also associated with the increasing conception rate in the process of artificial insemination. Therefore, accurately predicting fertility potential for a semen sample from donor bull for artificial insemination is crucial for consistently high reproductive efficiency. The present study performed a genome-wide sequencing analysis of microRNAs and mRNAs between immature and mature testes of Chinese Red Steppes. These results provide novel candidate microRNAs and functional genes related to bull reproduction traits and the networks between microRNAs and target genes, which will provide a useful genetic mechanism and epigenetic information for marker-assisted selection of bulls with excellent sperm quality in the future. This study aims to screen potential regulators and regulate fecundity networks between microRNAs (miRNAs) and target genes. The bovine testes of immature and mature Chinese Red Steppes were performed by genome-wide analysis of mRNAs and miRNAs. Compared with testicular tissues of newborns, 6051 upregulated genes and 7104 downregulated genes in adult cattle were identified as differentially expressed genes (DEGs). The DEGs were significantly enriched in 808 GO terms (p < 0.05) including male gonad development, male genitalia development, spermatogenesis, and sperm motility. Moreover, DEGs were also significantly enriched in 105 KEGG pathways (p < 0.05), including cGMP-PKG signaling pathway and calcium signaling pathway. To explore the expression of miRNA-regulated gene expression, 896 differentially expressed target genes negatively regulated with the expression levels of 31 differentially expressed miRNAs (DERs) were predicted and analyzed, and a network-integrated analysis was constructed. Furthermore, real-time PCR was performed to verify the expression levels of DEGs and DERs. Our results identified novel candidate DEGs and DERs correlated with male reproduction and intricate regulating networks between miRNAs and genes, which will be valuable for future genetic and epigenetic studies of sperm development and maturity, as well as providing valuable insights into the molecular mechanisms of male fertility and spermatogenesis in cattle. [ABSTRACT FROM AUTHOR]

Simple Summary: Fertility is one of the essential reproduction traits of bulls, and accurate prediction of fertility potential using a semen sample from a donor bull for artificial insemination is crucial to achieve consistently high reproductive efficiency. Somatic cells, such as Sertoli cells and Leydig cells, are important in testis formation and provide a nurturing and regulatory environment for spermatogenesis. Furthermore, it was suggested that non-coding RNAs, such as microRNAs, long non-coding RNAs, circular RNAs, and Piwi-interacting RNA, function as important regulators of gene expression at post-transcriptional level in spermatogenesis. In this study, microRNA-34c was verified to specifically regulate the AXL gene by targeting a sequence in the 3' UTR; miRNA-34c can also influence the proliferation, apoptosis, and relative abundance of the transcript of male-reproduction-related genes. Therefore, microRNA-34c can be considered an essential regulator in the process of bull spermatogenesis. These results identify a key microRNA and functional genes in the process of cattle male reproduction, providing useful information for future marker-assisted selection of bulls with excellent sperm quality. MicroRNAs (miRNAs) play significant roles in mammalian spermatogenesis. Sertoli cells can provide a stable microenvironment and nutritional factors for germ cells, thus playing a vital role in spermatogenesis. However, few studies elucidate the regulation of bovine testicular Sertoli cells by miRNAs. Here, we have reported that miRNA-34c (miR-34c) regulates proliferation, apoptosis, and relative transcripts abundance gene in bovine Sertoli cells. In bovine Sertoli cells, overexpression of miR-34c inhibited proliferation and relative abundance of gene transcripts while promoting apoptosis of Sertoli cells, and the effects were the opposite when miR-34c was knocked down. Receptor tyrosine kinase (AXL) was identified as a direct target gene of miR-34c in Sertoli cells, validated by analysis of the relative abundance of AXL transcript and dual-luciferase reporter assay. The relative abundance of the transcript of genes related to male reproduction in Sertoli cells was changed after the AXL gene was overexpressed, as demonstrated by the RT2 Profiler PCR Array results. In summary, miR-34c specifically regulated the AXL gene by targeting a sequence in the 3′-UTR, which could influence proliferation, apoptosis, and relative abundance of the transcript of male reproduction-related genes. Therefore, miR-34c could be considered an essential regulator in the process of bull spermatogenesis. [ABSTRACT FROM AUTHOR]

Simple Summary: Delta-like non-canonical Notch ligand 1 (DLK1) is a candidate gene associated with lipid metabolism. In order to verify the function of the DLK1 gene on lipid metabolism in Chinese Simmental cattle, we identified the effect of DLK1 on lipid metabolism in bovine fetal fibroblast cells (BFFs). At the same time, we also detected the relationship between single-nucleotide polymorphism sites (SNPs) of bovine DLK1 with the economic traits and fatty acids composition in Chinese Simmental cattle, such as the carcass fat coverage rate, loin eye muscle area, and fat color score, etc. In the present study, we precisely constructed and transfected the overexpression and interference vectors of the DLK1 gene in BFFs. the results showed that the overexpression of the DLK1 gene could decrease the contents of intracellular triglycerides (TGs) while interference of the DLK1 gene could increase the TGs contents. Gas chromatography analysis of the fatty acid composition showed that the contents of octanoate acid and γ-linolenate acid were regulated as the expression of DLK1 was altered. We found two SNPs and genes associated with these traits in Chinese Simmental by the restriction fragment length polymorphism (RPLF-PCR) detection method. In summary, we verified the effects of bovine DLK1 on fatty acid metabolism from the cellular level to population genetic polymorphism, which also paved the way for further studying of the effects of the DLK1 gene on lipid metabolism in vivo. In this study, we precisely constructed and transfected the overexpression and interference vectors in BFFs to evaluate the role of DLK1 gene on lipid metabolism in vitro. The expression of of DLK1 in the mRNA and protein level tended to reduce, and TGs were significantly increased in the pGPU6-shDLK1 group compared to the control group (p < 0.05). The expression of DLK1 in the mRNA and protein level were increased in the pBI-CMV3-DLK1 group compared to the control group, and the TGs content showed a significant decrease in the pBI-CMV3-DLK1 group (p < 0.05). Meanwhile, we used the restriction fragment length polymorphism (RFLP-PCR) detection method to screen SNPs further to explore and analyze the relationship between the gene and the economic traits of 28-month-old Chinese Simmental and the fatty acids composition of cattle longissimus muscle. The result showed that two SNPs, IVS3 + 478 C > T and IVS3 + 609 T > G, were identified as being significantly associated with carcass and meat quality traits in Chinese Simmental, such as the carcass fat coverage rate, loin eye muscle area, and fat color score. In summary, our results indicated that DLK1 can affect lipid metabolism in bovine and these two SNPs might be applied as genetic markers of meat quality traits for beef cattle breeding. [ABSTRACT FROM AUTHOR]

Simple Summary: Promoting fat deposition in beef cattle has been a focus of modern animal breeding research. However, previous researchers have not examined the mechanism of adipogenesis in much detail. MicroRNAs (miRNAs) are small noncoding RNAs that play a pivotal role in adipogenesis. In this study, to explore the molecular regulatory mechanism of adipocyte differentiation and formation, bovine preadipocytes were isolated and induced into adipocytes, and then the expression patterns of miRNAs between preadipocytes and adipocytes were detected through RNA sequencing. Deep sequence analysis has identified 78, 71, and 48 novel miRNAs and 497, 491, and 524 known miRNAs in the preadipocytes, and 44, 54, and 47 novel miRNAs and 519, 522, and 504 known miRNAs in the adipocytes. Among the annotated miRNAs, 131 bovine miRNAs were upregulated in adipocytes, and 119 bovine miRNAs were downregulated in adipocytes, such as bta-miR-3604, bta-miR-23b-3p, bta-miR-26a, and bta-miR-129-3p. Bovine target gene prediction results of these miRNAs show that numerous genes are associated with lipid metabolism. These results can provide both technical support and a research basis for promoting bovine adipocyte fat deposition. The elucidation of the mechanisms of preadipocyte differentiation and fat accumulation in adipocytes is a major work in beef cattle breeding. As important post-transcriptional regulators, microRNAs (miRNAs) take part in cell proliferation, differentiation, apoptosis, and fat metabolism through binding seed sites of targeting mRNAs. The aim of this study was to isolate and identify bovine preadipocytes and screen miRNAs associated with adipogenesis. Bovine preadipocytes were isolated from subcutaneous fatty tissue and induced to differentiate into adipocytes. Verification of preadipocytes and adipocytes was performed by qRT-PCR (real-time quantitative reverse transcription PCR), Oil Red O staining, and immunofluorescence staining. Total RNA was extracted for small RNA sequencing. The sequencing data showed that 131 miRNAs were highly expressed in adipocytes, and 119 miRNAs were highly expressed in preadipocytes. Stem–loop qPCR (stem–loop quantitative real-time PCR) results showed that the expression patterns of 11 miRNAs were consistent with the sequencing results (miR-149-5p, miR-24-3p, miR-199a-5p, miR-33a, etc.). According to KEGG pathway and Gene Ontology (GO) analyses, multiple predicted target genes were associated with lipid metabolism. In summary, this study provides a protocol of isolating bovine preadipocytes and screening various differently expressed miRNAs during preadipocyte differentiation. [ABSTRACT FROM AUTHOR]

• Crisper/Cas9 mediated GPAM gene knock out for deciphering its role in lipid metabolism in BMECs of Chinese Holstein. • GPAM knock out resulted in decreased triglyceride, cholesterol, and fatty acid contents in BMECs. • The DEGs identified from RNA-seq were enriched for lipid metabolism related GO terms and KEGG pathways. Glycerol-3-phosphate acyltransferase mitochondrial (GPAM) is an enzyme in animal lipid metabolism pathways that catalyzes the initial and most committed step of glycerolipid biosynthesis. The present study mainly focused on exploring the relationship between the GPAM gene and the lipid metabolism of mammary epithelial cells and the effect of GPAM on the related pathways of lipid metabolism. The GPAM gene was knocked out entirely in bovine mammary epithelial cells(BMECs) using CRISPR/Cas9 technology, and the mechanism by which the GPAM gene regulates lipid metabolism in BMECs was confirmed. Furthermore, after the complete loss of GPAM , BMECs' triglycerides (TGs) and cholesterol (CHOL) levels were significantly decreased (p < 0.05). Concurrently, the content of octanoic acid, a medium-chain saturated fatty acid, increased substantially in BMECs. RNA-seq of GPAM −/− BMECs revealed that GPAM could affect the expression of genes related to lipid metabolism, downregulated the expression of Acyl-CoA synthetase long-chain family member 5 (ACSL5) , Fatty Acid Binding Protein 3 (FABP3) , Hormone-sensitive lipase (HSL), Protease, serine-2 (PRSS2), 1-Acylglycerol-3-Phosphate O Acyltransferase 4 (AGPAT4), and regulated the milk synthesis metabolism pathway.The findings revealed that a number of genes were expressed, a number of genes were differentially expressed genes (DEGs), and a number of GO terms were enriched, with a number of GO terms considerably increased. Further, the differentially expressed genes (DEGs) were significantly enriched in Fat digestion and absorption pathway, Fatty acid metabolic pathway, Biosynthesis of unsaturated fatty acids, Biosynthesis of unsaturated fatty acids and steroids, NF-kappa B signalling pathway, MAPK signalling pathway. In conclusion, the current research results show that GPAM is a crucial regulator of BMEC lipid metabolism. GPAM −/− BMEC may also become useful genetic materials and tools for future research on gene functions related to lipid and fatty acid metabolism. This study will contribute to the discovery of gene regulation and molecular mechanisms in milk fat synthesis. [ABSTRACT FROM AUTHOR]

The CD44 gene encodes a cell-surface glycoprotein that participates in a variety of biological processes such as cell interactions, adhesion, hematopoiesis, and tumor metastasis. We compared the transcriptome in bovine mammary epithelial cells (bMEC) of Chinese Holstein dairy cows producing milk of high and low fat contents. Our results suggest that CD44 might be a candidate gene affecting milk fat synthesis. In the present study, the overexpression of the CD44 gene increased the contents of intracellular triglycerides (TG) and cholesterol (CHOL), whereas knockdown of the CD44 gene decreased bMEC CHOL and TG contents. Gas chromatography analysis of fatty acid composition showed that the contents of α-linolenic acid, palmitic acid, and cis -8,11,14-eicosatrienoic acid were altered due to changes in the level of expression of the CD44 gene. Additionally, elaidic acid, palmitoleic acid, tridecanoic acid, and oleic acid were markedly reduced in the CD44 gene overexpression group compared with the control group. On the contrary, cis -5,8,11,14-eicosatetraenoic acid and stearic acid were markedly increased in the CD44 knockdown group compared with the control group. And RT2 Profiler PCR array (Qiagen, CLAB24070A Frankfurt, Germany) further suggested that overexpression or knockdown of the CD44 gene altered expression levels of functional genes associated with lipid metabolism. The present data indicate that CD44 plays a key regulatory role in lipid metabolism in bMEC. [ABSTRACT FROM AUTHOR]

Delta-like non-canonical Notch ligand 1 ( DLK1 ), which inhibits the differentiation of precursor adipocytes, is a recognized marker gene for precursor adipocytes. Lipids play a crucial role in energy storage and metabolism as a vital determinant of beef quality. In this study, we investigated the mechanism of the DLK1 gene in lipid metabolism by constructing adipose tissue-specific knockout mice. We examined some phenotypic traits, including body weight, liver coefficient, fat index, the content of triglyceride (TG) and cholesterol (CHOL) in abdominal white adipose tissue (WAT) and blood. Subsequently, the fatty acid content and genes related to lipid metabolism expression were detected in DLK1 -/- and wild-type mice via GC-MS/MS analysis and quantitative real-time PCR (qRT-PCR), respectively. The results illustrated that DLK1 -/- mice exhibited significant abdominal fat deposition compared to wild-type mice. HE staining and immunohistochemistry (IHC) results showed that the white adipocytes of DLK1 -/- mice were larger, and the protein expression level of DLK1 -/- was significantly lower. Regarding the blood biochemical parameters of female mice, DLK1 -/- mice had a strikingly higher triglyceride content ( p < 0.001). The fatty acid content in DLK1 -/- mice was generally reduced. There was a significant reduction in the expression levels of the majority of genes that play a crucial role in lipid metabolism. This study reveals the molecular regulatory mechanism of fat metabolism in mice and provides a molecular basis and reference for the future application of the DLK1 gene in the breeding of beef cattle with an excellent meat quality traits. It also provides a molecular basis for unravelling the complex and subtle relationship between adipose tissue and health.

Meat quality has a close relationship with fat and connective tissue; therefore, screening and identifying functional genes related to lipid metabolism is essential for the production of high-grade beef. The transcriptomes of the Longissimus dorsi muscle in Wagyu and Chinese Red Steppe cattle, breeds with significant differences in meat quality and intramuscular fat deposition, were analyzed using RNA-seq to screen for candidate genes associated with beef quality traits. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the 388 differentially expressed genes (DEGs) were involved in biological processes such as short-chain fatty acid metabolism, regulation of fatty acid transport and the peroxisome proliferator-activated receptor (PPAR) signaling pathway. In addition, crystallin alpha B ( CRYAB ), ankyrin repeat domain 2 ( ANKRD2 ), aldehyde dehydrogenase 9 family member A1 ( ALDH9A1 ) and enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase ( EHHADH ) were investigated for their effects on intracellular triglyceride and fatty acid content and their regulatory effects on genes in lipogenesis and fatty acid metabolism pathways. This study generated a dataset from transcriptome profiling of two cattle breeds, with differing capacities for fat-deposition in the muscle, and revealed molecular evidence that CRYAB , ANKRD2 , ALDH9A1 and EHHADH are related to fat metabolism in bovine fetal fibroblasts (BFFs). The results provide potential functional genes for maker-assisted selection and molecular breeding to improve meat quality traits in beef cattle.

Chitosan oligosaccharide (COS) is a variety of oligosaccharides, and it is also the only abundant basic amino oligosaccharide in natural polysaccharides. Chitosan oligosaccharide is a low molecular weight product of chitosan after enzymatic degradation. It has many biological effects, such as lipid-lowering, antioxidant and immune regulation. Previous studies have shown that chitosan oligosaccharide has a certain effect on fat synthesis, but the effect of chitosan oligosaccharide on milk fat synthesis of bovine mammary epithelial cells (BMECs) has not been studied. Therefore, this study aimed to investigate chitosan oligosaccharide's effect on milk fat synthesis in bovine mammary epithelial cells and explore the underlying mechanism. We treated bovine mammary epithelial cells with different concentrations of chitosan oligosaccharide (0, 100, 150, 200, 400 and 800 μg/mL) for 24 h, 36 h and 48 h respectively. To assess the effect of chitosan oligosaccharide on bovine mammary epithelial cells and determine the concentration and time for chitosan oligosaccharide treatment on cells, several in vitro cellular experiments, including on cell viability, cycle and proliferation were carried out. The results highlighted that chitosan oligosaccharide (100, 150 μg/mL) significantly promoted cell viability, cycle and proliferation, increased intracellular cholesterol content, and reduced intracellular triglyceride and non-esterified fatty acids content. Under the stimulation of chitosan oligosaccharide, the expression of genes downstream of Phosphorylated AMP-activated protein kinase ( P-AMPK ) and AMP-activated protein kinase ( AMPK ) signaling pathway changed, increasing the expression of peroxisome proliferator-activated receptor alpha ( PPARα ) and hormone-sensitive lipase ( HSL ), but the expression of sterol regulatory element-binding protein 1c ( SREBP1 ) and its downstream target gene stearoyl-CoA desaturase ( SCD1 ) decreased. In conclusion, these results suggest that chitosan oligosaccharide may inhibit milk fat synthesis in bovine mammary epithelial cells by activating the AMP-activated protein kinase signaling pathway, promoting the oxidative decomposition of fatty acids and inhibiting fatty acid synthesis.

Acyl-CoA synthetase family member 3 (ACSF3) carries out the first step of mitochondrial fatty acid synthesis II, which is the linkage of malonate and, to a lesser extent, methylmalonate onto CoA. Malonyl-coenzyme A (malonyl-CoA) is a central metabolite in mammalian fatty acid biochemistry that is generated and utilized in the cytoplasm. In this research, we verified the relationship between expression of the ACSF3 and the production of triglycerides (TGs) at the cellular level by silencing and over-expressing ACSF3 . Subsequently, through Sanger sequencing, five polymorphisms were found in the functional domain of the bovine ACSF3 , and the relationship between ACSF3 polymorphism and the economic traits and fatty acid composition of Chinese Simmental cattle was analyzed by a means of variance analysis and multiple comparison. The results illustrated that the expression of ACSF3 promoted triglyceride synthesis in bovine mammary epithelial cells and bovine fetal fibroblast cells. Further association analysis also indicated that individuals with the AG genotype (g.14211090 G > A) of ACSF3 were significantly associated with the fatty acid composition of intramuscular fat (higher content of linoleic acid, α-linolenic acid, and arachidonic acid), and that CTCAG haplotype individuals were significantly related to the fatty acid composition of intramuscular fat (higher linoleic acid content). Individuals with the AA genotypes of g.14211055 A > G and g.14211090 G > A were substantially associated with a larger eye muscle area in the Chinese Simmental cattle population. ACSF3 played a pivotal role in the regulation of cellular triacylglycerol and long-chain polyunsaturated fatty acid levels, and polymorphism could serve as a useful molecular marker for future marker-assisted selection in the breeding of intramuscular fat deposition traits in beef cattle., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 He, Fang, Lu, Liu, Li, Zhao, Li and Yang.)

The acyl-CoA dehydrogenase family of enzymes includes short/branched-chain acyl-CoA dehydrogenase ( ACADSB ), which catalyzes the dehydrogenation of acyl-CoA derivatives in fatty acid metabolism. Our previous findings suggested that ACADSB was a critical candidate gene affecting milk fat synthesis by comparing the transcriptome in bovine mammary epithelial cells (bMECs) from Chinese Holstein dairy cows producing high-fat and low-fat milk as well as gene functional validation studies on the cellular level. In the present study, ACADSB in bMECs was knocked out (KO) using a CRISPR/Cas9 system, and mRNA transcriptome was further sequenced to verify the function of the ACADSB gene and analyze its correlation with lipid metabolism. The findings revealed that 15,693 genes were expressed, 1,548 genes were differentially expressed genes (DEGs), and 6,098 GO terms were enriched, of which 637 GO terms were greatly enhanced, such as phospholipid-translocation ATPase activity (GO:0004012), lipoprotein lipase activity (GO:0004465), acyl-CoA desaturase activity (GO:0016215), and so on. The analysis by KEGG showed that DEGs were distributed over 247 pathogens, of which 49 were significantly enriched, including the metabolism of fatty acids (PATH: 01212), metabolism of glycerolipid (PATH: 00561), and signaling of adipocytokines (PATH: 04920). The CHOL, TGs and FFA contents in bMECs were reduced when the ACADSB gene was knocked out. The RT 2 Profiler PCR array also revealed that the loss of the ACADSB gene changed the expression levels of functional genes involved in lipid metabolism, including ACADL, ACOX2, ACAT2, and FABP3 . In conclusion, the current findings show that ACADSB is a key regulator of lipid metabolism in bMECs. The ACADSB -/- bMECs could also be useful genetic material and tools for future research into gene functions related to lipid and fatty acid metabolism. It will be valuable for revealing the gene regulatory roles and molecular mechanisms in milk fat synthesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jiang, Iqbal, Wang, Li, Fang, Yu and Zhao.)

MicroRNAs (miRNAs) play significant roles in mammalian spermatogenesis. Sertoli cells can provide a stable microenvironment and nutritional factors for germ cells, thus playing a vital role in spermatogenesis. However, few studies elucidate the regulation of bovine testicular Sertoli cells by miRNAs. Here, we have reported that miRNA-34c (miR-34c) regulates proliferation, apoptosis, and relative transcripts abundance gene in bovine Sertoli cells. In bovine Sertoli cells, overexpression of miR-34c inhibited proliferation and relative abundance of gene transcripts while promoting apoptosis of Sertoli cells, and the effects were the opposite when miR-34c was knocked down. Receptor tyrosine kinase ( AXL ) was identified as a direct target gene of miR-34c in Sertoli cells, validated by analysis of the relative abundance of AXL transcript and dual-luciferase reporter assay. The relative abundance of the transcript of genes related to male reproduction in Sertoli cells was changed after the AXL gene was overexpressed, as demonstrated by the RT2 Profiler PCR Array results. In summary, miR-34c specifically regulated the AXL gene by targeting a sequence in the 3'-UTR, which could influence proliferation, apoptosis, and relative abundance of the transcript of male reproduction-related genes. Therefore, miR-34c could be considered an essential regulator in the process of bull spermatogenesis.

Alternative splicing is a ubiquitous regulatory mechanism in gene expression that allows a single gene generating multiple messenger RNAs (mRNAs). Significant differences in fat deposition ability and meat quality traits have been reported between Japanese black cattle (Wagyu) and Chinese Red Steppes, which presented a unique model for analyzing the effects of transcriptional level on marbling fat in livestock. In previous studies, the differentially expressed genes (DGEs) in longissimus dorsi muscle ( LDM ) samples between Wagyu and other breeds of beef cattle have been reported. In this study, we further investigated the differences in alternative splicing in LDM between Wagyu and Chinese Red Steppes cattle. We identified several alternative splicing types including cassette exon, mutually exclusive exons, alternative 5' splice site, alternative 3' splice site, alternative start exon, and intron retention. In total, 115 differentially expressed alternatively spliced genes were obtained, of which 17 genes were enriched in the metabolic pathway. Among the 17 genes, 5 genes, including MCAT, CPT1B, HADHB, SIRT2 , and DGAT1 , appeared to be the novel spliced candidates that affect the lipid metabolism in cattle. Additionally, another 17 genes were enriched in the Gene Ontology (GO) terms related to muscle development, such as NR4A1, UQCC2, YBX3/CSDA, ITGA7 , etc. Overall, altered splicing and expression levels of these novel candidates between Japanese black cattle and Chinese Red Steppes revealed by RNA-seq suggest their potential involvement in the muscle development and fat deposition of beef cattle., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Fang, Xia, Yu, He, Bai, Qin, Jiang, Zhao, Zhao and Yang.)

In this study, we precisely constructed and transfected the overexpression and interference vectors in BFFs to evaluate the role of DLK1 gene on lipid metabolism in vitro. The expression of of DLK1 in the mRNA and protein level tended to reduce, and TGs were significantly increased in the pGPU6-shDLK1 group compared to the control group ( p < 0.05). The expression of DLK1 in the mRNA and protein level were increased in the pBI-CMV3-DLK1 group compared to the control group, and the TGs content showed a significant decrease in the pBI-CMV3-DLK1 group ( p < 0.05). Meanwhile, we used the restriction fragment length polymorphism (RFLP-PCR) detection method to screen SNPs further to explore and analyze the relationship between the gene and the economic traits of 28-month-old Chinese Simmental and the fatty acids composition of cattle longissimus muscle. The result showed that two SNPs, IVS3 + 478 C>T and IVS3 + 609 T>G, were identified as being significantly associated with carcass and meat quality traits in Chinese Simmental, such as the carcass fat coverage rate, loin eye muscle area, and fat color score. In summary, our results indicated that DLK1 can affect lipid metabolism in bovine and these two SNPs might be applied as genetic markers of meat quality traits for beef cattle breeding., Competing Interests: The authors declare no competing financial interest.

QSOX1 (quiescin-sulfhydryl oxidase 1) is involved in various processes, including apoptosis and the development of breast diseases. Here, we investigated the effect of QSOX1 on the meat quality of Simmental cattle by analyzing the correlation between QSOX1 single nucleotide polymorphisms (SNPs), I2 204 C>T and I2 378 C>T, and certain meat quality traits. The effects of QSOX1 on triglyceride synthesis and cell apoptosis were further validated by gene silencing or overexpression in bovine fetal fibroblasts and mammary epithelial cells. The results showed that I2 204 C>T and I2 378 C>T had significant correlations with loin thickness, hind hoof weight, fat coverage, liver weight, heart weight, marbling and back fat thickness (P<0.05). QSOX1 overexpression also increased triglyceride production and suppressed apoptosis. In summary, QSOX1 is an important factor for meat quality, lipid metabolism, and cell apoptosis, indicating that QSOX1 could be used as a biomarker to assist in breeding cattle with superior meat.

Mammary epithelial cells (MECs) affect milk production capacity during lactation and are critical for the maintenance of tissue homeostasis. Our previous studies have revealed that the expression of miR-152 was increased significantly in MECs of cows with high milk production. In the present study, bioinformatics analysis identified ACAA2 and HSD17B12 as the potential targets of miR-152, which were further validated by dual-luciferase repoter assay. In addition, the expressions of miR-152 was shown to be negatively correlated with levels of mRNA and protein of ACAA2, HSD17B12 genes by qPCR and western bot analysis. Furthermore, transfection with miR-152 significantly up-regulated triglyceride production, promoted proliferation and inhibited apoptosis in MECs. Furthermore, overexpression of ACAA2 and HSD17B12 could inhibit triglyceride production, cells proliferation and induce apoptosis; but sh234-ACAA2-181/sh234-HSD17B12-474 could reverse the trend. These findings suggested that miR-152 could significantly influence triglyceride production and suppress apoptosis, possibly via the expression of target genes ACAA2 and HSD17B12.