Polymorphisms of the SCD1 Gene and Its Association Analysis with Carcass, Meat Quality, Adipogenic Traits, Fatty Acid Composition, and Milk Production Traits in Cattle.

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.

Research Progress on the Structural and Functional Roles of hnRNPs in Muscle Development.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a superfamily of RNA-binding proteins consisting of more than 20 members. These proteins play a crucial role in various biological processes by regulating RNA splicing, transcription, and translation through their binding to RNA. In the context of muscle development and regeneration, hnRNPs are involved in a wide range of regulatory mechanisms, including alternative splicing, transcription regulation, miRNA regulation, and mRNA stability regulation. Recent studies have also suggested a potential association between hnRNPs and muscle-related diseases. In this report, we provide an overview of our current understanding of how hnRNPs regulate RNA metabolism and emphasize the significance of the key members of the hnRNP family in muscle development. Furthermore, we explore the relationship between the hnRNP family and muscle-related diseases.

Bta-miR-206 and a Novel lncRNA-lncA2B1 Promote Myogenesis of Skeletal Muscle Satellite Cells via Common Binding Protein HNRNPA2B1.

Skeletal muscle satellite cells (MuSCs) can proliferate, differentiate, and self-renew, and can also participate in muscle formation and muscle injury repair. Long noncoding RNAs (lncRNAs) can play an important role with the RNA binding protein and microRNAs (miRNAs) to regulate the myogenesis of bovine MuSCs, however, its molecular mechanism is still being explored. In this study, differentially expressed 301 lncRNAs were identified during the myogenic differentiation of cells based on an in vitro model of induced differentiation of bovine MuSCs using RNA sequencing (RNA-seq). Based on the ability of miR-206 to regulate myogenic cell differentiation, a new kind of lncRNA-lncA2B1 without protein-coding ability was found, which is expressed in the nucleus and cytoplasm. Subsequently, lncA2B1 inhibited cell proliferation by downregulating the expression of the proliferation marker Pax7 and promoted myogenic differentiation by upregulating the expression of the differentiation marker MyHC, whose regulatory function is closely related to miR-206. By RNA pulldown/LC-MS experiments, heterogeneous ribonucleoprotein A2/B1 (HNRNPA2B1), and DExH-Box Helicase 9 (DHX9) were identified as common binding proteins of lncA2B1 and miR-206. Overexpression of lncA2B1 and miR-206 significantly upregulated the expression level of HNRNPA2B1. Downregulation of HNRNPA2B1 expression significantly decreased the expression level of the differentiation marker MyHC, which indicates that miR-206 and lncA2B1 regulate myogenic differentiation of bovine MuSCs by acting on HNRNPA2B1. This study screened and identified a novel lncRNA-lncA2B1, which functions with miR-206 to regulate myogenesis via the common binding proteins HNRNPA2B1. The results of this study provide a new way to explore the molecular mechanisms by which lncRNAs and miRNAs regulate muscle growth and development.

TFEB coordinates autophagy and pyroptosis as hepatotoxicity responses to ZnO nanoparticles.

Zinc oxide nanoparticles (ZnO NPs) have drawn serious concerns about their biotoxicity due to their extensive applications in biological medicine, clinical therapeutic, daily chemical production, food and agricultural additives. In our present study, we clarified hepatotoxic mechanism of ZnO NPs through investigating the crosstalk between autophagy and pyroptosis, a remaining enigma in hepatocyte stimulated by ZnO NPs. Based on the effects of autophagy intervention by Rapamycin (Rap) and 3-Methyladenine (3-MA), and the observation of pyroptosis morphology and related indexes, the autophagy and pyroptosis simultaneously initiated by ZnO NPs were interrelated and the autophagy characterized by autophagosome production and increased expression of autophagy proteins was identified as a protective response of ZnO NPs against pyroptosis. According to the analysis of protein expression and fluorescence localization, the NLRP3 inflammasome assemble and the classical Caspase-1/GSDMD-dependent pyroptosis induced by ZnO NPs was modulated by autophagy. In this process, the adjustment of TFEB expression and nuclear translocation by gene knockout and gene overexpression, further altered the tendency of ZnO NPs-induced pyroptosis via the regulation of autophagy and lysosomal biogenesis. The knockout of TFEB gene exacerbated the pyroptosis via autophagy elimination and lysosome inhibition. While the alleviation of NLRP3 generation and pyroptosis activation was observed after treatment of TFEB gene overexpression. Additionally, the siRNA interference confirmed that TRAF-6 was involved in the TFEB-mediated global regulation of autophagy-lysosome-pyroptosis in response to ZnO NPs. Accordingly, pyroptosis induced by ZnO NPs in hepatocyte could be significantly avoided by TFEB-regulated autophagy and lysosome, further providing new insights for the risk assessment and therapeutic strategy.

Genome-Wide Association Analysis of Reproductive Traits in Chinese Holstein Cattle.

This study was to explore potential SNP loci for reproductive traits in Chinese Holstein cattle and identify candidate genes. Genome-wide Association Study based on mixed linear model was performed on 643 Holstein cattle using GeneSeek Bovine 50 K SNP chip. Our results detected forty significant SNP loci after Bonferroni correction. We identified five genes (VWC2L, STAT1, PPP3CA, LDB3, and CTNNA3) as being associated with pregnancy ratio of young cows, five genes (PAEP, ACOXL, EPAS1, GLRB, and MARVELD1) as being associated with pregnancy ratio of adult cows, and nine genes (PDE1B, SLCO1A2, ARHGAP26, ADAM10, APBB1, MON1B, COQ9, CDC42BPB, MARVELD1, and HPSE2) as being associated with daughter pregnancy rate. Our study may provide valuable insights into identifying genes related to reproductive traits and help promote the application of molecular breeding in dairy cows.

PFN1 Inhibits Myogenesis of Bovine Myoblast Cells via Cdc42-PAK/JNK.

Myoblast differentiation is essential for the formation of skeletal muscle myofibers. Profilin1 (Pfn1) has been identified as an actin-associated protein, and has been shown to be critically important to cellular function. Our previous study found that PFN1 may inhibit the differentiation of bovine skeletal muscle satellite cells, but the underlying mechanism is not known. Here, we confirmed that PFN1 negatively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Immunoprecipitation assay combined with mass spectrometry showed that Cdc42 was a binding protein of PFN1. Cdc42 could be activated by PFN1 and could inhibit the myogenic differentiation like PFN1. Mechanistically, activated Cdc42 increased the phosphorylation level of p2l-activated kinase (PAK), which further activated the phosphorylation activity of c-Jun N-terminal kinase (JNK), whereas PAK and JNK are inhibitors of myogenic differentiation. Taken together, our results reveal that PFN1 is a repressor of bovine myogenic differentiation, and provide the regulatory mechanism.

Effects and potential mechanism of Ca2+/calmodulin­dependent protein kinase II pathway inhibitor KN93 on the development of ovarian follicle.

Adequate regulation of the speed of follicular development has been reported to prolong the reproductive life of the ovary. The aim of the present study was to assess the potential effects and mechanism of the Ca2+/calmodulin­dependent protein kinase II (CaMKII) pathway on the development of ovarian follicle. In the present study, the expression of CaMKII was measured in the ovary of mice at different developmental stages by immunofluorescence, confirming that CaMKII has a role in follicular development. Subsequently, the 17.5 days post­coitus (dpc) embryonic ovaries were collected and cultured with KN93 for 4 days in vitro. It was revealed that KN93 inhibited the development of follicles, where it reduced the expression levels of oocyte and granulosa cell markers DEAD­box helicase 4 (DDX4) and forkhead box L2 (FOXL2). These results suggested that KN93 could delay follicular development. Proteomics technology was then used to find that 262 proteins of KN93 treated 17.5 dpc embryonic ovaries were significantly altered after in vitro culture. Bioinformatics analysis was used to analyze these altered proteins. In total, four important Kyoto Encyclopedia of Genes and Genome pathways, namely steroid biosynthesis, p53 signaling pathway and retinol metabolism and metabolic pathways, were particularly enriched. Further analysis revealed that the upregulated proteins NADP­dependent steroid dehydrogenase­like (Nsdhl), lanosterol synthase (Lss), farnesyl­diphosphate farnesyltransferase 1 (Fdft1), cytochrome P450 family 51 family A member 1 (Cyp51a1), hydroxymethylglutaryl­CoA synthase 1 (Hmgcs1), fatty acid synthase (Fasn) and dimethylallyltranstransferase (Fdps) were directly interacting with each other in the four enriched pathways. In summary, the potential mechanism of KN93 in slowing down follicular development most likely lies in its inhibitory effects on CaMKII, which upregulated the expression of Nsdhl, Lss, Fdft1, Cyp51a1, Hmgcs1, Fasn and Fdps. This downregulated the expression of oocyte and granulosa cell markers DDX4 and FOXL2 in the follicles, thereby delaying follicular development. Overall, these results provide novel insight into the potential mechanism by which KN93 and CaMKII can delay follicular development.

Myostatin Mutation Enhances Bovine Myogenic Differentiation through PI3K/AKT/mTOR Signalling via Removing DNA Methylation of RACK1.

Myostatin (MSTN) is a negative regulator of skeletal muscle development and plays an important role in muscle development. Fluctuations in gene expression influenced by DNA methylation are critical for homeostatic responses in muscle. However, little is known about the mechanisms underlying this fluctuation regulation and myogenic differentiation of skeletal muscle. Here we report a genome-wide analysis of DNA methylation dynamics in bovine skeletal muscle myogenesis after myostatin editing. We show that, after myostatin editing, an increase in TETs (DNA demethylases) and a concomitant increase in the receptor for activated C kinase 1 (RACK1) control the myogenic development of skeletal muscle. Interestingly, enhancement of PI3K/AKT/mTOR signaling by RACK1 appears to be an essential driver of myogenic differentiation, as it was associated with an increase in myogenic differentiation marker factors (MyHC and MyoG) during muscle differentiation. Overall, our results suggest that loss of myostatin promotes the myogenic differentiation response in skeletal muscle by decreasing DNA methylation of RACK1.

Proteomic Studies on the Mechanism of Myostatin Regulating Cattle Skeletal Muscle Development.

Myostatin (MSTN) is an important negative regulator of muscle growth and development. In this study, we performed comparatively the proteomics analyses of gluteus tissues from MSTN+/- Mongolian cattle (MG.MSTN+/-) and wild type Mongolian cattle (MG.WT) using a shotgun-based tandem mass tag (TMT) 6-plex labeling method to investigate the regulation mechanism of MSTN on the growth and development of bovine skeletal muscle. A total of 1,950 proteins were identified in MG.MSTN+/- and MG.WT. Compared with MG.WT cattle, a total of 320 differentially expressed proteins were identified in MG.MSTN cattle, including 245 up-regulated differentially expressed proteins and 75 down-regulated differentially expressed proteins. Bioinformatics analysis showed that knockdown of the MSTN gene increased the expression of extracellular matrix and ribosome-related proteins, induced activation of focal adhesion, PI3K-AKT, and Ribosomal pathways. The results of proteomic analysis were verified by muscle tissue Western blot test and in vitro MSTN gene knockdown test, and it was found that knockdown MSTN gene expression could promote the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells (BSMSCs). At the same time, Co-Immunoprecipitation (CO-IP) assay showed that MSTN gene interacted with extracellular matrix related protein type I collagen α 1 (COL1A1), and knocking down the expression of COL1A1 could inhibit the activity of adhesion, PI3K-AKT and ribosome pathway, thus inhibit BSMSCs proliferation. These results suggest that the MSTN gene regulates focal adhesion, PI3K-AKT, and Ribosomal pathway through the COL1A1 gene. In general, this study provides new insights into the regulatory mechanism of MSTN involved in muscle growth and development.

A novel lncRNA promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1.

Myogenesis, the process of skeletal muscle formation, is a highly coordinated multistep biological process. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are emerging as a gatekeeper in myogenesis. Up to now, most studies on muscle development-related lncRNAs are mainly focussed on humans and mice. In this study, a novel muscle highly expressed lncRNA, named lnc23, localized in nucleus, was found differentially expressed in different stages of embryonic development and myogenic differentiation. The knockdown and over-expression experiments showed that lnc23 positively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Then, TMT 10-plex labelling quantitative proteomics was performed to screen the potentially regulatory proteins of lnc23. Results indicated that lnc23 was involved in the key processes of myogenic differentiation such as cell fusion, further demonstrated that down-regulation of lnc23 may inhibit myogenic differentiation by reducing signal transduction and cell fusion among cells. Furthermore, RNA pulldown/LC-MS and RIP experiment illustrated that PFN1 was a binding protein of lnc23. Further, we also found that lnc23 positively regulated the protein expression of RhoA and Rac1, and PFN1 may negatively regulate myogenic differentiation and the expression of its interacting proteins RhoA and Rac1. Hence, we support that lnc23 may reduce the inhibiting effect of PFN1 on RhoA and Rac1 by binding to PFN1, thereby promoting myogenic differentiation. In short, the novel identified lnc23 promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1.

A novel lncRNA, lnc403, involved in bovine skeletal muscle myogenesis by mediating KRAS/Myf6.

Skeletal muscle, the most abundant and plasticity tissue in mammals, is essential for various functions such as movement, breathing, maintaining posture and metabolism. Myogenesis is a complex and precise process, which is regulated by the sequential expression of multiple transcription factors, and accumulating evidence have confirmed that multiple lncRNAs are involved in muscle development as the important transcriptional regulator. In this study, a novel lncRNA, named lnc403 was obtained, with a full-length 2689 bp, which had poor coding ability and was mainly expressed in the nucleus of myoblasts and myotubes. The expression of lnc403 was significantly different in the proliferation and differentiation stages of muscle cells. Then we successfully constructed lnc403 loss/gain-function cell models by transfecting silnc403 and pCDNA3.1-EGFP-lnc403 into satellite cells, respectively; and found that lnc403 inhibited skeletal muscle satellite cell differentiation but had no significant effect on cell proliferation, either in the case of lnc403 knockdown or overexpression. In order to further screen the target factors regulated by lncRNA in the process of myogenic differentiation, the RNA-pull down, mass spectrometry and bioinformatics analysis were performed. The results showed that lnc403 negatively regulated the expression of the adjacent gene Myf6 and positively regulated interaction proteins KRAS expression. The above results indicate that lnc403 affects skeletal muscle cell differentiation by affecting the expression of nearby genes and interacting proteins, implying lnc403 might participate in the bovine myoblasts differentiation through multi-pathway network regulation mode. This study provides a new perspective for further understanding of the regulation mechanism of lncRNAs on bovine myogenic process.

Proteomics insights into the effects of MSTN on muscle glucose and lipid metabolism in genetically edited cattle.

The molecular mechanism underlying myostatin (MSTN)-regulated metabolic cross-talk remains poorly understood. In this study, we performed comparative proteomic and phosphoproteomic analyses of gluteus muscle tissues from MSTN-/- transgenic cattle using a shotgun-based tandem mass tag (TMT) 6-plex labeling method to explore the signaling pathway of MSTN in metabolic cross-talk and cellular metabolism during muscle development. A total of 72 differentially expressed proteins (DEPs) and 36 differentially expressed phosphoproteins (DEPPs) were identified in MSTN-/- cattle compared to wild-type cattle. Bioinformatics analyses showed that MSTN knockout increased the activity of many key enzymes involved in fatty acid ß-oxidation and glycolysis processes in cattle. Furthermore, comprehensive pathway analyses and hypothesis-driven AMP-activated protein kinase (AMPK) activity assays suggested that MSTN knockout triggers the activation of AMPK signaling pathways to regulate glucose and lipid metabolism by increasing the AMP/ATP ratio. Our results shed new light on the potential regulatory mechanism of MSTN associated with metabolic cross-talk in muscle development, which can be used in animal breeding to improve meat production in livestock animals, and can also provide valuable insight into treatments for obesity and diabetes mellitus in humans.

A novel long non-coding RNA, lncKBTBD10, involved in bovine skeletal muscle myogenesis.

Accumulating evidence suggests that long non-coding RNAs (lncRNAs) play a crucial role in regulating skeletal muscle myogenesis, a highly coordinated multistep biological process. However, most studies of lncRNAs have focused on humans, mouse, and other model animals. In this study, we identified a novel lncRNA, named lncKBTBD10, located in the nucleus and involved in the proliferation and differentiation of bovine skeletal muscle satellite cells. Prediction of coding potential and in vitro translation system illustrated that lncKBTBD10 has no encoding capability. With the process of myogenic differentiation, the expression of lncKBTBD10 gradually increased. To elucidate the functions of lncKBTBD10 during myogenesis, the gain/loss-of-function experiments were performed. Results showed that the proliferation and differentiation of bovine skeletal muscle satellite cells were all suppressed whether lncKBTBD10 was knocked down or over-expressed. Furthermore, we found that lncKBTBD10 may affect its proximity gene KBTBD10 to involve in myogenesis. Results indicated that the protein level of KBTBD10 was all diminished after induced differentiation for 2 d in differentiation medium (DM2) whether lncKBTBD10 was knocked down or over-expressed. It may support why the altering of lncKBTBD10 can suppress the proliferation and differentiation of bovine skeletal muscle satellite cells. In short, our study elucidated that lncKBTBD10 could induce a decrease of KBTBD10 protein and further to affect bovine skeletal muscle myogenesis. The novel identified lncKBTBD10 may provide a reference for lncRNAs involved in myogenesis of bovine skeletal muscle.

Parallel comparative proteomics and phosphoproteomics reveal that cattle myostatin regulates phosphorylation of key enzymes in glycogen metabolism and glycolysis pathway.

MSTN-encoded myostatin is a negative regulator of skeletal muscle development. Here, we utilized the gluteus tissues from MSTN gene editing and wild type Luxi beef cattle which are native breed of cattle in China, performed tandem mass tag (TMT) -based comparative proteomics and phosphoproteomics analyses to investigate the regulatory mechanism of MSTN related to cellular metabolism and signaling pathway in muscle development. Out of 1,315 proteins, 69 differentially expressed proteins (DEPs) were found in global proteomics analysis. Meanwhile, 149 differentially changed phosphopeptides corresponding to 76 unique phosphorylated proteins (DEPPs) were detected from 2,600 identified phosphopeptides in 702 phosphorylated proteins. Bioinformatics analyses suggested that majority of DEPs and DEPPs were closely related to glycolysis, glycogenolysis, and muscle contractile fibre processes. The global discovery results were validated by Multiple Reaction Monitoring (MRM)-based targeted peptide quantitation analysis, western blotting, and muscle glycogen content measurement. Our data revealed that increase in abundance of key enzymes and phosphorylation on their regulatory sites appears responsible for the enhanced glycogenolysis and glycolysis in MSTN-/- . The elevated glycogenolysis was assocaited with an enhanced phosphorylation of Ser1018 in PHKA1, and Ser641/Ser645 in GYS1, which were regulated by upstream phosphorylated AKT-GSK3ß pathway and highly consistent with the lower glycogen content in gluteus of MSTN-/- . Collectively, this study provides new insights into the regulatory mechanisms of MSTN involved in energy metabolism and muscle growth.

Comparative muscle proteomics/phosphoproteomics analysis provides new insight for the biosafety evaluation of fat-1 transgenic cattle.

The biosafety of fat-1 transgenic cattle has been a focus of our studies since the first fat-1 transgenic cow was born. In this study, we used tandem mass tag labeling, TiO2 enrichment, and nanoscale liquid chromatography coupled with tandem mass spectrometry (nanol LC-MS/MS) to compare proteomic and phosphoproteomic profiling analyses of muscle between fat-1 transgenic cows and wild-type cows. A total of 1555 proteins and 900 phosphorylation sites in 159 phosphoproteins were identified in the profiling assessments, but only four differentially expressed proteins and nine differentially expressed phosphopeptides were detected in fat-1 transgenic cows relative to wild-type cows. Bioinformatics analyses showed that all of the identified proteins and phosphoproteins were mainly related to the metabolic processes of three major nutrients: carbohydrates, lipids, and proteins. All of these differentially expressed proteins might take part in DNA recombination, repair, and regulation of the immune system. In conclusion, most of the identified proteins and phosphoproteins exhibited few changes. Our results provide new insights into the biosafety of fat-1 transgenic cattle.

Insights into the function of n-3 PUFAs in fat-1 transgenic cattle.

The n-3 PUFAs have many beneficial effects on human health, including roles in immunity, neurodevelopment, and preventing cardiovascular disease. In this study, we established reliable model fat-1 transgenic cattle using transgenic technology and performed a systematic investigation to examine the function of n-3 PUFAs. Our results showed that expression of the fat-1 gene improved several biochemical parameters related to liver function and to plasma glucose and plasma lipid metabolism. Results of global gene and plasma protein expression analysis showed that 310 genes and 13 plasma proteins differed significantly in the blood of fat-1 transgenic cattle compared with WT cattle, reflecting their regulatory roles in the immune and cardiovascular systems. Finally, changes in the gut microflora were also noted in the fat-1 transgenic cattle, suggesting novel roles for n-3 PUFAs in the metabolism of glucose and lipids, as well as anti-stress properties. To the best of our knowledge, this is the first report using multiple parallel analyses to investigate the role of n-3 PUFAs using models such as fat-1 transgenic cattle. This study provides novel insights into the regulatory mechanism of fat-1 in the immune and cardiovascular systems, as well as its anti-stress role.

Microarray Analysis of the Gene Expression Profile and Lipid Metabolism in Fat-1 Transgenic Cattle.

Long-chain n-3 polyunsaturated fatty acids (n-3 PUFAs) are beneficial for human health. However, humans and mammals are unable to synthesize n-3 PUFAs because they lack the n-3 desaturase gene fat-1 and must therefore obtain this type of fatty acid through their diet. Through the production of fat-1 transgenic animals, it is possible to obtain animal products that are rich in n-3 PUFAs, such as meat and milk. The aim of this study was to analyze the gene expression profile and the mechanism of lipid metabolism in fat-1 transgenic cattle and to accumulate important basic data that are required to obtain more efficient fat-1 transgenic cattle. Transcriptome profiling of fat-1 transgenic and wild-type cattle identified differentially expressed genes that are involved in 90 biological pathways, eight pathways of which were related to lipid metabolism processes 36 genes of which were related to lipid metabolism. This analysis also identified 11 significantly enriched genes that were involved in the peroxisome proliferator-activated receptor signaling pathway. These findings were verified by quantitative polymerase chain reaction. The information obtained in this study indicated that the introduction of an exogenous fat-1 gene into cattle affects the gene expression profile and the process of lipid metabolism in these animals. These results may provide important insights into how an exogenous fat-1 gene synthesizes n-3 PUFAs in transgenic cattle and other mammals.

Analysis of the gut microbiota by high-throughput sequencing of the V5-V6 regions of the 16S rRNA gene in donkey.

Considerable evidence suggests that the gut microbiota is complex in many mammals and gut bacteria communities are essential for maintaining gut homeostasis. To date the research on the gut microbiota of donkey is surprisingly scarce. Therefore, we performed high-throughput sequencing of the 16S rRNA genes V5-V6 hypervariable regions from gut fecal material to characterize the gut microbiota of healthy donkeys and compare the difference of gut microbiota between male and female donkeys. Sixty healthy donkeys (30 males and 30 females) were enrolled in the study, a total of 915,691 validated reads were obtained, and the bacteria found belonged to 21 phyla and 183 genera. At the phylum level, the bacterial community composition was similar for the male and female donkeys and predominated by Firmicutes (64 % males and 64 % females) and Bacteroidetes (23 % males and 21 % females), followed by Verrucomicrobia, Euryarchaeota, Spirochaetes, and Proteobacteria. At the genus level, Akkermansia was the most abundant genus (23 % males and 17 % females), followed by Sporobacter, Methanobrevibacter, and Treponema, detected in higher distribution proportion in males than in females. On the contrary, Acinetobacter and Lysinibacillus were lower in males than in females. In addition, six phyla and 15 genera were significantly different between the male and female donkeys for species abundance. These findings provide previously unknown information about the gut microbiota of donkeys and also provide a foundation for future investigations of gut bacterial factors that may influence the development and progression of gastrointestinal disease in donkey and other animals.

Upregulation of triglyceride synthesis in skeletal muscle overexpressing DGAT1.

The gene encoding diacylglycerol acyltransferase (DGAT1) is a functional and positional candidate gene for milk and intramuscular fat content. A bovine DGAT1 overexpression vector was constructed containing mouse MCK promoter and bovine DGAT1 cDNA. MCK-DGAT1 transgene in FVB mice was researched in present study. The transgene DGAT1 had a high level of expression in contrast to the endogenous DGAT1 in posterior tibial muscle of the transgenic mice, but a low expression level in the cardiac muscle. Compared with wild-type mice, triglyceride and DGAT1 content were approximately fourfold and 50% increased in posterior tibial muscle of the transgenic mice, respectively, while a little increase in cardiac muscle.

Modifications of chemically induced-enucleated nuclear transfer technique by reverse-order nuclear transfer in mouse.

To improve the developmental potential of somatic cell cloned embryos derived from demecolcine (DC) induced-enucleated nuclear transfer (INT), we modified the INT procedures by transferring donor nuclei into recipient cytoplasts prior to the induced enucleation of the recipient cytoplasts, and we called this modified INT technique as reverse-order and induced-enucleated nuclear transfer (RINT). Standard nuclear transfer (SNT) and INT were performed as controls. The dynamic changes of maternal and transferred donor nuclei in the RINT oocytes were monitored to evaluate the feasibility of this new nuclear transfer (NT) technique by timed immunofluorescence. Timed immunofluorescence showed that RINT is feasible because none of the transferred donor nuclei were expelled with the second polar body (Pb) in the RINT oocytes, while 42.2% of the oocytes showed extrusion of all maternal chromosome and spindles with the second Pb at 60 min after activation and DC treatment. Although there was no difference in cleavage rate (86.6% vs. 82.1%), the rates of successful enucleation and blastocyst formation were significantly increased in RINT compared with INT (44.1% vs. 27.5% and 43.3% vs. 12.8%, respectively; p < 0.01). Compared with SNT, there was no difference in cleavage rate (86.6% vs. 78.4%), but the blastocyst developmental rate was significantly increased in the RINT group (43.3% vs. 25.3%; p < 0.01). Blastocysts derived from RINT had a higher total cell number than those from SNT (45.1 +/- 3 vs. 37.6 +/- 4; p < 0.05). Our results provide evidence that RINT is feasible and may provide a more efficient and simple method for NT than INT.