Alternative splicing(AS) refers to the process of selective splicing across exons or splice junctions during individual development or cell differentiation to produce tissue-or development-stage specific mRNA. Through AS process, organisms can produce many different protein isomers from a single gene and finally increase the proteome diversity. Therefore, AS plays an important role in the regulation of animal growth, development and physiological metabolism. This article not only summarized the types and the detection methods of alternative splicing, but also highlighted its impact on various economic traits of livestock and poultry, and prospected its application prospects in livestock and poultry breeding. This article could provide new ideas for the improvement of livestock and poultry breeding.

The meat quality is influenced by many factors, among which intramuscular fat (IMF) is one of the most prominent factors. IMF content is closely related to the tenderness and flavor of meat. Numbers of studies suggested that gut microbiota and its functional metabolites (such as short fatty acids, bile acids, lipopolysaccharides, trimethylamines, tryptophan and their derivatives) played an important role in host fat metabolism. In this review, we present the role of gut microbiota and its functional metabolites in regulating fat metabolism and IMF deposition. This paper would provide new insights and feasible ways through nutrition regulation to increase IMF deposition and improve meat quality.

Lipids are a kind of substance which can store and provide energy for animals. A large number of studies have confirmed that the change of lipids in diets can alter the expression of microRNAs (miRNAs) and thus affect the biological activities of the bodies. This review mainly summarizes the regulation of fatty acids on the expression of miRNAs in mammary gland of dairy cows, from the aspects of bovine mammary miRNAs expression characteristics, miRNAs that regulate milk fat synthesis, and the changed bovine mammary miRNAs in response to exogenous addition of fatty acids. All these works may be beneficial for the establishment of the connection between nutrients and mammary function, and provide information for the related research in the future.

Under laboratory conditions, the number of cultured microorganisms accounts for about 1% of the total number of microorganisms in nature, which limits people's understanding and utilization of 99% of the unknown microorganisms. However, relevant researches show that those "uncultured microorganisms" can be developed and utilized, and the uncultured microorganisms are the main body of the unknown microorganisms. The microbial culturomics explored the application of multiple culture conditions and long-term culture, it was combined with Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and 16S ribosomal RNA (rRNA) sequencing to identify all kinds of microorganisms on a large scale. At the same time, whole-genome sequencing (WGS) and Metagenomics sequencing technology were used to analyze unknown microorganisms in depth. In this paper, the latest progress of culturomics in the ruminant gastrointestinal tract, poultry cecum, and livestock nasal microflora in recent years was reviewed, and the feasibility of applying the method of microflora culturomics in animal disease prevention and control was discussed. As a new research idea, culturomics has some immature aspects, but its development prospect is very broad. The complementary of microflora culturomics and other research methods have gradually become a breakthrough in the development of veterinary microbiology.

Streptococcus, Staphylococcus aureus, and Escherichia coli are the three major pathogens causing mastitis in dairy cows, and Streptococcus agalactiae (S.agalactiae) in Streptoco-ccus is one of the important pathogens causing mastitis in dairy cows, which accounts for around 56.25% of the incidence of sub-clinic mastitis. The process of S.agalactiae invasion mammary glands of dairy cows mainly includes:infection, adhesion, invasion, and then damage the mammary tissue and entail the inflammation. The virulence factor of S. agalactiae has the function of attaching and invading the mammary gland epithelial cells so that the bacteria could form the biofilm on the surface. After infection, the irritation of the mammary tissues by infectious processes serves to the immune response. In this paper, we summarized the types, mechanism, and regulation process of S. agalactiae virulence factors which plays a major role in the invasion of mastitis, aimed to show the activity of S. agalactiae by inhibiting the activity of its related virulence factors in preventing dairy cow mastitis and provide new ideas for the treatment.

Staphylococcus aureus(SA)bovine mastitis is a chronic and subclinical infectious disease, which is hard to control. It brings unpredictable losses to dairy-farming industry. Immune escape effect of SA plays an important role in the occurrence and development of mastitis, and some virulence factors are mostly associated with the immune evasion of bacteria. In this paper, some important immune evasion mechanisms of SA in recent researches were reviewed in order to provide theoretical reference for further prevention and treatment of mastitis in dairy cows.

The objective of this study was to identify Sus scrofa circular RNAs (circRNAs) systematically and analyze their molecular characteristics. The samples from 16 developmental stages of Landrace, Tongcheng and Wuzhishan pigs (Embryonic 33, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 and 105 days as well as postnatal days 0 and 10) and 9 different tissues (longissimus dorsi, heart, spleen, lung, liver, kidney, stomach, small intestine, and leg muscle) were collected. RNA was extracted from samples and then mixed with equal quantities. A strand-specific RNA-seq library was constructed and sequenced on the Illumina Genome Analyzer II sequencing platform. A total of 118 million reads were obtained. The circRNAs were identified using CIRI2 software and their molecular characteristics were analyzed using bioinformatics tools. A total of 8 486 circRNAs were identified. These circRNAs mainly located on chromosome 1, 6 and 13, and 91% of them were located on the exonic regions in genome. The average length of these circRNAs was 350 nt and GC content was 47.11%. The number of circRNAs was significantly positively correlated with the number of mRNAs (R=0.84, P=1.285×10^-5) on each chromosome. Finally, three circRNAs (sus-MYH2_0025, sus-CDK13_0002, and sus-FANCL_0003) that were associated with skeletal muscle growth and development were identified. The results suggested that sus-MYH2_0025 had higher tissue-specificity, sus-CDK13_0002 was highly conserved among swine, human and mouse, and sus-FANCL_0003 was highly conserved between swine and rat. The transcriptome data was used to systematically identify circRNAs in pig genome and their molecular characteristics were analyzed, which provided rich information for the study of circRNA functions and mechanisms, and provided new potential candidate genes for molecular breeding of pigs.

The purpose of this study was to investigate the effect of SRD5A2 gene on the expression of genes related to goat lambing traits. In this study, 3 healthy (2-3 years old) and female Qianbei Ma goat with a weight of approximate 32 kg were selected. Ovary tissue was collected and granulosa cells were successfully cultured. Four pairs of siRNA interference sequence of SRD5A2 gene sequence and a pair of negative control sequence were designed by online software. After connecting pGPH1/GFP/Neo vector, the constructed interference vectors were transfected into ovarian granulosa cells of Qianbei Ma goats. The interference vector with optimal interference efficiency was screened by qRT-PCR, and its effect on the expression of SRD5A2 gene was detected. The effects of silencing SRD5A2 gene on the expression of bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9), bone morphogenetic protein-1B (BMPR-1B) and follicle-stimulating hormone lactin (FSHβ) were investigated. In this experiment, the interference vector of SRD5A2 gene was successfully constructed on the basis of cultivating ovarian granulosa cells of Qianbei Ma goats. shRNA-SRD5A2-1 interference vector with the optimal interference efficiency was screened out (P<0.01). After inhibiting SRD5A2 gene expression, the expression levels of BMP15, BMPR-1B, GDF9 and FSHβ were significantly reduced. The expression level of BMP15 was extremely significantly lower than that in shRNA-NC control group (P<0.01), and the expression levels of BMPR-1B, GDF9 and FSHβ were significantly lower than those in shRNA-NC control group (P<0.05). In this study, the constructed SRD5A2 gene interference vector was successfully transfected into ovarian granulosa cells. The silencing of SRD5A2 gene can inhibit the expression of genes related to lambing traits, which indicate that SRD5A2 gene is closely related to lambing traits of Qianbei Ma goats, the result will provide a basis for further studies on the regulatory mechanism of SRD5A2 gene on lambing traits in Qianbei Ma goats.

The purpose of this study was to screen the genome-wide selection signals of Liaoning cashmere goat and Inner Mongolia cashmere goat. Based on the Illumina Caprine 50K SNP chip, Liaoning cashmere goat, Inner Mongolia cashmere goat and Huanghuai goat were genotyped, and the common 50 010 SNPs were obtained after quality control. Using population differentiation coefficients Fst and XP-EHH, Huanghuai goats were used as reference populations to detect selection signals for Inner Mongolia cashmere goats and Liaoning cashmere goats. The top 5% of Fst and XP-EHH values was used as the threshold. The result showed that 501 candidate genes were selected by Fst and 145 candidate genes were selected by XP-EHH in cashmere goats. Based on these SNPs, we detected 12 SNPs under strong selection by Fst and XP-EHH. After gene annotation, 21 candidate genes were identified, such as EXOC4, ASIC2, PCDH9, RHBDD1, IRS1 and PDE10A. These genes were found to be mainly enriched in PI3K-Akt signaling pathway, protein digestion and absorption and relaxin signaling pathways. The study indicated that genes associated with cashmere traits were subjected to strong artificial selection pressure during domestication of the cashmere goats. These findings also help us better understand the selection progress of cashmere goats and provide a new theoretical basis for the protection and utilization of germplasm resources of Chinese cashmere goat breeds.

This study aimed to analyze the microbiota composition and diversity and the distribution of Bacteroides in duodenal, jejunal, ileal and caecal contents in healthy ducks. The contents of duodenum, jejunum, ileum and cecum were collected aseptically from 20 healthy adult Gaoyou ducks (70-day-old, half male and female), and the bacterial DNA of intestinal contents were extracted. The IonS5TMXL platform was used for 16S rRNA high-throughput sequencing, the microbiota structure and abundance, as well as the distribution of Bacteroides were analyzed. The results showed that the microbiota abundance in duodenal and jejunal contents was significantly higher than that in ileal and caecal contents (P<0.05), the microbiota diversity in ileal contents was the lowest. The microbiota structure in duodenal and jejunal contents was similar, they were different from that in ileum, and especially cecum. The dominant phyla in the intestinal contents of healthy duck were Bacteroidetes, Firmicutes, Proteobacteria and Fusobacteria. The relative abundance of these phyla were different in different intestinal segment contents. The different microbial species were colonized in different intestinal segments, and the differential phyla in duodenum, jejunum, ileum and cecum were Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes, respectively. There were 28 kinds of Bacteroides species in duck intestine, among them, B. acidifaciens, B. barnesiae, B. caccae, B. caecicola, B. coprocola, B. sp and B. luti were significantly clustered in the cecum, and B. caecigallinarum, B. plebeius and B. barnesiae were dominantly colonized in duck cecum. The results indicate that intestinal space significantly affects the microbiota abundance and diversity, and different intestinal segments were colonized by differential microorganism, which is consistent with the intestinal segment niche and function. In the cecum, the dominant colonization of Bacteroides may be related to its physiological and biochemical functions.

The purpose of this study was to obtain the structure and tissue expression of the StAR gene, and to initially explore its effect on the development of testis in waterfowl. The hypo-thalamus and testis of Shanma duck and Shitou goose were selected as materials to clone the StAR genes, and their expression patterns in different tissues of Shanma duck and Shitou goose were detected by real-time fluorescent quantitative PCR, and the differential expression of the genes in goose testis tissues in different periods were further compared. In this study, three transcripts of the duck StAR gene (dSTAR-A, dSTAR-B, and dSTAR-C) were obtained. The coding sequences of dSTAR-A and dSTAR-B were the same, encoding 283 amino acids, while dSTAR-C encoded 238 amino acids. Two transcripts of goose StAR gene (gSTAR-A and gSTAR-B) were obtained, both encoding 283 amino acids. The comparative analysis showed that the amino acid sequence of waterfowl StAR was highly homologous with other birds and highly conserved among species. The StAR gene was expressed in various tissues of ducks and geese, of which the highest expression level was in the testis, and it had higher expression levels in abdominal fat, breast muscle and leg muscle. Comparing the testis of male geese in different periods, it was found that the expression level of StAR gene was extremely significantly higher at age 3 years than that at age 1 and 52 days(P<0.01), and was extremely significantly higher in breeding period of adult male geese than that in restoration period (P<0.01). The results indicates that StAR gene may be a key gene for goose testis development and participate in the regulation of reproductive performance of adult male geese.

The objective of this study was to explore the influence factors of SCC change pattern of Holstein cows in Jiangsu. Based on the division of 9 SCC change patterns, 253 706 DHI records of Holstein cows from 2017 to 2019 in 12 dairy farms in Jiangsu province were analyzed, and the least squares model was used to explore the influence of different farm size, parities, calving seasons, calving intervals, and 305-days milk production on SCC change pattern. The results showed that the distribution of SCC patterns at different levels of each factor was significantly different (P<0.01). Among them, the percentage of lower to maintain pattern was the highest, and lower to infection pattern was the lowest for cows in farm with more than 5 000 cows. The percentage of infected to maintain pattern was the highest, and lower to maintain pattern was the lowest for cows in farm with less than 1 000 cows. The percentage of lower to maintain pattern was the highest, and infected to healed pattern was the lowest for cows with parity 1. The percentage of lower to infection pattern was the highest, and lower to maintain pattern was the lower for cows with parity 5. The percentage of lower to infection pattern was the highest, and lower to maintain pattern was the lowest for cows calving in spring. The percentage of susceptible to reduce pattern was the highest, lower to infection pattern was the lowest for cows calving in summer. The percentage of lower to infection pattern was the highest for cows with calving interval more than 441 d, and lowest for cows with calving interval with 300-400 d. The percentage of lower to infection pattern was the highest for cows with 305-days milk production of 3 000-5 000 kg. The percentage of lower to maintain pattern was the highest, and infected to healed pattern was the lowest for cows with 305-days milk production was 9 001-11 000 kg. The results indicate that different farm sizes, parities, calving seasons, calving interval, and levels with 305-days milk production have certain influences on the distribution of the SCC change patterns in Holstein cows. The results provide references for the prediction of subclinical mastitis in Holstein cows.

This study aimed to investigate the effects of AMPK activators metformin (Met) and acadesine (AICAR) on sheep semen cryopreservation. Firstly, Met and AICAR with different concentrations (0, 100, 200, 300, 400, 500 μmol·L^-1) were added into the frozen diluent. After freezing and thawing, the optimal concentration (400 μmol·L^-1 Met, 200 μmol·L^-1 AICAR) were selected based on sperm motility, motion performance and membrane integrity; Secondly, the collected semen was froze using different frozen diluents (control group:diluent; Met group:diluent + 400 μmol·L^-1 Met; AICAR group:diluent + 200 μmol·L^-1 AICAR). After thawing, the sperm AMPK protein expression, acrosomal enzyme activity, metabolic index, mitochondrial function and antioxidant enzyme activity were examined. The results showed that the addition of 400 μmol·L^-1 Met and 200 μmol·L^-1 AICAR could significantly improve sperm motility, motion performance and membrane integrity(P<0.05) after thawing. In 400 μmol·L^-1 Met group, the total sperm motility, acrosome integrity rate and plasma membrane integrity rate were 43.20%, 91% and 46%, respectively. Compared with the control group, the level of AMPK phosphorylation in the sperm of the Met and AICAR groups was significantly increased after thawing (P<0.05), the acrosome enzyme activity of sperm was significantly increased(P<0.05); the pyruvate level was significantly decreased (P<0.05), the lactate dehydrogenase activity, lactic acid content, and ATP content were significantly increased(P<0.05). Compared with the control group, the dilution of Met group and AICAR group was more conducive to maintain mitochondrial membrane potential (P<0.05), increase ATPase and antioxidant enzyme activity (P<0.05). The addition of appropriate concentrations of Met and AICAR could improve semen quality of cryopreservation in sheep.

The aim of this study was to screen the interaction proteins and receptor of CART (cocaine and amphetamine regulated transcript peptide), which was a key negative regulatory protein of follicular development in bovine. The both ovaries of 3 healthy cows were collected, after sepa-rating follicles, the GCs (granulosa cells) were scraped and mixed. The membrane proteins were extracted by transmembrane protein extraction kit. The protein G agarose magnetic beads were used to incubate with recombinant CART protein and Anti-CART antibody (n=3). CART-protein complex was eluted and analyzed by LC-MS/MS. The obtained proteins were filtered, and function cluster was analyzed by bioinformatics technology, subcellular localization and transmembrane region analysis of these proteins were performed by CELLO V2.5 software. The intracellular and extracellular structural segments of GPCRs (G-protein-coupled receptors) with 7 times transmembrane were obtained and analyzed by ProtScale software. The GPCRs were used as candidate receptors of CART, their structural characteristics were consistent with the structural characteristics of neuropeptide and receptor binding. The 149, 557 and 298 interaction proteins were identified by immunoaffinity chromatography (n=3), respectively, and total 620 proteins were identified after screening and elimating repetitive proteins. Functional enrichment analysis of multiple database sets showed that GNAS, GNG8 and JUP involved in signal transduction activity, HSD3B involved in steroid biosynthesis, OPA1 and S100A11 involved in regulation of cell apoptosis or proliferation, and ERH involved in Notch signaling pathway, they were interaction proteins and closely related to follicular development in bovine. A total of 156 membrane proteins were obtained by subcellular localization and transmembrane domain analysis, which was 20.53% of total protein. Out of 156, 8 proteins with 7 times transmembrane were considered as GPCRs. The TMHMM analysis showed that the characteristics of ZMPSTE24, HM13 and GPR108 was consistent with the structural characteristics of neuropeptide receptor, their N-terminal was outside and C-terminal was inside. The analysis of PredictProtein binding site showed that there was a G-protein binding site in the intracellular loop of 5-6 transmembrane region of ZMPSTE24. As a candidate receptor for CART, ZMPSTE24 needs to be further verified by molecular interaction and functional tests. This study has laid a foundation for the identification of CART receptors in bovine follicles, and was of great significance to enrich the theory of regulation of follicular development in bovine.

This study aimed to explore the expression patterns of autophagy regulators Atg5 and Beclin1 in the early embryonic development and the effects of different embryonic production methods on the expression of the two factors. Female mice aged 6-8 weeks were subjected to superovulation and divided into 2 groups. The mouse oocytes of one group were collected, and cultured in vitro after parthenogenetic activation. The other group of female mice were caged with male mice (1:1), and the next day, the mouse fertilized eggs were collected for in vitro culture. Parthenogenetic activated embryos and naturally fertilized embryos were collected at 2 cell stage, 4-8 cell stage, mulberry embryo stage and blastocyst stage, respectively. RNA and protein were extracted, real-time fluorescence quantitative PCR, Western blot and other methods were used to detect the expression of key autophagy factors Atg5 and Beclin1. And indirect immunofluorescence was used to detect the expression and location of Atg5 and Beclin1 in mouse blastocysts. The results showed that Atg5 and Beclin1 were expressed in all development stages of naturally fertilized and parthenogenetic activated embryos in mice, and showed a high level in the early stage of embryonic development. The expression of Atg5 and Beclin1 were gradually reduced from the 2 cell stage in mouse naturally fertilized embryos. The expression levels of Atg5 and Beclin1 in parthenogenetic activated embryos were the highest in the 4-8 cell stage, which was extremely significantly different from the naturally fertilized embryos of the same period (P<0.01). From the 4 cell stage, the expression levels of Atg5 and Beclin1 in parthenogenetic activated embryos were higher than naturally fertilized embryos at all subsequent stages, the difference was extremely significantly different (P<0.01). In mouse blastocysts, the fluorescence of Atg5 and Beclin1 protein could be detected in the trophoblast cells and the inner cell mass, but the fluorescence intensity in the inner cell mass was higher than that in the trophoblast cells. In addition, the fluorescence intensity of Beclin1 protein in the inner cell mass of parthenogenetic activated embryos was higher than that in naturally fertilized embryos. Atg5 and Beclin1, the key autophagy factors, are expressed at different levels in the early development of mouse embryos from different sources. It is suggested that the regulation of autophagy on early embryonic development is related to embryo production modes. The results will provide a theoretical basis for further exploring the role of autophagy in the physiological regulation of mammalian embryo development.

Ovarian granulosa cells provide a special microenvironment for follicle formation and maturation through interaction with oocytes and their own secretion. A variety of harmful stimuli can cause granulosa cell apoptosis and metabolic disorders, reduce the quality of oocytes and have a negative impact on embryo formation. Zearalenone (ZEA) is a common cause of ovarian granulosa cells injury in the livestock industry, which is produced by mycotoxins, and lack of effective treatment drug. Therefore, in the current study zearalenone was used to induce ovarian granulosa cell injury and to explore the protective effect of caffeic acid on zearalenone-induced ovarian granulosa cell apoptosis in mice. Mouse ovarian granulosa cells were isolated by mechanical method, and indirect immunofluorescence was used to identify the isolated cells. MTT assay was used to determine the effect of caffeic acid on the activity of normal mouse ovarian granulosa cells.After granulosa cells were co-treated with caffeic acid (200, 100 and 50 μg·mL^-1) and ZEA for 24 hours, and control and ZEA group were set up at the same time, cell morphology and adherence were observed under a microscope. MTT was also used to detect cell viability. Caspase-3 mRNA expression level was detected by qRT-PCR. Cleaved-caspase-3 and cleaved-PARP protein expre-ssion levels were determined by Western blot. The results showed that positive FSHR staining appeared in cell cytoplasm of the test group, which confirmed that the isolated cells were mouse ovarian granulosa cells. The cell viability was above 90% which showed that caffeic acid had no toxic effect on granulosa cells. Compared with control group, ZEA group had smaller cell size, poor adherence, increased cell gap, and significant reduction in cell viability (P<0.001). Furthermore, the relative expression of caspase-3 mRNA, and cleaved-caspase-3 and cleaved-PARP protein level were significantly increased (P<0.001) compared with the control group. After caffeic acid treatment, cell gap was reduced, adherence was tight, cell viability was significantly increased (P<0.001). Caffeic acid significantly reduced zearalenone-induced increase in caspase-3 mRNA, and cleaved-caspase-3 and cleaved-PARP protein expression level (P<0.001). This study indicated that caffeic acid can restore granulosa cell viability by inhibiting ZEA-induced apoptosis.

Hepatocytes injury is the main pathological feature of fatty liver disease in cows during the perinatal period. The aim of this study was to investigate the preventive effect of monoammonium glycyrrhizinate (MAG) on cell damage induced by sodium oleate in hepatocytes of cows. Primary hepatocytes of cows cultured in vitro were divided into 3 groups according to different treatments:control group (untreated primary hepatocytes) and 2 experimental groups (model group, MAG group:add 0 mg·L^-1, 0.25 mg·L^-1 MAG in culture media respectively, for 12 h and then use sodium oleate at a concentration of 0.25 mmol·L^-1 to induce hepatocytes steatosis), and then the activity of hepatocytes were detected by CCK-8, the cell apoptosis rate was calculated by DAPI fluorescence staining, the content of ALT and AST in the culture solution were detected by ultraviolet colorimetry, the levels of intracellular fat deposition were analyzed by oil red O staining and imageJ. Real-time fluorescence quantification (RT-qPCR) was used to detect the mRNA expression of lipid metabolism-related genes PPARα,SREBP-1c,ChREBP,CPT1,CPT2,MTP and inflammation-related genes TNF-α,NF-κB,IL-1β,IL-6,IL-8. The experimental results showed that hepatocytes preconditioning with 0.25 mg·L^-1 MAG could improve the activity of model cells (P<0.05), and inhibit the apoptosis rate of model cells effectively (P<0.05). The release levels of ALT and AST in the cell culture supernatant of the MAG group were lower than those of the model group (P<0.05). At the same time, compared with the model group, the number of intracellular lipid droplets and the average lipid droplet area of hepatocytes treated with MAG were significantly reduced (P<0.05). And then the levels of lipid metabolism genes ChREBP, PPARα, MTP and inflammation related genes TNF-α, IL-1β, NF-κB, IL-6, IL-8 mRNA were significantly down-regulated in MAG group than the model group (P<0.05). The results showed that MAG could alleviate the impact and relieve lipid deposition induced by sodium oleate in hepatocytes by inhibiting the expression of genes related to lipid metabolism and inflammatory cytokines, which indicated that it could provide protection for the liver.

The experiment was performed to evaluate the effects of dietary metabolic energy (ME) and crude protein (CP) levels on production performance, nutrient digestibility, serum biochemical parameter, and organ indexes in furring male mink. One hundred eighty healthy male mink of (145±5) d were randomly divided into 6 groups with 30 replicates, one mink for each replicate. We adopted a completely randomized 2×3 factorial experiment, and the mink were fed 6 experimental diets with two CP levels (30.45% and 34.58%) and three ME levels (13.11, 14.94 and 16.76 MJ·kg^-1), the feeding experiment was completed from September 29, 2018 to December 10, 2018. The results showed that:1) During 175 to 213 days old, the body weight (BW) of the mink in the 34.58% CP group was significantly higher than those in the 30.45% CP group (P<0.01), and the BW of the mink in the 13.11 MJ·kg^-1 ME group was significantly lower than those in the other ME groups (P<0.01). At the age of 175-190 and 213 days, the BW of the mink in A (CP 30.08%, ME 13.03 MJ·kg^-1) group was significantly lower than those in other groups (P<0.01). At 205 day, the maximum value of BW of mink was observed in E (CP 34.86%, ME 15.08 MJ·kg^-1) group. 2) The fresh skin length and weight of mink in 34.58% CP group were significantly greater than those in the 30.45% CP group (P<0.01), and the fresh skin length of mink in 14.94 MJ·kg^-1 ME group was significantly greater than those in other ME groups (P<0.05).However, the fresh skin weight of mink in the 16.76 MJ·kg^-1 ME group was significantly greater than those in 13.11 MJ·kg^-1 group (P<0.01). The fresh skin length and weight of mink in E (CP 34.86%, ME 15.08 MJ·kg^-1) group was significantly higher than those in A (CP 30.08%, ME 13.03 MJ·kg^-1) and B (CP 30.46%, ME 14.80 MJ·kg^-1) groups (P<0.01). 3) The fat digestibility of mink in the 34.58% CP group was significantly higher than those in the 30.45% CP group (P<0.01). The fat digestibility of mink in the 13.11 MJ·kg^-1 group was significantly lower than those in other groups (P<0.01).The fat digestibility of mink in the group A (CP 30.08%, ME 13.03 MJ·kg^-1) and D (CP 34.17%, ME 13.19 MJ·kg^-1) was significantly lower than those in other groups (P<0.01). 4) The content of serum LDL in 30.45% CP group was significantly higher than those in 34.58% CP group (P<0.01), but the HDL concentration in serum was significantly lower than that in 34.58% CP group (P<0.01).The concentration of serum Alb, GLU, CHO, TG and HDL of mink in the 13.11 MJ·kg^-1 ME group were significantly lower than 16.76 MJ·kg^-1 ME group (P<0.01). The concentrations of serum Alb, GLU of mink in the group C (CP 30.80%, ME 16.99 MJ·kg^-1), E (CP 34.86%, ME 15.08 MJ·kg^-1), and F (CP 34.72%, ME 16.53 MJ·kg^-1) were significantly higher than other groups (P<0.05). The concentration of serum HDL of mink in group E (CP 34.86%, ME 15.08 MJ·kg^-1) and F (CP 34.72%,ME 16.53 MJ·kg^-1) was significantly higher than other groups (P<0.01). 5) The heart and liver indexes of mink in 30.45% CP group were significantly higher than those in 34.58% CP group (P<0.05).The heart index of mink in the 13.11 MJ·kg^-1ME group was significantly higher than those in other groups (P<0.05).The heart and liver indexes of mink in group A (CP 30.08%, ME 13.03 MJ·kg^-1) were significantly higher than those in group E (CP 34.86%, ME 15.08 MJ·kg^-1) (P<0.05). In conclusion, when the dietary CP level was 34.86% and the ME level was 15.08 MJ·kg^-1, mink could obtain better growth performance and fur quality, higher digestibility of nutrients.

The zoonotic protozoa Toxoplasma gondii is an opportunistic pathogen and distributes worldwide. Acute Toxoplasma infection causes serious pathological damages. Dense granule protein 1(GRA1) secreted by dense granule is an important component of Circulating antigen, which is an indication of acute toxoplasmosis. We aimed to use a monoclonal antibody against TgGRA1 to establish an enzyme-linked immunosorbent assay that targets antigen GRA1 in serum for acute toxoplasmosis diagnosis. First, the spleens of TgGRA1-His immunized mice were fused with SP2/0 cells,then we screened hybridomas that can constantly secret monoclonal antibody to the supernatant and injected them into mice to produce a large amount of mAbs. After the identification and purification of ascites, we choose one mAb as a capture antibody, HRP conjugated mouse anti-TgGRA1 polyclonal antibody as a detection antibody to develop sandwich ELISA. This method was used to detect samples from swine and mice artificially infected with Toxoplasma gondii. Besides, the results were compared with that of nPCR and two commercial kits to evaluate the efficiency of sandwich ELISA. We successfully got 4 mAbs with ascitic titers of 10⁶-10⁷, their subtypes are IgG1. Indirect fluorescent assay and Western blot showed that all of them can react specifically with TgGRA1.1G2 mAb and HRP conjugated mouse anti-TgGRA1 polyclonal antibody were used subsequently to establish sandwich ELISA for diagnosing acute infection. After optimization, sandwich ELISA can specifically detect 1.563 ng·mL^-1 GRA1 or 100 ng·mL^-1 ESA in serum. When detecting experimental animal samples, the sandwich ELISA exhibited the high consistency with the results of nPCR and showed higher efficiency than the commercial kits. In summary, we established a sandwich ELISA for acute toxoplasmosis diagnosis that captures one certain toxoplasma antigen GRA1, samples of artificially infected animals can be detected by this method, which makes acute toxoplasmosis diagnosis more reliable. It has guiding significance for clinical treatment of acute toxoplasmosis.

Coccidiosis infected by Eimeria spp. has a major economic impact on the poultry industry worldwide, resulting in high mortality, low growth, low productivity, and high control costs. Drug resistance of Eimeria spp. is a serious problem with the widespread use of anticoccidial drugs. The focus of coccidiosis prevention and control tends to research on subunit vaccines that are convenient, safe, and easier to produce than live vaccines. Therefore, new methods are urgently needed to effectively control coccidiosis, including the search for specific molecular targets. In this study, cDNA of E. tenella sporulated oocysts (SO) as a template, we amplified the ORF sequences of the elongation factor 1α of Eimeria tenella (EtEF1α). Bioinformatics analyses shows that the gene encodes 450 amino acids with a predicted molecular mass of 49.1 kDa. The isoelectric points were 4.7. Sequence analysis found that the proteins encoded by EtEF1α have no signal peptides and transmembrane domains, but contain N-myristoylation sites. The transcription and translation levels were analyzed using real-time quantitative PCR and Western blot. The results showed that the mRNA transcription levels of EtEF1α were higher in the sporozoite (Spz) and merozoite (Mrz) than unsporulated oocysts (UO) and SO, and the translation levels were high in UO and Mrz. Indirect immunofluorescence localization showed EtEF1α was mainly located at anterior of Spz and the whole cytoplasm of Mrz. As the parasites developed in the cell, the fluorescence intensity gradually brightens. The secretion assay indicated that EtEF1α was a secretion protein, but not secreted from micronemes. Invasion inhibition assay showed that rabbit anti-rEtEF1α polyclonal antibodies can inhibit sporozoite invasion of DF-1 cells. These results indicated that EtEF1α may play a role in the growth and development of the parasite in the host cell and participate in the process of sporozoite invasion into the host cell.

To development monoclonal antibodies against cOmpT of avian pathogenic Escherichia coli (APEC), the recombinant cOmpT of APEC origin expression plasmid pET-28a-compT was employed, and cOmpT protein with a molecular weight about 36 kD in the form of inclusion bodies was obtained after induction with IPTG, and then renatured by urea gradient dialysis. BALB/c mice were immunized with the purified cOmpT. An indirect enzyme-linked immunosorbent assay (iELISA) was developed, the optimal coating concentration of the antigen was 0.625 μg·mL^-1 and the optimal serum dilution was 1:6 400. After the fourth immunization, the spleen of immunized mice was collected for cell fusion, three monoclonal hybridomas that can secrete antibody specific to cOmpT were obtained after multiple screenings, named 1G8, 2C3 and 2G3 respectively. And all of their immunoglobulin subclasses were IgG2b. The titers of monoclonal antibodies in the cell culture supernatant were 1:200, 1:3 200 and 1:3 200 determined by iELISA, respectively. All three monoclonal antibodies were confirmed to react with cOmpT in Western blot, without cross reaction with other tested bacteria. The antigenic epitopes recognized by the three monoclonal antibodies were identified by using a series of E. coli strains harboring expression plasmids recombined with truncated fragments from compT gene. The results revealed that the antigenic epitope required for reactivity with the 1G8 was ⁸³DQDWMDS⁸⁹, and ⁹⁰SNPGTW⁹⁵, ¹⁹⁷TFKYSGW²⁰³were recognized by 2C3 and 2G3, respectively. In this study, three monoclonal antibodies against cOmpT were successfully developed and the antigenic epitopes recognized by the antibodies were identified. The cOmpT specific monoclonal antibodies obtained in this study are potentially useful tools for both the functional study of cOmpT and the development of APEC epitope vaccines.

This study was conducted to establish an indirect ELISA method for the detection serological antibody of bluetongue virus (BTV). The purified BTV recombinant NS4 protein obtained from the prokaryotic express system was used as the coated antigen, and then an indirect ELISA antibody detection method of BTV was developed by optimizing the reaction conditions. SDS-PAGE results showed that the recombinant NS4 protein with a size of about 52 kDa was obtained, which mainly existed in the supernatant. Western blot results showed that the purified recombinant NS4 protein had good antigenicity. The ELISA reaction conditions were optimized by the square matrix test. The optimal coating amount of recombinant NS4 protein antigen was determined to be 3.0 μg per well, and the optimal dilution ratio of serum to be tested was 1:200, and the optimal dilution concentration of HRP-labeled rabbit anti-cow IgG secondary antibody was 1:4 000, and the critical values were 0.29 and 0.35, respectively. The detection sensitivity of the BTV antibody was up to 1:1 600. The intra-assay repeatability and the inter-assay repeatability coefficient of variation were less than 10%. The positive coincidence ratio and negative coincidence ratio were 98% and 100% respectively. The indirect ELISA method established in this study laid a foundation for clinical serum antibody detection and serum epidemiological investigation of BTV.

To investigate the epidemic situation of viral diarrhea in East China in recent years, and provide epidemiological survey data for comprehensive prevention, 549 fecal samples were collected in 2017-2019 from diarrhea pigs in 5 cities of East China and RT-PCR were used to detect porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), porcine transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV) and porcine Sapelovirus (PSV), and statistical analysis of its positive rate and mixed infection was performed.The results showed that during 2017 and 2019, PEDV,PDCoV,PoRV,TGEV and PSV positive rate were 62.6% (372/594),27.9% (166/594),16.8% (100/594),13.3% (79/594), and 3.87% (23/594), respectively.In the double mixed infection, the average mixed infection rate of PEDV + PDCoV and PEDV + PoRV were the main ones, which were 22.4% (133/594) and 13.3% (79/594), respectively. Among the triple infections, the mixed infection rate of PEDV + PoRV + PDCoV was dominated by 7.58% (45/594), and there were no mixed infections of four and five pathogens. The highest positive detection rate of PEDV was during 2017 and 2019, and mixed infection with PoRV and PDCoV, which was currently the focus of prevention and control of viral diarrhea in East China. TGEV and PDCoV accounted for a large proportion of the detection rate of healthy fattening pigs and sows, indicating that recessive infections had become common and deserve attention from farmers. In addition, the detection rate of PSV was more lower than the other four diarrhea viruses. On the whole, since August 2018 that the farms strengthened biosecurity prevention and control measures, the detection rate of various pathogens and the detection rate of sites have shown a downward trend year by year.

Rab is a family of molecules that mediate the transport of active proteins in cells. The invariant chain (Ii) has the immunological functions of assisting antigen peptide transport, B cell maturation, and acting as the receptor of cytokines. Previous studies showed that Rab5a of chicken (Gallus gallus) is bound to Ii in the early endocytosis, but it is not clear whether there are other Rab molecules with this property. Based on the localization of Rab7b in late endocytosis and its relationship with intracellular molecular transport, this study explored the molecular structure of Rab7b binding to Ii. First, Rab7b genes of chicken and mouse (Mus musculus) were amplified with self-designed primers, and the amino acid homology was analyzed; the prokaryotic and eukaryotic recombinant plasmids containing Rab7b and its 67 amino acid mutant Rab7bQ67L were constructed. Secondly, the recombinant plasmid containing red fluorescent protein and target gene was transfected into mouse macrophage line Raw264.7. After culture, the recombinant plasmid was stained with FITC labeled mouse anti late endosomal protein 1 (LAMP1) antibody to observe the localization of the target protein. Furthermore, chicken Rab7b and Rab7bQ67L were co-transfected with Ii to observe their co-localization with Ii. Finally, the binding of Rab7b and its mutants to Ii were detected by pull-down technique and Western blotting. The results showed that the Rab7b gene was the same size as expected, and its open reading frame was 624 bp, encoding 208 amino acids. The homology of Rab7b protein structure between chicken and mouse was 74%. Although both Rab7b and Rab7bQ67L could bind to Ii, it was Rab7b rather than Rab7bQ67L could locate in the late endocytosis. In conclusion, Rab7b and Ii were not only co-located in the late endocytosis, but also combined with each other; the 67th amino acid of Rab7b affected its location in cells but not with Ii. These results suggest that Rab molecules are involved in the transport of Ii in intracellular organelles, which provides a new way to further study the mechanism of Ii transport in cells.

Defensin is a small cationic peptide widely distributed in animals and plants. It is an important component of the innate immune defense system of mammals. It has a broad spectrum of antibacterial, antiviral and antifungal activities. Porcine β-defensin-2 (PBD-2) is one of the natural defensins expressed in pigs and has good antibacterial activity in vitro. The purpose of this study is to evaluate the therapeutic effect of PBD-2 in the treatment of Streptococcus suis infection in animals and to provide more insights for evaluating the value of PBD-2 as a therapeutic drug. Firstly, the bactericidal activity of PBD-2 against S. suis clinical isolate SC-19 was measured in vitro, and the cytotoxicity of PBD-2 was tested on mouse-derived macrophages (RAW264.7). Subsequently, C57 female mice aged 4-6 weeks and weighing between 18-22 g infected with S. suis SC-19 were treated with PBD-2 or PBS (n ≥ 6). The survival rate of mice, and the bacteria loads, inflammatory cytokine levels, and pathological changes of tissues were determined. The results showed that PBD-2 (25-200 μg·mL^-1) could significantly inhibit the growth of S. suis SC-19 in a concentration-dependent manner (P<0.05). At the same time, PBD-2 had no significant toxic effect on RAW cells (P>0.05). The in vivo study revealed that PBD-2 treatment could effectively reduce the mortality of mice infected with S. suis SC-19. At the same time, PBD-2 treatment significantly reduced bacteria loads in brain, lung, spleen and blood of mice, and decreased the levels of inflammatory cytokines IL-6, IL-12 and TNF-α in mouse sera (P<0.05). At the same time, PBD-2 treatment also significantly alleviated the degree of pathological damages in lung and spleen tissues of mice infected with bacteria (P<0.05). These results indicate that PBD-2 confers great resilience to S. suis in vitro without significant cytotoxicity, while it also exhibits a therapeutic effect on S. suis infection in mice, suggesting that the use of PBD-2 as a therapeutic drug and substitutes for antibiotics is of an excellent prospect.

Swainsonine (SW) can cause disorders of reproductive hormones. Gonadotropins are glycoprotein hormones, so they are regulated by N-glycosylation modifications. ɑ-mannosidase, a key enzyme that accelerates the processing of N-glycosylation modifications, can be inhibited by SW. So how does SW affect the structure of N-glycan and the secretion performance of reproductive hormone is unclear. Thus, this test was conducted by intraperitoneal injection of SW exposed mice to establish models of poisoning. The changes of N-glycan structure in their pituitary tissues were detected by MALDI-TOF-MS mass spectrometry; the activity of glycosylase, the level of reproductive hormone, the quantity of reproductive hormone receptors were analyzed. With the extension of injection time, the five composite glycosides of the pituitary glycoprotein in the poisoned group disappeared, and three hybrid glycosides were added. The activities of glycosyltransferase and glycosidase in the poisoned group were significantly decreased. There were further found that the expression levels of gonadotropin receptor, estradiol and progesterone receptor proteins in the poisoned group were significantly lower than those in the control group, and the secretion levels of reproductive hormones were also significantly decreased. SW can significantly inhibit the activity of N-glycan glycosylase and cause changes of normal N-glycan structure; it has a negative influence on the activities of gonadotropins and their receptors, causing the regulation of downstream steroid hormone secretion out of balance, and eventually, reproductive hormone regulation can be disrupted.

Porcine reproductive and respiratory syndrome virus (PRRSV) mutates continuously, and it is increasingly difficult to control it. To monitor the genetic evolution characteristics of PRRSV timely under the condition of natural infection, a novel PRRSV variant named SHpd1/2018 was isolated from PRRSV positive clinical samples. The results showed that the SHpd1/2018, with a total genome length of 15 018 bp (excluding poly A), showed similar proliferation characteristics to HP-PRRSV strain HuN4. However, it could not be recognized by the monoclonal antibody against HuN4-Nsp2. The sequence analysis indicated that SHpd1/2018 had the greatest homology with HP-PRRSV-like strains, reaching 94.3% with HuN4 strain. The results of recombination analysis showed that SHpd1/2018 was recombined from HP-PRRSV-like strain (main parent strain) and NADC30 strain (secondary parent strain), and both the two recombination breakpoints nt2002 and nt3205 are located in the hypervariable regions of the Nsp2 gene. In short, we confirmed that the isolated strain SHpd1/2018 is a recombinant PRRSV, which may be the main cause of the disease in the pig farm in Shanghai.

This experiment was conducted to establish a droplet digital PCR (ddPCR) method for the detection of Newcastle disease virus (NDV). A ddPCR method was developed, which the primers and probes were designed based on the conservative regions of F gene of NDV. The concentration of primer and probe, the annealing temperature in ddPCR reaction were optimized. The sensitivity, specificity and reproducibility of ddPCR method were evaluated. In results, the optimal primer concentration and the probe concentration were 900 and 250 nmol·L^-1, the optimum annealing temperature was 55℃. The detection limit of ddPCR method was 1.8 copies·μL^-1 with a good linear response, it had no cross reaction with other six viruses (include IBV), the coefficient of variation of sample repetition was 2.4%. All the results showed that NDV ddPCR was sensitive and specific, it was suitable for quantitative detection of clinical samples infected with NDV.

To detect the drug resistance genes, the multiplex PCR method for drug resistance genes of β-lactams, tetracyclines, aminoglycosides, amphenicols, and sulfonamides were developed. Based on the sequences of drug resistance genes from GenBank, 17 pairs of specific primers were designed. Then, 4 multiple PCR assays were established through the optimization of PCR reaction conditions and primers concentrations (cat+floR+tetB+tetC; dfrA12+sul2+sul1+bla_CTX-M+bal_TEM-1; aac3+aph3+aadA1+strB; tetA+cmlA+strA+sul3). The sensitivity and specificity of these assays were determined. The multiplex PCR assays were used to detect the drug resistance genes of 42 avian pathogenic Escherichia coli (APEC). The drug sensitivity of these APEC strains were also determined, which were compared to the distributions of drug resistance genes in these strains. The results showed that the 17 drug resistance genes were effectively and specifically amplified in these 4 optimized multiplex PCR assays. The detection limits of the 4 multiplex PCR were 10³, 10⁴, 10⁴ and 10⁵CFU of bacteria, respectively. The established multiplex PCR assays are specific and rapid for the detection of drug resistance genes in APEC strains, which showed 92.86% coincident with the drug resistance for these strains. The developed 4 multiplex PCR are simple and rapid assays for drug resistance genes detection, which can be used for the epidemiologic study for drug resistance genes.

This study aimed to explore the metabolic differences in sera of antemortem neonatal goats based on an epidemiological perspective. A case-control design was used to discover the metabolic differences in serum between the dead newborn goats suffered from a disease and healthy goats at the same age by using UPLC-MS/MS. A total of 13 compounds were significantly different between healthy and dead newborn goats. KEGG analysis showed that these differential produced metabolites mainly involved the synthesis and catabolism of amino acids. The differential produced metabolites in serum are important for us to understand the agonal reaction at the metabolic level when neonatal goats suffering from a disease. This study also provides an advanced research basis for the establishment of disease-related biomarkers for neonatal goats which may suffer from a disease.