To investigate the targeting relationship between miR-370-3p and inhibin subunit beta B (INHBB), SMAD family member 4 (SMAD4) and fibroblast growth factor 18 (FGF18) genes that were associated with oviductal function in sheep, in this study, the sequences of miR-370-3p in multiple species (Ovis aries, Homo sapiens, Mus musculus, Rattus norvegicus, Macaca mulatta, Cavia porcellus and Oryctolagus cuniculus) were compared and RNAhybrid was used to predict the potential binding sites of ovine miR-370-3p to the 3'UTR of INHBB, SMAD4 and FGF18 genes. Then wild type and mutant type dual-luciferase reporter vectors for INHBB, SMAD4 and FGF18 gene 3'UTR were constructed and co-transfected with miR-370-3p mimics and mimics NC into HEK293T cells to detect dual-luciferase activity, respectively. The results showed that the ovine miR-370-3p sequence was different from the other six species but was somewhat conserved, and was predicted by RNAhybrid to have binding sites to the 3'UTRs of the INHBB, SMAD4 and FGF18 genes. The electrophoresis results and sequencing results indicated that the wild type and mutant type vectors of INHBB, SMAD4 and FGF18 gene 3'UTR were successfully constructed. The dual-luciferase activities of the co-transfected INHBB, SMAD4 and FGF18 wild type vectors and miR-370-3p mimics were extremely significantly or significantly lower than the corresponding controls (P<0.01;P<0.05). While the dual-luciferase activities of the three mutant type vectors and miR-370-3p mimics co-transfected were not significantly different from the corresponding controls (P>0.05). It was shown that the 3'UTR region of INHBB, SMAD4 and FGF18 genes could all bind to miR-370-3p and inhibit dual-luciferase activity, which verified that INHBB, SMAD4 and FGF18 genes were both target genes of miR-370-3p and provided a basis for further study on the molecular mechanism of oar-miR-370-3p affecting oviductal function and sheep fecundity.

This study was aimed to excavate the microRNAs that regulate the reproduction of Xiang pigs from the small RNA sequencing data of Xiang pigs ovaries, it is of great significance fo analyze the molecular mechanism of miRNAs regulating ovarian function and reproductive traits in pig. In this study, four estrus pigs and four diestrus pigs were used as the test animals. The ovary tissues were collected from Xiang pigs after slaughter. The total RNA was extracted from ovary tissues for small RNA sequencing, the profile of miRNAs expression were calculated with bioinformatics selection, and the different expression target genes were predicted and the GO function enrichment and KEGG pathway enrichment analysis of the target genes were performed. The results showed that miRNAs were no uniformly distributed on chromosomes, mainly on chromosome 1 and chromosome X. Total of 627 known porcine miRNAs were expressed in the ovaries of Xiang pigs, of which 34 were differently expressed miRNAs. The top five expression levels of miRNAs were miR-23, let-7i-5p, miR-103, miR-30E-5p and miR-1271-5p. The results of GO function analysis showed that the main biological processes involved in target genes were cellular processes mainly distributed in cells and the main molecular function was binding. KEGG pathway analysis significantly enriched gonadotropin-releasing hormone receptor pathway, insulin-like growth factor pathway and EGF receptor signaling pathway, which were associated with oocyte development and maturation. Therefore, it was speculated that miR-23b, let-7i-5p, miR-103, miR-30e-5p and miR-1271-5p could be used as candidate miRNAs affecting the reproductive regulation of Xiang pigs. In this study, five miRNA molecules that might regulate the reproduction of Xiang pigs were preliminarily screened, which could provide a theoretical basis for improving the litter size of Xiang pigs at molecular level.

The expression changes of casein content, triglyceride content and fatty acid-related genes were detected by transfecting small interfering-hydroxysteroid 17-beta dehydrogenase Ⅳ(si-HSD17B4) in buffalo mammary epithelial cell, which were revealing the effects of interfering with the HSD17B4 gene on milk fats and caseins in buffalo mammary epithelial cell. The relative expressions of HSD17B4 mRNA in various tissues of buffalo were detected by Real-time quantitative PCR. 3 HSD17B4 siRNA fragments of buffalo and one control si-HSD17B4-nc (si-nc) fragment were synthesized and then the optimal fragments and time of the interference were selected. Caseins were detected by casein kits after si-HSD17B4 transfected buffalo mammary epithelial cell. Then triglycerides were detected by triglyceride kit and oil red O staining, and the effects of HSD17B4 gene interference on the gene expression of the triglycerides synthesis and fatty acid synthesis and oxidation. The results showed that HSD17B4 gene was relatively highly expressed in buffalo heart and ovary, and was significantly higher than other tissue(P<0.05). 3 HSD17B4 siRNA fragments could effectively decrease the expression of the HSD17B4 gene, and the si-HSD17B4-1 was the best interference fragment, the relative expression of HSD17B4 mRNA was extremely significantly lower than that in the control group (P<0.01). The optimum interference time was 24 h. Casein test showed no effect of interfering HSD17B4 on casein synthesis in buffalo mammary epithelial cell. Triglyceride detections showed an increase in triglyceride content in cells after interfering with the HSD17B4 gene with significant differences (P<0.05). The oil red O tests showed a significant increase in fat droplets in cells after interfering with HSD17B4 gene. The results of Real-time quantitative PCR showed that interference with the HSD17B4 gene, the relative expressions of FABP3, ACSL1, DGAT1, DGAT2, PLIN2 and BTN1A1 gene were increased by 9.28 (P<0.01), 1.36 (P<0.05), 1.17 (P<0.05), 1.83 (P<0.01), 1.60 (P<0.01) and 2.17 (P<0.01) times, respectively. The relative expressions of PPARA, SREBP1C, SCD, ACSS2, ACACA, AGPAT6, LPIN1, XDH, ABCD1, ACOX2, EHHADH and SCP2 genes decreased to 50.55% (P>0.05), 61.15% (P<0.01), 84.91% (P<0.01), 89.34% (P<0.01), 21.88% (P<0.01), 86.48% (P<0.01), 25.35% (P<0.01), 27.24% (P<0.01), 22.17% (P<0.01), 62.70% (P<0.01) and 70.33% (P<0.01), respectively. The results showed that the HSD17B4 gene was widely expressed in buffalo tissue. si-HSD17B4 effectively reduced the expression of the HSD17B4 gene in buffalo mammary epithelial cell, and it had no effect of interfering with the HSD17B4 gene on the caseins in mammary epithelial cell, but it would increase the expressions of FABP3, ACSL1, DGAT1, DGAT2 genes to promote the synthesis of triglycerides while decrease the expression of ABCD1, ACOX2, EHADH, SCP2 genes to reduce the β-oxidation of fatty acids.

The aim of this experiment was to obtain the full-length coding sequence (CDS) of PDGFD gene in sheep, and understand its expression in adipose tissue deposited in different parts of the body. The full-length CDS sequence of PDGFD gene was cloned from Altay ewes and Chinese Merino ewes, and the bioinformatics analysis of the sequence was carried out. The expression of PDGFD gene in adipose tissues of the tail, mesentery, back and kidney of the two breeds of sheep was quantitatively analyzed by Real-time quantitative PCR. The results showed that the total CDS region sequence of PDGFD gene was 1 113 bp, which encoded 370 amino acids. The sequence of CDS region and its coding sequence of PDGFD gene had the highest similarity with Capra hircus, followed by Bos taurus, Sus scrofa, Equus caballus and Homo sapiens, and the lowest similarity with Mus musculus, which was consistent with the result of phylogenetic tree analysis. It was predicted that PDGFD protein was a stable hydrophilic protein, no transmembrane structure, had signal peptide sequence. PDGFD protein contained a glycosylation site, a CUB domain and a PDGF domain, and the secondary structure of PDGFD protein were composed of alpha helix, beta turn, extended chain and random coil, and tertiary structure was basically consistent with the analysis results of domain and secondary structure. Real-time quantitative PCR results showed that the expression of PDGFD gene in four adipose tissues of Altay sheep was higher than that of Chinese Merino sheep, especially the expression in tail fat and back fat of Altay sheep was significantly higher than of Chinese Merino sheep (P<0.05). The results of this study provided reference for further research on the mechanism of PDGFD gene in adipose deposition in sheep.

The purpose of this experiment was to amplify the Snail2 gene of Qinchuan cattle, and construct the temporal and spatial expression pattern of Snail2 gene in different developmental stages of tissues and different differentiation stages of adipocytes, which laid a foundation for further study on the function and regulation of Snail2 gene in bovine fat deposition. Qinchuan cattle were taken as the research object, the CDS region sequence of Snail2 gene was amplified by PCR, and its functional structure was predicted by bioinformatics softwares. At the same time, Real-time quantitative PCR was used to detect the temporal and spatial expression profile of Snail2 gene in the process of adipogenic differentiation of newborn and adult bovine tissues and adipocytes. The results showed that the full length of Snail2 gene coding sequence of Qinchuan cattle was 952 bp. The phylogenetic tree results of different species showed that bovine Snail2 protein was highly conserved in cattle, buffalo, goat and sheep. 41 potential phosphorylation sites were found by phosphorylation site analysis, in which cyclin dependent kinase 1 (CDK1), CDK5, CKⅡ, CKⅠ and other cell cycle-related kinases were involved in the phosphorylation of Snail2 protein. Protein domain prediction found that there were 8 similar Motif among 8 species including cattle, human and mouse, etc. The secondary structure of Snail2 protein was mainly composed of irregular curls, and the prediction results of secondary and tertiary structures were consistent. Sequence analysis of the promoter region of Snail2 gene revealed a CpG island and E2F, C/EBPα(CCAAT/enhancer binding proteins alpha), AP2 and Sp1 transcription factor binding sites related to adipogenesis. The results of Real-time quantitative PCR showed that Snail2 gene was highly expressed in bovine adipose tissue, and showed an upward trend with adipogenic differentiation and bovine growth and development, indicating that Snail2 gene played an important role in bovine fat deposition. The results laid a foundation for further revealing the mechanism of Snail2 gene affecting fat deposition in beef cattle by affecting the proliferation and differentiation of adipocytes.

The purpose of this study was to clone the CDS nucleotide sequences of two subtypes (DGAT1 and DGAT2) of diacylglycerol acyltransferase (DGATs) in Yanbian Yellow cattle, analyze the amino acid sequences of two subtypes according to bioinformatics, and explore the expression patterns of two subtypes in Yanbian Yellow cattle. RT-PCR and Real-time quantitative PCR were used to amplify and clone DGATs gene CDS and detect its mRNA expression in 8 tissues of Yanbian Yellow cattle, respectively. BLAST in NCBI was used to analyze its similarity with other species and construct phylogenetic tree. A series of online tools were used to predict the physicochemical properties, primary structure and higher structure of the proteins encoded by it. The results showed that the CDS of DGAT1 and DGAT2 genes were 1 470 and 1 086 bp, coding 489 and 361 amino acids, respectively. Both of them were stable hydrophobic proteins. There were 25 potential phosphorylation sites, 1 N-terminal glycosylation site and 8 transmembrane domains for DGAT1, and 28 potential phosphorylation sites, 2 N-terminal glycosylation sites and 1 transmembrane domain for DGAT2. There was no signal peptide in both of them, so they were not secretory proteins. DGAT1 was dominated by alpha helix and random coil, while DGAT2 was dominated by alpha helix, random coil and extended chain. The expression levels of DGAT1 and DGAT2 genes in small intestine and adipose tissue of Yanbian Yellow cattle were the highest, which were significantly higher than other tissues (P<0.05). The results of this study were of great significance to further study the DGATs gene of Yanbian Yellow cattle and explore its mechanism in the process of fat deposition.

This study was aimed to indentify the members of ATP-binding cassette (ABC) transporter D (ABCD) gene and its expression in buffalo. The chromosome mapping, gene structure, conserved domains and physicochemical properties of ABCD gene family in buffalo were analyzed using bioinformatics. The expression of ABCD1 gene in buffalo tissues and mammary glands at different lactation stages was detected by Real-time quantitative PCR. The effects of 3 hormones on the expression of ABCD1 gene in mammary epithelial cells of buffalo were also detected. The results indicated that 9 ABCD gene were identified from the whole genome of buffalo, which included ABCD1, ABCD2, ABCD3 and ABCD4, and ABCD4 gene contained 6 different transcripts(ABCD4X1-ABCD4X6). ABCD1-ABCD4 were identified on chromosome X, 4, 6 and 11, respectively. ABCD gene included 9-22 introns, which coded 444-741 amino acid residues with the molecular weight ranging from 49.63-83.41 ku, and the isoelectric points ranging from 6.43-9.64.3 motifs were identified from ABCD protein, including ABC_membrane_2, Endonuclease_5 and ABC_tran. ABCD gene family could be divided into 4 subfamilies, and the evolutionary tree of different species showed that ABCD gene was highly conserved. Collinearity analysis results showed that there were 7 pairs of ABCD gene family with homologous lineages between buffalo and cattle. ABCD1 gene was expressed in 15 tissues of buffalo with a relatively high level in fat. The expression of ABCD1 gene was variable during lactation and exhibited a trend of low-high-low with the highest level at 50 days after parturition. The mammary gland cells in buffalo were treated with different concentrations of prolactin, estradiol and progesterone, the expression of ABCD1 gene was significantly higher than that of control group when the concentrations of prolactin, estradiol and progesterone were above 2.0, 0.3 and 4.5 μg/mL, respectively. The results showed that there were 9 genes in ABCD gene family of buffalo, among which ABCD1 gene was involved in the regulation of lactation. This study provided a reference for further exploring the function of ABCD family genes in mammary gland of buffalo.

The objective of this study was to investigate the effects of prenatal exercise on the indices of physiological metabolism in perinatal dairy cows, and provide scientific guidance and theoretical basis for the health management of perinatal dairy cows. 24 multiparous Holstein cows were randomly and equally selected and divided into test group (TRT group) and control group (CON group), fed and managed uniformly. The experiment started at 21 days before the due date, the cows in TRT group walked twice a day (09:00 and 16:00) at a speed of less than 3 km/h and the total daily exercise volume of each cow was (4.0±0.2) km/d, CON group moved freely. The blood samples were collected at 21 and 7 days before confinement, parturition day, 7 and 30 days after confinement (noted as －21, －7, 0, ＋7 and +30 d, respectively) before morning meal. Blood was collected before feeding in the morning, and the plasma contents of non-esterified fatty acid (NEFA), β-hydroxybutyric acid (BHBA), glucose (GLU), triglyceride (TG), cholesterol (CHOL), creatinine (CREA) and urea (UREA) were detected. The results showed that the concentration of NEFA and BHBA in TRT group exhibited a declining trend compared with CON group during the experiment (0.05<P<0.1), and the concentration of NEFA of TRT group at －7 d was significantly lower than CON group (P<0.05). The change of plasma GLU concentration in TRT group was more stable than CON group, and was extremely significantly higher than that of CON group at ＋30 d (P<0.01). The plasma TG concentration of the two groups were decreased sharply on parturition day and the prenatal concentration were extremely significantly higher than that of all time points after delivery (P<0.01), while the plasma concentration of TG in TRT group was extremely significantly higher than CON group at ＋30 d (P<0.01). There was no significant difference in plasma UREA and CREA concentrations between the two groups (P>0.05). The results indicated that the exercise at a speed of less than 3 km/h (total amount of exercise was 4.0 km/d±0.2 km/d could reduce the plasma NEFA and BHBA concentrations of perinatal dairy cows effectively and attenuate the stress of negative energy balance(NEB).

Skeletal muscle is an important part of biological body, accounting for about 40% of the body of meat producing animals. It is closely related to sports, development, meat production performance and other important economic traits of livestock and poultry. Multinucleated muscle fiber is the basic unit of skeletal muscle which is composed with parallel myofibrils, and the numbers, types and the transform of muscle fiber reflect the individual muscle development and physiological conditions directly which lead it to act an important economy trait being concerned in breeding. The study to muscle fibers in-depth will contribute in breeding process and helpful for exploring and handles of the causes of myopathies that frequently appear in recent years, some extent, to alleviate the economic losses caused by myopathies to livestock and poultry industry, and meet the demand of consumers for high-quality meat products. In recent years, the understanding of muscle fiber and the research has achieved great progress, including in accordance with the structure and function of the muscle fiber characteristics to predict and verify the biological functions of the skeletal muscle and the relationships between muscle fiber types with meat quality, on the other hand, the important regulation molecules of muscle fibers during occurred and regeneration repair were digged out and defined their functions with the development of technology and the application of animal models. In this paper, the structure and function of muscle fiber were systematically described according to the existing researches and the occurrence and development of muscle fiber during embryonic period as well as the regeneration and repair after birth were sorted out from the molecular level, so as to provide theoretical reference for improving meat production performance of meat animals and breeding new varieties or strains of high-quality meat producing animals in the future.

The aim of this study was to separate exosomes from the plasma of buffalo calves, analysis and identify molecular biological characteristics of the isolated exosomes. The differential centrifugation was used to separate exosomes from plasma of three buffalo calves, and the morphology of extracted exosomes was observed under a transmission electron microscope. The particle size of the extracted exosomes was analyzed using nanoparticle size and Zeta potentiometer, and the expression of exosome marked proteins, including Calnexin, tumor susceptibility gene 101 (TSG101), CD9 and CD81, were detected by Western blotting method. The results showed that plasma exosomes of buffalo calves were mostly round and elliptical, with a diameter between 30 to 150 nm. The Western blotting results showed that the specific proteins Calnexin, TSG101 and CD81 were positive expressed in the extracted plasma exosomes, but protein CD9 was negative. Based on morphology and molecular characteristics, it was confirmed that the obtained extract were exosomes, indicating that the differential centrifugation could be used to successfully isolate plasma exosomes of buffalo calves. The results provided an important technical basis and reference for further research on buffalo exosome.

Alternative splicing occurs after DNA is transcribed into precursor mRNA, and multiple mRNA isomers and protein subtypes can be produced from a single gene through this process, which increases the information diversity and protein abundance of genes during post-transcriptional processing. Spliceosomes, splicing factors and other RNA-binding proteins catalyse the recognition of splicing sites and alternative splicing exons, and participate in the regulation of alternative splicing. From the perspective of evolution, alternative splicing provides a driving force for biological evolution to some extent. As a tissue-specific regulator, alternative splicing is involved in the whole process of muscle development. During myoblast differentiation, polypyrimidine tract-binding protein (PTB) and RNA-binding motif protein 4 (RNA-binding motif protein 4, RBM4) binds to the intron polypyrimidine sequence of tropomyosin and the CU-rich intron, respectively, and modulate the activity of muscle cell-specific exon selection of alpha-tropomyosin. RBM4 plays a synergistic role in the specific splicing of muscle cell proliferation and differentiation by down-regulating the expression of PTB and antagonizing the activity of PTB exon selection. Myosin Ⅰ subtype produces proteins of four different length lever arms through alternative splicing, which affect muscle tension and muscle stretching activation. Alternative splicing may be an important mechanism for the generation of muscle type specificity. Selective splicing produces myosin heavy chain isoforms with different catalytic kinetics, which regulate contractiity differently, thus affecting muscle fiber type and muscle function. By expounding the main roles of alternative splicing events in muscle development and muscle fiber formation, the author revealed the role of alternative splicing on muscle development, in the hope of providing a reference for the follow-up research on muscle development regulation mechanism.

In this experiment, the appearance quality, energy and protein concentrations, digestive enzyme activitties, immunoglobulin levels and growth factor concentrations of pigeon milk in squabs at different periods were determined, and the dynamic changes of these factors at different days were analyzed, so as to provide theoretical basis for systematically understanding the nutritional and physiological needs of squabs. Twelve healthy White King pigeons aged 0, 7, 14, 21 and 28 days were randomly selected respectively. After being fed by parent pigeons, squabs were slaughtered and all contents of their crops were taken out. One part was diluted with normal saline, then the supernatant was isolated for the determination of digestive enzyme activity, immunoglobulin activity and growth factor concentration. The other part was made into air-dried samples for detection of routine nutrient levels. The results showed that:The pigeon milk of 0-day-old showed a milky, oily, cheesy-like appearance, and it was a mixture of cheesy-like crop milk and feed at 7-day-old. After 14 days of age, the pigeon milk was gradually transformed into softened feed. The energy and protein concentrations of pigeon milk were affected by age significantly (P<0.05), and they were significantly higher at 0-day-old than those in later periods (P<0.05). The energy and protein concentrations of pigeon milk after 7 days of age were very similar. The activities of amylase, trypsin and chymotrypsin in pigeon milk at different periods were significantly different (P<0.05), and showed a linear increasing relationship (P<0.05). The levels of immunoglobulin A (IgA), immunoglobulin M (IgM) and immunoglobulin G (IgG) in pigeon milk at different periods had significant differences (P<0.05), and showed a quadratic curve relationship that first decrease and then increase (P<0.05). The levels of insulin growth factor-1(IGF-1) and epidermal growth factor(EGF) in pigeon milk at different periods were significantly different (P<0.05), and showed a significant trend of decreasing first and then increasing (P<0.05). In conclusion, during 0-7 days, the exfoliation and cell debris of the mucosa epithelial cells from the parents’ were the main contents of pigeon milk, and the energy, protein, immune factors and growth factors were at the highest levels. From 8-14 days, the energy and protein levels of pigeon milk tended to be flat, while the immune factors and growth factors decreased to the minimum. The nutrients levels of pigeon milk of squabs from 15 to 28 days were similar to the feed of parent pigeons, and the digestive capacity of squabs gradually increased.

This experiment was conducted to study the effects of lignocellulose as an insoluble fiber source on gastrointestinal development and gut function of young layers. A total of 375 28-day-old Hy-line Brown commercial young layers were randomly divided into 3 groups with 5 replicates, and each replicate had 25 chickens. Each group was fed corn-soybean meal diet supplemented with 0 (control group), 8 (0.8% lignocellulose group), and 10 g/kg lignocellulose (1.0% lignocellulose group), respectively. The feeding experiment lasted for 56 days. At the ending of the experiment, the average daily gain, average daily feed intake, and feed to gain ratio were calculated. Additionally, the organ index, ileal digestive enzyme activities, tight junction-related gene expression, and morphological changes were measured. The results showed that, compared with the control group, the addition of lignocellulose had no significant effect on the average daily gain, average daily feed intake, and feed to gain ratio(P>0.05), but significantly increased the weight of muscular stomach and muscular stomach organ index of young layers (P<0.05). Adding lignocellulose significantly increased the villus width and height of chickens (P<0.05), and the villus height of the 1.0% lignocellulose group was higher than that of the 0.8% lignocellulose group (P<0.05). The activities of ileum α-amylase and trypsin and the mRNA levels of tight junction ZO-1 and ZO-2 in the 0.8% lignocellulose group were higher than those in the control group (P<0.05). In conclusion, the addition of 8 g/kg lignocellulose could stimulate the development of muscular stomach, increase intestinal digestive enzyme activities, and improve the integrity of intestinal morphology that maintaining gut health of young layers.

The aim of this study was to research the optimal fermentation conditions and culture medium component for sublancin, an antimicrobial peptide produced by Bacillus subtilis YT168-6, in order to increase the content of sublancin in fermentation broth. A single factor test was used to screen out the optimal fermentation conditions, inorganic salts, carbon and nitrogen sources of fermentation medium. The optimum fermentation medium component was determined by the orthogonal test, the steepest climbing test and the response surface analysis method of Box-Behnken design. The results showed that the optimal fermentation conditions for Bacillus subtilis YT168-6 to produce the antimicrobial peptide sublancin were 1.0% bacterial inoculum, fermentation temperature 37 ℃ and initial pH 7.0, the optimum fermentation medium was K₂HPO₄ 5 g/L, KH₂PO₄ 5 g/L, MgSO₄ 10 g/L, soluble starch 30.2 g/L, peptone 23.6 g/L and corn steep liquor 18.2 g/L. After optimization, the yield of sublancin was increased from 756 μg/mL to 1 581 μg/mL, which was 2.09 times as high as before optimization. In summary, the optimal fermentation conditions and culture medium selected in this research laid the technical foundation for the subsequent industrial production of Bacillus subtilis YT168-6.

This study was aimed to evaluate the effects of Astragalus membranaceus (AP) and dextran sulfate sodium (DSS) on hindgut fermentation and microbial composition of lambs. The experiment was divided into two stages:Before and after DSS stimulation. In the first stage, 36 healthy, weaned Xiangdong Black goats with similar body weight (5.2 kg±2.0 kg) at 42 days old were randomly assigned to control group (CON, n=16;basic diet) and Astragalus group (AP, n=20;basic diet supplemented with 10 g/d). Lambs were slaughtered (except for 6 lambs selected in each group in the second stage, the rest were all slaughtered) after 36 days of feeding (including 7 days of pre feeding period), cecal tissue and chyme were collected to evaluate the effect of supplementing AP on hindgut fermentation and microbial composition. In the second stage, on the 36th day of the experiment, six lambs in CON group and AP group were randomly selected for DSS stimulation (4% body weight dose group). 8 days after stimulation, all lambs were slaughtered, and cecal tissue and chyme were collected to evaluate the hindgut fermentation and microbial composition changes after DSS stimulation. The results showed that:①Compared with CON group, the caecum fermentation parameters and microbial composition of AP group were not significantly different (P>0.05). ②Compared with the pre-DSS, acetate concentration was decreased while the butyrate was significantly increased in the DSS (P<0.05). ③Compared with the pre-DSS, the copy number of total bacteria and Clostridium cluster XIVa in tissue in the DSS was increased (P<0.05), but the copy number of Clostridium cluster XIVa and Lactobacillus in the chyme were decreased (P<0.05). ④The 16S rDNA amplicon sequencing results showed that the relative abundance of Firmicutes and Bacteroidetes was more than 80% in all groups, followed by Proteobacteria and Tenericutes. No significant difference was observed in hindgut microbial composition between different treatment groups (P>0.05). ⑤A significant correlation between short chain fatty acids concentration and carbohydrate utilizing bacteria were observed. In conclusion, these results provided evidence that AP inclusion in the diet of lambs had no significant effect on the hindgut fermentation and microbial composition (P>0.05). In contrast, the addition of DSS in the diet could reduce acetate concentrations and the number of beneficial bacteria, change the relative abundance of bacteria at the genus level.

Milk fever is a nutritional metabolic disease caused by the decrease of blood calcium level at cow’s lactation stage, which has a great impact on the health and production performance. It increases the risk of reproductive inflammation, reproductive disorders and endocrine, digestion and other diseases in cows, causing great losses to the farming industry. There are many factors that affect milk fever in cows, including breed, age, physical condition scores, previous case of milk fever and other factors. Besides, the influence factors of feeding management containing previous lactation length, dry period length, calving interval and the control of cow feed nutrition level, and external factors such as climate and environment, also affect the incidence rate of milk fever. These factors often intertwined with each other, increasing the difficulty of prevention and treatment of milk fever in dairy cows. This paper summarized the pathogenesis of milk fever in cows, analyzed the mechanism of influence of various factors on milk fever in cows, and introduced several methods to prevent and control milk fever in cows. It mainly included the management of dairy farming environment, the control of the interval and lactation of cows, and the regulation of feed nutrition levels at different stages of cows, so as to provide a theoretical basis for reducing milk fever in cows.

In order to study the effect of Ziziphus jujuba polysaccharide (ZJP) on the growth performance and immune indexes of immunosuppressive egg-type chicks, nine hundred one-day-old Jinghong No. 1 egg-type chicks of healthy and similar body weight were randomly divided into 6 groups with 6 replicates in each group and 25 chickens in each replicate. Group Ⅰ was blank control group, fed with basal diets. The immunosuppressive model was established by injecting 100 mg/(kg·BW) cyclophosphamide once a day at 7-9 days old in groups Ⅱ-Ⅵ, the basic diets were supplemented with 600 mg/kg Astragalus polysaccharides (APS) and 0, 400, 800 and 1 600 mg/kg ZJP, respectively. The trial period was 6 weeks. The results showed that ZJP could significantly alleviate the decrease of body weight at 42 days old, average daily gain at 22-42 days old and 1-42 days old, thymus and bursa index, polymeric immunoglobulin receptor, secretory immunoglobulin A (sIgA) and serum immunoglobulin A (IgA) and IgG, and the increase of F/G at 22-42 days, and the F/G and IgA and IgG reached the level of APS group. Under the conditions of this experiment, adding appropriate level of ZJP to the basal diet could improve the growth retardation and immune dysfunction caused by cyclophosphamide in varying degrees.

The aim of the experiment was to screen out the lytic bacteriophages of Campylobacter and to explore their rational application in broiler production. Campylobacter bacteriophage screening samples were collected from broiler slaughterhouse, and specific Campylobacter bacteriophages were obtained by co-incubating, proliferating and purifying with local Campylobacter epidemic strains. The biological characteristics (host range, lethal curve, morphological characteristics, etc.) of Campylobacter bacteriophage were identified. As a feed additive, it was added to the diets of 38-day-old male Ross 308 broilers with 5×10⁷, 1×10⁸, 5×10⁸, 1×10⁹ and 5×10⁹ PFU/d 5 different amounts to observe the scavenging effect of Campylobacter in cecal contents of broilers. Finally, by comparing the effects of different phage ratio on the clearance rate of Campylobacter and the growth performance of broilers, the application scheme was established. The results showed that a total of 12 strains of Campylobacter jejuni bacteriophages were isolated from local broiler slaughterhouses using Campylobacter jejuni L26 as host bacteria. The bacteriophages BP11 and BP12 which had wide lytic spectrum and large plaque were chose by co-incubating, purifying and proliferating with 7 strains of Campylobacter jejuni from broiler farms. By morphological identification, the two bacteriophages were in accordance with the characteristics of Myoviridae and had a wide lytic spectrum, but their lytic ability of the same host bacteria was different. The bactericidal effect was significant when the BP11 and BP12 addition amount were 1×10⁹ and 5×10⁸ PFU/d, respectively. Bacteriopages BP11 and BP12 mixed addition group could significantly reduce the carrying rate of Campylobacter in broiler production without affecting the slaughter index. These results provided theoretical foundation for the prevention and control of Campylobacter contamination and the rational utilization of bacteriophages in broilers.

This study was aimed to analyze the metabolites of fermented Astragalus by Lactobacillus plantarum using liquid chromatograph-mass spectrometer (LC-MS) metabonomics technology, and explore the mechanism of its interaction fermentation. The fermented (FT group) and unfermented (CT group) Astragalus solid powders were collected, respectively, and find out different metabolites and metabolic pathways through the sample pretreatment, LC-MS analysis and bioinformatics analysis. The result showed that the peak map of the total ion current map had good reproducibility. The main metabolites of fermented Astragalus had identified 183 metabolic components. The PCA map of the positive ion and negative ion mode samples could be distinguished well. The volcanic map analysis showed the metabolite changes were different between FT and CT groups, there were 1 416 metabolites up-regulated in positive ion model were enriched to 83 metabolic pathways, 935 metabolites down-regulated were enriched to 83 metabolic pathways;And 1 040 metabolites up-regulated in negative ion model were enriched to 52 metabolic pathways, 809 metabolites down-regulated were enriched to 45 metabolic pathways. The differential metabolites of fermented Astragalus acid, lipids, ketones and other amino acids were significantly increased, and olefins and other amino acids were significantly down-regulated, among them, 15 were up-regulated metabolites, and 2 were down-regulated metabolites. The key metabolites were mainly α-diethyl sulfate, 2-methyl citric acid, 3-isopropenyl-6-oxoheptanoic acid, etc., involving pyruvate metabolism, propionic acid metabolism, galactose metabolism and other pathways. The results provided a theoretical basis for the metabolites, fermentation interaction mechanism and clinical application of fermented Astragalus.

This experiment was conducted to compare and analyze the fecal microflora composition and gene function prediction of normal and diarrhea yak calves under grazing conditions, and screen out the bacteria as the basis for diarrhea evaluation, so as to provide the theoretical basis for the development of probiotics for yak calves in the next step. Seven fecal samples were collected from normal and diarrhea yak calves in Yushu area of Qinghai province under grazing conditions, respectively, and DNA was extracted. 16S rRNA sequencing technology was used to study the OTU clustering, relative abundance difference at phylum level and genus level, Alpha diversity, Beta diversity and functional prediction level information of intestinal microflora of yak calves between normal and diarrhea groups. The results showed that at phylum level, Bacteroidetes and Firmicutes were the dominant phyla in normal and diarrhea groups, and the Fusobacterium in diarrhea group was significantly higher than that in normal group (P<0.05). At genus level, the relative abundance of dominant bacteria in the feces of normal group calves from high to low were Bacteroides, Alloprevotella and unidentified_Ruminococcaceae. The relative abundance of dominant bacteria in the feces of diarrhea group calves from high to low were Bacteroides, Fusobacterium, unidentified_Enterobacteriaceae and unidentified_Ruminococcaceae, and the abundance of Fusobacterium in diarrhea group was significantly higher than that in normal group (P<0.05). At function prediction level, the main contents of glycan biosynthesis and metabolism, transportation and catabolism, aging, classification and degradation and lipid metabolism in diarrhea group were significantly higher than that in normal group (P<0.05), while the main contents of amino acid metabolism, signal transduction, cell movement and genetic information in normal group were significantly higher than that in diarrhea group (P<0.05). According to this experiment, the difference of dominant flora between normal and diarrhea groups of grazing yak calves was analyzed, the diarrhea of calves in this experiment was caused by Fusobacterium.

This study was aimed to investigate the effect of pine needle powder on production performance, egg quality, serum biochemical and antioxidant indices of laying hens, in order to provide a reference for applying pine needle powder in laying hens production. A total of 120 healthy 40-week-old No. 1 Jingfen laying hens were selected as experimental animals, which were randomly divided into 2 groups. There were 6 replicates per group and each replicate contained 10 individuals. The hens in control group were fed a basal diet, and those in experimental group were fed the diet adding 3% pine needle powder in the basal diet to replace wheat bran. The pre-experimental period lasted for 7 days for adaption and the experimental period lasted for 56 days. In the course of experiment, the feed intake, total egg production and total egg weight were recorded per repeat each day. And then, the average daily feed intake, average egg weight, daily egg production and feed egg ratio were computed in groups. Furthermore, two eggs were taken from each replicate to gauge eggshell strength, eggshell thickness, egg yolk color and Haugh unit at the 28th and 56th days of the experiment. The last two days of the experiment, 10 eggs were selected from each replicate and stored in refrigerator at 4 ℃. Then, two eggs were selected from each replicate to measure Haugh units of eggs at the 15th, 20th, 60th and 105th days of storage. At the 56th day of the experiment, one laying hen was selected from each replicate after fasting for 12 h to extract 5 mL of blood from the inferior wing vein. The contents of total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), total protein (TP), albumin (ALB), uric acid (UA), urea and glucose (GLU), and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were tested by automatic biochemical analyzer. The content of malondialdehyde (MDA), the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH) and catalase (CAT), and total antioxidant capacity (T-AOC) in serum were tested by reagent kit method. The results showed that:①Compared with control group, dietary pine needle powder significantly increased the laying rate of laying hens (P<0.05), and significantly decreased the average egg weigh and F/E (P<0.05), but there was no significant differences in average daily feed intake, average daily egg production and qualified egg rate (P>0.05). ②At the 28th day of the experiment, dietary pine needle powder significantly increased the egg yolk color of laying hens (P<0.05), there was no significant differences in egg yolk color between two groups at the 56th day of the experiment (P>0.05). There was no effects on the Haugh unit, eggshell thickness and eggshell strength of eggs (P>0.05). ③ Compared with control group, dietary pine needle powder had no effects on the contents of serum TC, TG, HDL-C, LDL-C, TP, ALB, GLB, urea, UA, GLU and the activities of serum ALT, AST and ALP (P>0.05). Dietary pine needle powder significantly increased the content of serum MDA and the activity of GSH-Px of laying hens (P<0.05), but there were no effects on the contents of GSH and the activities of SOD and CAT, and T-AOC (P>0.05). In conclusion, dietary supplementation of 3% pine needle powder could significantly increase the egg production rate of laying hens, improve F/E and egg yolk color, and significantly increase the content of serum MDA and the activity of GSH-Px of laying hens, but there was no effect on T-AOC of laying hens. The results indicated that it’s feasible to replace the equal proportion of wheat bran with 3% pine needle powder in the diet of laying hens.

The aim of this study was to elucidate the polymorphisms of g. 13504098 C>T of diacylglycerol acyltransferase 1 (DGAT1) gene, g. 11204707 A>C of nuclear receptor subfamily 6 group A member 1 (NR6A1) gene and g. 18795097 C>T of alpha-mannosidase (MAN1A1) gene, and its association with the growth traits in Ujumqin sheep, providing new genetic markers for high productivity molecular breeding of Ujumqin sheep. g. 13504098 C>T, g. 11204707 A>C and g. 18795097 C>T of DGAT1, NR6A1 and MAN1A1 genes in Ujumqin sheep were detected by Sequenom MassARRAY^®SNP assay, and the linear mixed model was used to analyze the association between genotypes and the growth trait in Ujumqin sheep at 4 and 6 months of age. The results showed that three genotypes were detected at three loci. Specifically, g. 13504098 C>T included CC, CT and TT genotypes, CC genotype was dominant genotype (0.49), C was the dominant allele (0.70). g. 11204707 A>C included AA, CA and CC genotypes, AA genotype was dominant genotype (0.54), A was the dominant allele (0.73). g. 18795097 C>T included CC, TC and TT genotypes, TC genotype was dominant genotype (0.51), C was the dominant allele (0.55). χ² test indicated that g. 13504098 C>T, g. 11204707 A>C and g. 18795097 C>T of Ujumqin sheep were in Hardy-Weinberg equilibrium (P>0.05). Population genetic analysis showed that g. 13504098 C>T, g. 11204707 A>C and g. 18795097 C>T were at moderate polymorphisms in Ujumqin sheep population (0.25<PIC<0.50). Association analysis revealed that g. 13504098 C>T was extremely significantly associated with the body weight, body height, chest girth, shin circumference of Ujumqin sheep at 4 and 6 months of age, and the chest width at 6 months of age (P<0.01);g. 11204707 A>C was extremely significantly associated with the body height and body length of Ujumqin sheep at 4 and 6 months of age, the body weight at 4 months of age, and the chest girth at 6 months of age (P<0.01), and it was significantly associated with the body weight and shin circumference at 6 months of age (P<0.05);g. 18795097 C>T was extremely significantly associated with the body height at 4 months of age and the chest girth at 6 months of age (P<0.01), and significantly associated with the body weight and chest girth at 4 months of age, and the chest width at 6 months of age (P<0.05). Therefore, it could be concluded that g. 13504098 C>T of DGAT1 gene, g. 11204707 A>C of NR6A1 gene and g. 18795097 C>T of MAN1A1 gene had significantly correlation with the growth traits of Ujumuqin sheep, indicating that 3 SNPs could be used as candidate molecular markers for genetic breeding in Ujumuqin sheep.

In order to explore the population characteristics and influencing factors of rest time in Holstein dairy cows in Beijing, the rest time records in 838 Holstein dairy cows from a large-scale farm, and the corresponding dairy herd improvement records, environmental temperature and humidity, management records were collected from April 2018 to April 2020. The GLM procedure in SAS 9.2 software was used to analyze the impacts of year, season, herd, parity and lactation month on rest time in Holstein dairy cows. The relationship between production performance and rest time was also analyzed. The results showed that the average rest time in Holstein dairy cows was 397.39 min/d, ranging from 87.74 to 707.03 min/d, and the coefficient of variation was 26%. The rest time firstly decreased and then increased with change of season, with the lowest in summer and the highest in winter, and it gradually decreased with the increase of environmental temperature and humidity index. The rest time in dairy cows reached a peak high level when they calved or suffered from health events, and then it gradually decreased to base levels. Measuring year, season, herd, parity and lactation month had extremely significant impacts on rest time (P<0.01). The milk production showed a decrease trend with the increase of rest time in Holstein dairy cows. This study provided a theoretical basis for the use of automatic recording equipment to explore the behavior of dairy cows and the use of continuously measured rest time data to improve the accurate management level of dairy cows.

In order to explore the candidate loci affecting the lambing traits in the breeding of high-quality meat sheep in Ningxia, 5 sheep populations of Dorper sheep, Tan×Han hybrid sheep, hybrid generation 1, hybrid generation 2 and cross generation 1 were studied. Flight mass spectrometry technology was used to detect the polymorphism of beta-1, 4-N-acetyl-galactosaminyl transferase 2 (B4GALNT2) gene g. 36933082 G>A and g. 36946470 G>A and estrogen receptor 1 (ESR1) gene g. 75378892 A>T in 5 sheep populations and analyze their association with litter size in sheep. The secondary and tertiary structures of proteins before and after B4GALNT2 and ESR1 genes mutation were analyzed by bioinformatics online software. The results showed that there were three genotypes (AA, AG and GG) in g. 36933082 G>A and g. 36946470 G>A of B4GALNT2 gene, and GG genotype was the dominant genotype. There were three genotypes (AA, TA and TT) in g. 75378892 A>T of ESR1 gene, and AA genotype was the dominant genotype. g. 36933082 G>A and g. 36946470 G>A showed low polymorphism in 5 sheep populations (PIC<0.25), g. 75378892 A>T showed moderate polymorphism in 5 sheep populations (0.25<PIC<0.5), and the three loci were in Hardy-Weinberg equilibrium in 5 sheep populations. The correlation analysis of litter size results showed that g. 36933082 G>A and g. 36946470 G>A of different genotypes were not significantly different from those in 5 sheep populations (P>0.05), but g. 75378892 A>T in hybrid generation 1 sheep population, the litter size of individuals with TA genotype was significantly higher than that of TT genotype (P<0.05). Bioinformatics results showed that all three sites caused changes in the secondary and tertiary structures of the corresponding protein. In conclusion, g. 36933082 G>A and g. 36946470 G>A of B4GALNT2 gene were not suitable for the breeding of multiple lambing traits in 5 sheep populations, while g. 75378892 A>T of ESR1 gene could be considered as a molecular assistant marker for multiple lamb traits in hybrid generation 1 sheep population.

This study was aimed to use homologous recombination mediated by Tild-CRISPR/cas9 system to acquire homozygous hornless cell lines. sgRNA were designed for Pc target site controlling the polledness in cattle, and its mutation efficiency was detected by T7E1 digestion and sequencing. pUC57 vector was used as the skeleton to produce homologous recombinant fragment through connecting the Pc fragment and homology length of 1 600 bp (800 bp homology on each arm), then the homologous recombinant fragment was obtained by double enzyme digestion and sgRNA were co-transfected into the fibroblasts of the auricular margin in Holstein bulls, the single cell clone was screened and identified. The results showed that six sgRNAs_(1-6) were successfully constructed and sgRNA₁ with the highest mutation efficiency of 32.6% was obtained. The homologous recombination fragment of 1 901 bp was successfully obtained. A total of 147 single cell clones were obtained, and 8 single cell clones had correct single allele Pc site insertion, and 1 single cell clone had corrected double allele Pc site insertion. The results of foreign gene residue test showed that there was no Cas9 gene residue in single cell clone, which could be used as the nuclear donor cells for subsequent cloning and production of excellent hornless homozygous embryos. This study successfully used Tild-CRISPR/Cas9 site-specific editing method to obtain the ear margin fibroblast cell line without exogenous Cas9 gene residue with homozygous insertion at PC site, which provided a good material reserve and technical platform for the breeding of excellent bulls and the research of main functional genes in the future.

In order to explore the effects of metformin on the proliferation and differentiation of bovine skeletal muscle satellite cells, bovine skeletal muscle satellite cells cultured in vitro were treated with 0 (control group), 1, 2 and 4 mmol/L metformin, and using CCK-8 method to screen the optimal concentration of metformin on bovine skeletal muscle satellite cells. The effect of metformin treatment on the proliferation of bovine skeletal muscle satellite cells was detected by EdU staining method, and the metformin-treated bovine skeletal muscle satellite cells were myogenically induced and differentiated in vitro, and the cell status of bovine skeletal muscle satellite cells during the differentiation period was observed. Then Western blotting was used to detect the expression of the differentiation markers myosin heavy chain (MyHC) and myogenin (MyoG) of bovine skeletal muscle satellite cells at 24, 48 and 72 h of differentiation. The results showed that the optimal concentration of metformin on bovine skeletal muscle satellite cells was 2 mmol/L. After treating bovine skeletal muscle satellite cells with 2 mmol/L metformin, the cell proliferation rate were significantly reduced (P<0.05), indicating that metformin could inhibit the proliferation of bovine skeletal muscle satellite cells. The number and diameter of myotubes formed by bovine skeletal muscle satellite cells after induced differentiation showed a decreasing trend, and the expressions of myogenic differentiation markers MyHC and MyoG of bovine skeletal muscle satellite cells were significantly lower than 0 mmol/L (control) group (P<0.05) at 24, 48 and 72 h after differentiation, indicating that 2 mmol/L metformin could inhibit the myogenic differentiation process of bovine skeletal muscle satellite cells. The results of this study showed that metformin could significantly inhibit the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells, providing a theoretical basis for the application of metformin in muscle development regulation and muscle injury repair.

This study was aimed to investigate the association of the polymorphism of NR5A2 gene with reproductive traits in Songliao Black pigs. 130 Songliao Black pigs were selected as the research objects. The SNP of 6 exons of NR5A2 gene were found by PCR sequencing. The polymorphism of SNPs was analyzed by HRM typing technology. The correlation between SNPs of NR5A2 gene and total number born, number born alive, birth weight, 3-week weight, weaning weight, weaning piglet number and nipple number was analyzed by SPSS 19.0 software. The results showed that there was an SNP (C99T) in exon 6 of NR5A2 gene in Songliao Black pig, and three genotypes were detected:CC, CT and TT. The results of genetic parameter analysis and Chi square test showed that Songliao Black pig population was in Hardy-Weinberg equilibrium (P>0.05), but the heterozygosity of C/T mutation site was relatively low, and had a low variation in Songliao Black pig population, which belonged to moderate polymorphism (0.25<PIC<0.5). In the C99T site of NR5A2 gene, the total number born, number born alive and weaning piglets of CC genotype were the highest, and significantly higher than TT genotype (P<0.05), the number born alive and weaning piglet number were also significantly higher than CT genotype (P<0.05). There was no significant difference in birth weight, 3-week weight, weaning weight and nipple number among different genotypes (P>0.05). In conclusion, the mutation site C99T in exon 6 of NR5A2 gene was significantly associated with number born of Songliao Black pigs. However, whether this mutation site could be used as a genetic marker of number born of Songliao Black pigs needed further study in a larger population.

The aim of this study was to explore the population characteristics of the dry period length (DPL) and its impacts on the production performance in Chinese Holstein cattle in Beijing, including milk production, reproduction performance, colostrum quality and udder health. In this study, dry dates, production performance records and reproduction records from 136 231 Chinese Holstein cattles born between 2009 to 2018 were collected from 34 herds in Beijing. Based on dry dates recorded from these farms, the population characteristic of DPL of the 1st and 2nd parity were revealed. A fixed effect model was used to analyze the impacts of parity, calving season, herd-calving year and age at first calving on DPL. Furthermore, a fixed model was employed to analyze the impacts of variation in DPL on test-day milk yield, fat percentage, protein percentage, interval from the first to last inseminations (IFL), interval from calving to first inseminations (ICF) and colostrum quality. Logistic regression model was used to analyze the impacts of variation in DPL on conception rate of first insemination (CR), 56-days non-return rate for first insemination (NRR56), calving ease and udder health. It was found that the average DPL was 58.81 d in Chinese Holstein cattle in Beijing, and parity, calving season, herd-calving year and age at first calving had significant impacts on DPL (P<0.05). The variation of DPL had significant effects on daily milk yield, milk protein rate, ICF, CR, NRR56, dystocia rate and breast health (P<0.05). The cows with short DPL had higher test-day milk protein percentage, CR and NRR56. The cows with moderate DPL had better test-day milk yeild and udder health, shorter ICF and lower dystocia risk. This study revealed the population pattern of DPL of Chinese Holstein cattle by using large-scale herd data in China, and could provide reference for improving the management level of large-scale dairy farms in China.

The aim of this study was to investigate the effect of melatonin (MLT) on lipopolysaccharide (LPS)-induced inflammatory response in skeletal muscle satellite cells in Tan sheep. In this study, the 30-day-old fetus of Tan sheep was selected. The skeletal muscle satellite cells were isolated by collagenase Ⅳ, and cultured in vitro, the corresponding inductive reagent was added to induce differentiation of skeletal muscle satellite cells, and the expression of surface markers CD29, CD44, CD73 and Vimentin were analyzed by immunofluorescence. The skeletal muscle satellite cells were cultured at different doses (0, 5, 8 and 10 μg/mL) of LPS in the medium without fetal bovine serum, and the mRNA levels of inflammatory related factors such as interleukin-6 (IL-6), IL-8 and tumor necrosis factor-α (TNF-α) were detected by Real-time quantitative PCR (qRT-PCR), and the optimal concentration of LPS was selected. Skeletal muscle satellite cells were co-cultured with different concentrations 0.1 and 0.5 μmol/L of MLT and LPS of optimal concentration, and the mRNA levels of IL-6, IL-8 and interferon-γ (IFN-γ) were detected by qRT-PCR. The immunofluorescence results showed that the expression of the surface markers CD29, CD44, CD73 and Vimentin of skeletal muscle satellite cells were positive, and the cells had the properties of myogenic, lipogeneic and osteogenic differentiation. qRT-PCR results showed that different concentrations of LPS treatments could significantly increase the genes expression of inflammatory related factor when compared with control group (P<0.05), and the mRNA expression of inflammatory factors was the strongest when treated with 8 μg/mL LPS. When the cells were co-cultured by MLT and LPS, 0.5 μmol/L MLT could significantly decrease the expression of IL-6 induced by LPS when compared with LPS group (P<0.05). In conclusion, MLT could alleviate LPS-induced inflammatory response in skeletal muscle satellite cells in Tan sheep. The results laid a foundation for further research on the regulatory mechanism of MLT alleviating the inflammatory response of skeletal muscle satellite cells induced by LPS.

This study was aimed to explore the site of G protein-coupled receptor 50 (GPR50) expression and localization in yak oocytes during in vitro maturation (IVM), and to provide the basis for better understanding the molecular mechanism of oocyte maturation and the specificity of yak reproduction. After yak oocytes were matured in vitro, fluorescence staining was used to monitor the changes of spindle morphology and nuclear phase at different time points (0-24 h). The time points of the four critical periods of maturity for yak oocytes, including germinal vesicle(GV), germinal vesicle break down(GVBD), metaphase Ⅰ(M Ⅰ) and metaphase Ⅱ(M Ⅱ) were determined accurately. On this basis, the dynamic expression of GPR50 gene during the maturation of yak oocytes was detected by Real-time quantitative PCR, and the subcellular dynamic site of GPR50 protein during the maturation of oocytes was detected by immunofluorescence staining. The results showed that 90% of the in vitro matured yaks oocytes were at GV stage at 0 h, 94% at GVBD stage at 6 h, 92% at MⅠ stage at 16 h, 94% at MⅡ stage at 24 h. The results of Real-time quantitative PCR showed that GPR50 gene was expressed in GV phase of yak oocytes, and it gradually increased during the maturation of GVBD, MⅠ and MⅡ stages, and reached the peak at MⅡ stage, which was extremely significantly higher than GV and GVBD stages (P<0.01). GPR50 protein was mainly expressed on the membrane at GV stage, and then dispersed to cytoplasm gradually with the development of maturation, and high brightness dispersion expression was found at MⅡ stage. The above results indicated that GPR50 gene might play an important role in the meiosis of yak oocytes, and provided for the study on the function and mechanism of GPR50 in yak oocytes.

The aim of this study was to investigate the effects of hormone dose, interval of superovulation and times of repeated superovulation on superovulation in Wagyu cattle, in order to provide evidence for establishing a stable and efficient repeated superovulation protocol. 63 Wagyu cattles aged 15-18-month-old were selected as superovulation donors. Among them, 17 donors were randomly divided into two groups, injected with a total dose of 200, 200, 200 mg FSH and a total dose of 200, 220, 240 mg FSH, respectively. Repeated superovulation was performed for 3 times with an interval of 30 days, to study the effect of hormone dosages on superovulation. 24 donors were randomly divided into three groups, each group was injected 200 mg dose of FSH for 3 consecutive times superovulation for 60, 45 and 30 days treated intervals, respectively, to study the effect of interval time of hormone injection on superovulation. 22 donors were injected with 200 mg FSH each time for 7 consecutive repetitions with an interval of 30 days, to study the effect of repetition times on superovulation. The results showed that the total number of embryos and the total number of available embryos obtained by the donors received 200, 220 and 240 mg doses of FSH were significantly higher than those obtained by the donors subjected to 200 mg dose of FSH treatment (P<0.05). By comparison with the superovulation interval of 60 days group, the number of available embryos produced at an interval of 45 days was significantly decreased (P<0.05), and the number of available embryos obtained by the donors in the 30 days interval group was also decreased, but there was no significant difference (P>0.05). There was no significant difference in the number of available embryos when repeated superovulation was less than 6 times (P>0.05). In summary, the effect of 200, 220 and 240 mg doses of FSH on repeated superovulation was better than that of 200, 200 and 200 mg dosage group. Longer superovulation interval was benefit to the recovery of reproductive tract and the available of available embryos, but the repetitious superovulation at 30 days interval could also get satisfactory results. The suitable number of superovulation times was 5 to 6 when the interval was 30 days for high intensive repeated superovulation.

In terms of genome research, the whole genome sequencing technology has developed from the first generation sequencing technology to the third generation sequencing technology. Compared with traditional methods, whole genome sequencing has the advantages of more comprehensive, more accurate and more efficient. With the development of sequencing technology and the reduction of cost, whole genome sequencing (WGS) technology has gradually become the most commonly used technology in genome research. The whole genome sequencing has made many achievements in the origin and evolution of livestock and poultry, gene mining of important economic traits, molecular breeding and so on. Through whole genome re sequencing, the regions where copy number variation (CNV) occurs and single nucleotide polymorphism (SNP) variation can be found, which can enrich the existing CNV and SNP database, and provide reference for disease resistance, growth, appetite, metabolic regulation, phenotype, environmental adaptation mechanism and important economic trait genes. This paper focused on the research progress of whole genome sequencing technology in major livestock and poultry, summarized the application of whole genome sequencing in the research of variety genetic diversity, population evolution mechanism and functional gene mining of livestock and poultry, and discussed the existing problems of whole genome sequencing, in order to provide reference for the protection of germplasm resources and molecular breeding practice of livestock and poultry.

The aim of this study was to investigate the effect of Hericium erinaceus polysaccharide (HEP) pretreatment of RAW264.7 cells on the replication of Muscovy duck reovirus (MDRV) and its mechanism, and dissect the mechanism of HEP in regulating MDRV-induced immunosuppression in Muscovy ducks from the Toll-like receptor 3/interferon-β TIR domain adaptor protein (TLR3/TRIF) signaling pathway. The trial included blank control group (BCG), viral infection control group (VCG), HEP control group (HCG) and HEP prevention viral infection group (HPG). The protein expression levels of TLR3, TRIF and tumor necrosis factor receptor-associated factor 6 (TRAF6) in RAW264.7 cell at 12 and 24 h of MDRV infection was detected by Western blotting. After virus infection for 24 h, the contents of tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), IL-6, IL-1β and interferon β (IFN-β) in the cell supernatant culture medium of each group were detected by ELISA. The TCID₅₀ detection results showed that the TCID₅₀ of MDRV was 10^3.46. At 12 h after infection, the cells did not change significantly, 24 h after infection, the cells became round, and 36 h after infection, a large number of cells died. The replication results of MDRV in RAW264.7 cells showed that within 12-24 h of virus infection, the expression of σNS increased, and at 24-36 h, the expression of σNS was maintained at a high level. Western blotting results showed that compared with blank control group, the expression levels of TLR3, TRIF and TRAF6 proteins in VCG group were significantly increased (P<0.05) after 12 and 24 h infection, and TRIF protein in HCG group were significantly increased (P<0.05) after 24 h infection. When compared with infected group, the expression of σNS, TLR3, TRIF and TRAF6 proteins were significantly decreased (P<0.05) after 12 and 24 h infection in the HPG group. ELISA results showed that compared with blank control group, the contents of TNF-α, IL-6, IL-1β and IFN-β in the culture medium of VCG group were significantly increased (P<0.05). When compared with infected group, the contents of TNF-α, IL-6 and IL-1β in the cell culture medium of HPG group were significantly decreased (P<0.05), while the content of IFN-β was significantly increased (P<0.05). The results showed that HEP could regulate MDRV infection to induce TLR3 signal transduction pathway activation, inhibit the overexpression of TNF-α, IL-10, IL-6 and IL-1β of downstream products of TLR3 signal transduction pathway, and up-regulate the expression of IFN-β in RAW264.7 cells, thereby inhibiting the replication of MDRV in RAW264.7 cells.

To obtain the monoclonal antibodies against Newcastle disease virus (NDV) M protein, the gene encoding the protein was cloned, the recombinant protein was expressed and purified. Mice were immunized with recombinant M protein, and an appropriate ELISA screening method was established. After the serum titer of mice was determined by ELISA, the mouse spleen cells with the highest serum antibody titer were screened and fused with SP2/0 cells. Hybridoma cell lines that could stably produce monoclonal antibodies against NDV M protein were obtained. Immunofluorescence, Western blotting, subclass identification, chromosome count of hybridoma cells, preparation of mouse ascites, and titer determination of monoclonal antibody in ascites were carried out. The results of PCR, recombinant plasmid sequencing and double enzyme digestion showed that this study successfully amplified the NDV M gene, which was about 1 095 bp in size. SDS-PAGE and Western blotting analysis showed that the recombinant M protein was successfully expressed with about 60 ku molecular weight, and it could react with positive serum of NDV. In the established ELISA screening method, the optimal working concentration or dilution of recombinant M protein, His tagged protein and antibody were 0.5 μg/mL, 0.5 μg/mL and 1∶256 000, respectively. Immunofluorescence, Western blotting and subclass identification of the antibody showed that the hybridoma cells produced antibody could specifically bind to NDV SG10 strain and recombinant M protein, and its light chain was κ and heavy chain was IgG2A. Chromosome counts of C9-G2 and D3-F2 hybridioma cells were 97 and 101, respectively. The ELISA titers of the supernatant of the hybridioma cell lines were both 1∶6 400. And the ELISA titers of ascites were 1∶409 600 and 1∶102 400, respectively. In this study, monoclonal antibodies against NDV M protein were successfully prepared, which could provide tools for further study of the function of M protein.

Influenza virus is a kind of RNA virus which is harmful to human and animal health. Its effective replication in host cells cannot be achieved without the assistance and support of host protein acidic nuclear phosphoprotein 32 family member A (ANP32A) and virus RNA polymerase. Viral RNA polymerase consists of three proteins:PB1, PB2 and PA, and the strongest interaction between ANP32A and viral RNA polymerase requires the participation of these three proteins. ANP32A is a member of the acidic leucine-rich nuclear phosphoprotein 32 (ANP32) family. It has been identified as a key host factor supporting the activity of viral RNA polymerase in the nucleus and plays an important role in Influenza virus replication. The species-specific difference of ANP32A determines the host range of viral RNA polymerase:A unique 33 amino acid sequence exists in avian ANP32A (avANP32A), but lacks this amino acid sequence in mammalian ANP32A. The 33 amino acid sequences unique to avANP32A can enhance the function of ANP32A, thus increasing the polymerase activity of Avian influenza virus (AIV). AIV cannot make effective use of short ANP32A (that is, ANP32A lacking a unique 33 amino acid sequence), so mammalian ANP32A cannot support avian characteristic polymerase activity. However, inserting these 33 amino acids into human ANP32A (huANP32A) can promote its support for AIV polymerase. Adaptive mutation of Influenza virus can also enhance the transmission and pathogenicity of AIV in mammals. E627K mutation often occurs when AIV adapts to mammals to enhance its replication ability in mammals. This review mainly introduced the effect of host protein ANP32A on replication and transcription of Influenza virus and the mechanism of adaptive mutation of Influenza virus, and briefly discussed the molecular mechanism of the interaction between ANP32A and polymerase on Influenza virus cross-species infection.

The purpose of this study was to obtain recombinant G protein of Bovine respiratory syncytial virus (BRSV) and identify its reactogenicity. The nucleotide sequence of G gene was found from NCBI, its antigenic region was analyzed, and a pair of specific primers was designed with Primer Premier 5.0 software. The BRSV G gene fragment was amplified by RT-PCR and ligated to the cloning vector pMD19-T, and then it was identified by double enzyme digestion and sequenced. The gel was recovered from the target fragment digested by double enzyme, then the recombinant plasmid pET-32a-G was constructed and transformed into E. coli BL21(DE3) competent cells. The protein induced by IPTG was purified by Ni-IDA affinity chromatography and its concentration was determined, then the protein was analyzed and identified by SDS-PAGE and Western blotting. The results showed that the G gene with the size of 567 bp was successfully cloned and the soluble recombinant protein with molecular weight of 40 ku was obtained. After optimizing the induction conditions, the expressed product was identified by SDS-PAGE. The protein expression was the highest at 16 ℃, IPTG concentration 1.2 mmol/L and induction for 4 h. Through Western blotting detection, it found that the recombinant protein could react with goat BRSV standard positive serum specifically, indicating that the protein had reactivity. In summary, BRSV G protein was successfully expressed and purified in this study, which laid a foundation for the establishment of indirect ELISA detection of BRSV antibody and the development of BRSV subunit vaccine.

H9N2 subtype Avian influenza virus (AIV) is a low pathogenic AIV. However, it is widely prevalent and spreads quickly, and can decline the performance of infected poultry, bringing great economic losses to the poultry industry. H9N2 subtype AIV can cause severe immunosuppression in the infected poultry, making it easier to be co-infection with upper respiratory tract bacteria and digestive tract bacteria. The co-infection with bacteria results in increasing of pathogenicity of H9N2 subtype AIV, and enhanced ability of bacterial adhesion and colonization, leading to a significant elevation in poultry mortality. Besides, H9N2 subtype AIV can co-infect with avian Infectious bronchitis virus, avian Infectious bursal disease virus, and Newcastle disease virus, etc., and there may be synergistic or antagonistic effects when the virus invades, thereby mutually promoting or inhibiting viral replication and shedding. Moreover, H9N2 subtype AIV is prone to mutation or genetic recombination with other subtypes of Influenza viruses to produce new influenza strains that may infect humans, posing a great threat to public health worldwide. This paper summarized the research advance of mixed infection of H9N2 subtype AIV and other pathogens. By expounding the synergistic or antagonistic effects between H9N2 subtype AIV and bacteria or other viruses, it is hoped to provide a reference for the prevention and treatment of H9N2 subtype AIV associated co-infection in the clinic.

The aim of this study was to construct a lactic acid bacteria (LAB) expression vector to develop a live bacterial vaccine of Avian leukosis virus subgroup A (ALV-A) for the prevention of avian leukemia. In this experiment, an anchored expression vector was used to construct two recombinant Lactobacillus plantarum (L. plantarum) expressing gp85 and gp85-DCpep genes. Using the genomic DNA of DF-1 cells infected with ALV-A as a template, PCR was used to amplify the encoding gene gp85 of ALV-A Gp85 protein, then the DC-targeting peptide (DCpep) encoding gene and gp85 gene were fused by the same method to obtain the gp85-DCpep gene. Then the recombinant plasmids pSIP409-pgsA’-gp85-DCpep and pSIP409-pgsA’-gp85 were constructed by seamless cloning. After sequencing identification, the plasmids were electroporated into L. plantarum NC8 competent cells to obtain recombinant L. plantarum. After induction by SppIP, the cells in logarithmic growth phase were collected. The protein samples of cell membrane were obtained by reverse freezing and thawing. Afterwards, the expression products were analyzed and identified by Western blotting. The results showed that the gene fragments gp85-DCpep and gp85 were successfully obtained while there was no mutation or loss in the recombinant plasmids pSIP409-pgsA’-gp85-DCpep and pSIP409-pgsA’-gp85. All of them were successfully transferred into L. plantarum NC8 competent cells. The expression products were detected by SDS-PAGE and Western blotting, and the bands were visible at 48.3 ku for pgsA’-gp85-Dcpep and at 47 ku for pgsA’-gp85. In this study, the recombinant plasmids pSIP409-pgsA’-gp85-DCpep and pSIP409-pgsA’-gp85 were successfully constructed. The recombinant L. plantarum NC8-pSIP409-pgsA’-gp85-DCpep and NC8-pSIP409-pgsA’-gp85 were obtained and successfully expressed as well as had the reactionogenicity, which laid a good foundation for further study on the protective mechanism of the two recombinant L. plantarum strains against ALV-A infection.

In order to clarify the main bacterial pathogens causing respiratory symptoms of calves in a large-scale dairy farm in Shihezi area and their biological characteristics, 39 nasal swabs and 39 anal swabs of 2-6 months old calves were collected. The biological characteristics of the isolated strains were analyzed by bacterial isolation and culture, morphological observation, biochemical analysis, PCR amplification of 16S rRNA and khe genes, drug sensitivity test and mouse pathogenicity test. The results showed that 3 of the isolated strains formed purplish red colonies with sedimentation rings on the MIAC plate (1 nasal swab and 2 anal swabs), and were Gram-negative Brevibacterium and suspected to be Klebsiella pneumoniae by microscopic examination. The automatic microbiological analysis system showed that the similarity between isolates and Klebsiella pneumoniae was 96%. PCR amplified 16S rRNA sequencing results showed that the nucleotide homology between the isolates and Klebsiella pneumoniae in the GenBank database was between 96.5% and 99.8%, and the specific gene khe of Klebsiella pneumoniae was positive and the similarity was over 99%. The results of drug sensitivity showed that the isolated strains were resistant to penicillins, cephalosporins, first-generation and second-generation aminoglycosides, tetracyclines, first-generation macrolides, sulfonamides, polyenes and lincamides. They were sensitive to the third generation of aminoglycosides, the second generation of macrolides, chloramphenicols, polypeptides and quinolones, and showed different degrees of multi-drug resistance. The pathogenicity test showed that the isolated strains could lead to the death of mice in varying degrees, and the nasal swab isolates were highly pathogenic. This study successfully isolated and identified three strains of Klebsiella pneumoniae causing respiratory symptoms of calves in large-scale dairy farms in Shihezi area, clarified some biological characteristics of the isolated bacteria, and provided technical support for detection, diagnosis and clinical treatment of bovine Klebsiella pneumoniae in Xinjiang.

In order to explore the effects of hypertonic stress on gene expression and growth of efflux pump and outer membrane protein of multidrug-resistant bacteria, growth curve and relative adaptability were used to compare the effects of different salt stress conditions on resistant Escherichia coli QL15 and sensitive Escherichia coli ATCC 25922. RT-PCR was used to investigate the difference of gene expression of efflux pump and outer membrane protein under salt stress. The results showed that Escherichia coli QL15 was a high-level multidrug-resistant bacterium, and the most serious resistance to spectinomycin and enrofloxacin was 32 times of the standard MIC. When cultured independently, Escherichia coli QL15 was more sensitive to salt stress than Escherichia coli ATCC 25922, and under 6.0% NaCl stress, Escherichia coli QL15 grew slowly within 10 h, but its concentration was higher at 24 h. When cultured in mixture, Escherichia coli QL15 had higher adaptability to 6.0% NaCl stress, and had more competitive advantage in vitro culture. Escherichia coli QL15 carried 15 drug resistance related genes in 5 categories, including 8 efflux pump genes and 3 outer membrane protein genes. The expression levels of 5 genes in Escherichia coli ATCC 25922 at 6.0% NaCl concentration were about 50% lower than that in 3.5% NaCl concentration (P<0.05). However, the expression of Escherichia coli QL15 were significantly up-regulated at 6.0% NaCl concentration except acrB and ompF genes (P<0.05). It was speculated that the higher adaptability of Escherichia coli QL15 to salt stress might be related to the expression of membrane associated proteins.

Canine pyoderma is a purulent skin disease caused by methicillin-resistant Staphylococcus pseudintermedius (MRSP) infection. Staphylococcus is a kind of bacteria that can infect both human and animals, and cause various suppurative diseases. Among them, MRSP as an animal derived Staphylococcus, can be a reservoir of drug-resistant genes and it can transmit drug-resistant genes to human beings via environmental factors or food chain. In recent years, the cases of skin diseases caused by MRSP have increased significantly which challenges infection control. In this paper, the drug resistance and disinfectant resistance of pathogenic bacteria of canine pyoderma were reviewed. In terms of the pathogenic mechanism of canine pyoderma, the mechanism of MRSP leading to infection by destroying the function of cellular immune system were summarized, the significant drug resistance and related drug resistance genes of MRSP in many areas were mainly described, such as mecA and cat genes. The resistance to disinfectants and its mechanism in MRSP, including efflux pump, were introduced. In order to avoid the common resistance interfering with the treatment, the relationship between the resistance to disinfectants and the resistance to antibiotics from the acquired resistance mediated by mobile genetic elements and inherent resistance depending on bacterial cell structure in pathogenic bacteria were systematically analyzed, in order to find a scientific and reasonable treatment for canine pyoderma, and provide reference and theoretical support for clinical medication of canine pyoderma.

In order to explore the ultrastructural characteristics of pathological damage in mice induced by Escherichia coli(E. coli) highly pathogenic virulence island (HPI) from Saba pig, the E. coli HPI positive strain (HPI⁺) and E. coli HPI gene deletion strain (^ΔHPI) preserved in the laboratory were resuscitated and cultured. Then the Kunming mice were infected with E. coli HPI⁺ and E. coli ^ΔHPI strains by intraperitoneal inoculation, and the 50% lethal dose (LD₅₀) of the strains was detected. The HE staining and transmission electron microscopy were used to observe and analyze the ultrastructural characteristics of pathological damage in mice. Immunohistochemistry was used to mark the distribution of interleukin-1β(IL-1β) positive cells in the liver and kidney tissues of infected mice to reflect the difference in inflammation levels caused by the strain E. coli HPI⁺ and E. coli ^ΔHPI. The results showed that the LD₅₀ of E. coli HPI⁺ and E. coli ^ΔHPI strains were 1×10^7.39 and 1×10^8.62 CFU/mL, respectively. HE staining showed that after E. coli infection in mice, pathological changes, such as swelling and degeneration of liver cells, hepatic sinus congestion, renal interstitial congestion, renal tubular epithelial cells degeneration and shedding, were observed. The ultrastructural changes showed that the complete morphology of liver cells disappeared, the nuclei showed irregular morphology, mitochondrial abnormalities, ribosome shedding on rough endoplasmic reticulum, and smooth endoplasmic reticulum hyperplasia. Most renal tubular epithelial cells showed nucleus pyknosis, some of the nucleoli were shifted and the volume increased, the foot processes were fused, and the mesangial cell gap was widened. In addition, the edema in liver and kidney of E. coli HPI⁺ infection group was more obvious than that of mice infected with E. coli ^ΔHPI strain. The results of immunohistochemistry showed that IL-1β was mainly expressed in hepatocytes and pericentral veins, renal interstitial cells and renal tubular epithelial cells after E. coli infection in mice, and IL-1β expression in E. coli HPI⁺ infection group was higher than that of the E. coli ^ΔHPI infection group. To sum up, E. coli HPI from Saba pig could regulate the pathogenicity of E. coli to mice. The regulation of HPI could make the pathological changes and ultrastructural changes of liver and kidney of mice more obvious, and increase the inflammatory response of mice.