Circular RNA (circ-RNA) is a class of closed circular RNA molecules formed by reverse splicing of the initial transcriptional RNA, which are expressed abundantly in eukaryocytic cells. circ-RNA exists stably for a long period because of the resistance to RNA exonuclease. The expression of genes is regulated by cornpetitive binding of miRNA via sponge absorption. Recent studies demonstrated that circ-RNA molecular identification and physiological regulation had become the focus of life science research. In this review, we summarized the biogenesis of circ-RNA, the role in regulating early embryonic development, and the quantity and effects during the gamete development.

Pigeon milk (PM) is a semi-solid nutrient substance produced and secreted by the crop of pigeon and it is the only nutritional source of squab. Squabs should be fed by their parents because they could not eat by themselves, so PM is very important for their growth and development. The production and secretion of PM is a special physiological process, which is rare in birds and different from the lactation process of mammals. PM is mainly composed of proteins, lipids, carbohydrates and minerals. Synthesis and secretion of PM are regulated by numerous hormones such as prolactin, insulin and relaxin. The signaling pathways such as JAK/STAT, PI3K/Akt/TOR, AMPK and Wnt are activated by these hormones and then the expression of related genes are regulated. The processes of crop epithelial cell proliferation, degeneration, nutrient synthesis and enrichment are regulated. The composition of pigeon milk and its production regulatory mechanism are reviewed, we compare these characteristics of PM with mammalian milk, and the physiological mechanism of pigeon milk production by crop and its similarities and differences with mammalian mammary lactation are further clarified. Research on the composition of PM will provide reference for the formulating of breeding pigeon nutrition requirements and research of artificial PM. The regulation mechanism of PM production will further find out genes regulating lactation and select breeding populations with high yield of PM by using molecular marker-assisted breeding technology.

Long non-coding RNAs (lncRNAs) are designated as the transcripts longer than 200 nt without protein-coding capacity, which play a crucial role in various biological processes. Current evidence demonstrates that lncRNAs, an important class of novel regulatory RNAs, are induced by viruses and mediate host-virus interactions. They can regulate pathogen recognition receptors and inhibit or activate natural immune responses to feedback viral infections through different mechanisms. In this paper, we focus on the regulation of lncRNAs in virus-host interactions and highlight the regulatory networks of host antiviral responses. This review provides a valuable reference for studying the antiviral effect and mechanism of lncRNAs, and lays the foundation for revealing the pathogenesis mechanism of the virus and discovering new antiviral targets.

Genomic data is more and more widely used in livestock breeding. Genotype imputation is an important tool to handle missing values in genotypic data, and the quality of imputation results directly affects the subsequent analysis. To obtain good imputation results, a comprehensive imputation strategy needs to be formulated. We studied on the effects of several factors on genotype imputation by simulation. The factors included reference population size, genetic relationship (distance) between the target population and the reference population, the number of target sites (proportion), the minimum allele frequency (MAF), and the imputation algorithm. The results showed that the number of target sites was the main factor affecting the genotype imputation, and it showed significantly positive correlation with the quality of imputation(P<0.05). The reference population size was the main factor affecting the imputation error rate in Beagle5.1. Correspondingly, the number of target sites was the main factor affecting the imputation error rate in Minimac4. Genetic distance between the target population and the reference population had a more significant effect on the imputation quality of Beagle5.1 than Minimac4. In general, the imputation error rate increased as the increases of MAF in a site. When the number of individuals in the reference population was small and the number of target sites was large, the speed of Minimac4 was superior to Beagle5.1, but there was a reverse trend as the reference population size increased. On the premise of ensuring the imputation quality, Beagle5.1 had relatively lower requirements for the above factors. In contrast, when the number of target sites was low and reference population size was large, the imputation effect of Beagle5.1 was better, while Minimac4 was more suitable for the imputation of a small reference population size and a higher number of target sites. In this study, different strategies were formulated for different imputation purposes, and the study results would provide a reference for genotype imputation.

The aim of this study was to detect the copy number variation (CNV) in the genome of Bama Xiang pigs and investigate the effect of marker density on the efficiency and accuracy of CNV detection. PennCNV and R-Gada were employed to detect CNVs using the 1.4M high-density SNP chip data of 319 (160 hogs and 159 gilts) Bama Xiang pigs, and the CNV region (CNVR) was constructed by merging overlapping CNVs. Only the CNVR with higher frequency than 5% was verified by the genome-wide association study (GWAS). Finally, according to the marker densities, a certain number of SNPs were evenly extracted, and the effect of marker density on CNV detection efficiency and accuracy was explored. There were 6 327 CNVs detected by PennCNV and 3 489 CNVs detected by R-Gada, which made up of 795 and 340 CNVRs, respectively, including 226 CNVRs identified by both programs. Among the 226 CNVRs, the shortest was 3.98 kb, the longest was 1 297.78 kb, and their total length was 33.27 Mb, of which 102 (45%) overlapped the CNVRs reported previously. Among the 795 CNVRs detected by PennCNV, 135 had a higher frequency than 5%, 20 of which had been verified by GWAS, and the verification rate was 15%. With the SNP density increasing, the efficiency and accuracy of CNV detection were increased, especially for the small size CNVs. A CNVR sketch of Bama Xiang pigs had been drawn using 1.4M SNP chips, which was helpful to identify CNVRs associated with important economic traits in the future. At the same time, we revealed the positive effect of marker density on the efficiency and accuracy of CNV detection, and the results provided a reference of choosing marker density for the follow-up research of CNV detection.

At present, genomic selection (GS) has been applied in pigs breeding, but some implementation strategies, such as the determination of genotyping ratios or early selection rates for piglets, are required to obtain a higher benefit using this technology. The Large White pigs born from 2011 to 2016 at WENS Foodstuff Group Co.，Ltd were chose as the research objects, including more than 45 000 growth measurement records, more than 70 000 reproduction records and 2 090 individuals with genotyping-by-sequencing (GBS) data. The 440 individuals born from July to December in 2016 were used as the candidate individuals. The traits included two growth traits, age at 100 kg and backfat thickness at 100 kg, and one reproduction trait, number of total born. To compare the prediction effects, four prediction scenarios were designed according to including or ignoring the phenotypic or genotypic information of candidate individuals when predicting their breeding values. The predictive reliability of different scenarios and rankings of selection indices of individuals would be compared. The results showed that the results using the phenotypic and genotypic information was more reliable than ignoring them to predict the breeding values of candidate individuals. When genomic selection indices were calculated before and after performances testing for the growth traits, the individuals ranking in the top 30% of indices after testing were all found in the individuals ranking in the top 60% of indices before testing. If the piglets with the top 60% of traditional BLUP indices were only selected, around 15% of individuals with good genetic potentials would be omitted. This study suggests that all healthy piglets after birth are genotyped and their genomic selection indices are calculated, and then the individuals ranking in the top 60% of indices are chose to perform growth measurement.

The objective of this study was to investigate the influence of miR-499-5p injection on gastrocnemius muscle fiber diameter and gene expression, and improve the molecular regulation mechanism of miR-499-5p in skeletal muscle development in broiler. Sixteen healthy Daheng cocks aged 14 days were randomly assigned into 2 groups with 8 replicates per group. agomiR-NC(5 nmol) were injected into the chicks in control group, and agomiR-miR-499-5p(5 nmol) were injected into the chicks in treating group, respectively. agomiR-NC and agomiR-miR-499-5p were injected once every 3 days for a total of 6 times. The gastrocnemius muscle in the 2 groups were collected after slaughter for the measurement of muscle fiber diameter and RNA-seq analysis. The results showed that the muscle fiber diameter of gastrocnemius of chicks in treating group was significantly shorter than that of chicks in control group (P<0.05). 308 263 164 and 316 696 152 clean reads were obtained from the treating group and control group, respectively. The ratios of these reads to chicken reference genome were 90.29% and 89.57%, respectively. Compared with the chicks in the control group, there were 18 differentially expressed genes in gastrocnemius muscle of miR-499-5p treated chicks, of which 12 up-regulated genes and 6 down-regulated genes (P<0.05). The top 2 differentially expressed genes were FOS proto oncogene (FOS) and early growth response gene-1 (EGR1). GO enrichment analysis showed that the differentially expressed genes were mainly enriched in the transmembrane receptor protein serine/threonine kinase signaling pathway, transcriptive regulation, lipid biosynthetic process and skeletal muscle regeneration. KEGG analysis showed that the differentially expressed genes were mainly involved in the MAPK signaling pathway and Wnt signaling pathway. A total of 78 target genes of miR-499-5p were predicted by bioinformatics software. GO enrichment analysis showed that the 78 target genes were also mainly enriched in the negative regulation of protein serine/threonine kinase activity and transcription factor activity. In conclusion, the results showed that miR-499-5p could change the gastrocnemius muscle fiber diameter and affect characteristics of muscle fiber. FOS, EGR1, CYR61 and WNT7A, which were significantly associated with the alteration of muscle fiber diameter, might be involved in the regulation of fat deposition and muscle development through the pathways mentioned above in chicks after miR-499-5p injection.

The aim of this study was to investigate the effect of discoidin domain receptor 1 (DDR1) gene on the proliferation, apoptosis and cell cycle of dairy cow mammary epithelial cells through interfering and overexpressing DDR1. The small RNA interference fragment of DDR1 gene and overexpression vector pcDNA3.1-DDR1 were transfected into dairy cow mammary epithelial cells, qRT-PCR and Western blot were used to detect the interference and overexpression efficiency of DDR1; CCK-8 was employed to detect cell proliferation; Flow cytometry was performed to detect the changes of cell cycle and apoptosis (early apoptosis and late apoptosis). qRT-PCR was used to detect the expression of genes related to proliferation and apoptosis. After interfering DDR1 gene in dairy cow mammary epithelial cells, mRNA and protein expression levels of DDR1 were down-regulated by 94% and 30%, respectively; While after overexpressing DDR1 gene, its mRNA and protein expression were up-regulated 68.24 and 1.38 times, respectively. Interfering DDR1 extremely significantly inhibited the proliferation of cells (P<0.01), the ratio of early and late apoptotic cells was extremely significantly increased (P<0.01); the ratio of G1 phase cells in the interference group was extremely significantly increased (P<0.01), and the proportions of S and G2 phase cells were significantly decreased (P<0.01 and P<0.05), which suggested that the cells were blocked in G0/G1 phase. Conversely, overexpression of DDR1 could significantly promote cell proliferation (P<0.05), significantly inhibit the late apoptosis of cells (P<0.05), the proportion of G1 phase cells was extremely significantly decreased (P<0.01), and the proportion of S phase cells was extremely significantly increased (P<0.01), suggesting the enhancement of transition from G1 to S phase. The results of qRT-PCR detection showed that after interfering DDR1, the expressions of BAX and Caspase9 genes were significantly up-regulated (P<0.05), and the expressions of P53 and FAS genes were extremely significantly up-regulated (P<0.01), while the expressions of PCNA and CyclinB1 genes were significantly down-regulated (P<0.05). After overexpression of DDR1, the expressions of Cytc and BAX genes were significantly (P<0.05) and extremely significantly down-regulated(P<0.01), respectively, and the expression of CyclinB1 gene was extremely significantly up-regulated (P<0.01). In summary, DDR1 can regulate the proliferation, apoptosis and cycle of dairy cow mammary epithelial cells, this result will provide a reference for elucidating the molecular mechanism of DDR1 involved in the growth and development of dairy cow mammary epithelial cells.

The purpose of this study was to screen the regulatory factors that regulate the expression of GIPR from the downstream Akt and PKA signal pathways of GIP/GIPR, and to analyze the regulatory mechanism of GIPR expression. In this study, mouse insulinoma cell line Min6 was used as experimental material, under the condition of blocking Akt and PKA signal pathways, transcription factor T cytokine 4 (TCF4) related to GIPR expression was screened by Western blot, the effect of TCF4 on the regulation of GIPR expression was determined by double luciferase reporter system, and the relationship between TCF4 and GIPR was further verified by knockout or overexpression of TCF4. The proliferation-promoting effect mediated by TCF4 was detected by CCK8 method, and the ability of insulin secretion was detected by ELISA. The results showed that GIP could activate Akt phosphorylation and promote the expression of GIPR; when GIP was activated and Akt and PKA signal pathways were blocked, the expression trend of GIPR protein was consistent with that of TCF4; TCF4 could bind to the core promoter of GIPR and regulate its expression; when TCF4 was overexpressed, the mRNA and protein expression of GIPR were up-regulated, and β-cell proliferation and insulin secretion were promoted. Interfering with TCF4 significantly decreased the mRNA and protein expression of GIPR under the action of GIP, and inhibited the proliferation of β-cell. In conclusion, after GIP is combined with GIPR, TCF4 is upregulated through Akt signal pathway to enhance the expression of GIPR, which forms a positive feedback to enhance GIP signal, and enhance the function of β-cell proliferation and insulin secretion to maintain blood glucose homeostasis. Therefore, when insulin resistance blocks Akt and upstream signal pathway, the enhancement of GIPR expression by transcription factor TCF4 can be used as a target to improve islet β-cell dysfunction.

The purpose of this experiment was to study the growth and developmental ability and reproductive performance of cloned Wujin pigs fire hair line by somatic cell cloning technology, and provide a theoretical basis for the application of somatic cell cloning technology in the conservation of local excellent pig breeds. In this study, the ear samples were collected from 3 male and 12 female pigs which met the characteristics of Wujin pigs. The 3 boars were 3, 6 and 11 months old, respectively. The sows had a large age gap, from 2 months old to 10 years old, the three sows and one boar of 3-month-old had been castrated and the sow of 10-year-old had no reproductive capacity. The ear fibroblast cell lines were successfully established from 15 Wujin pigs. Then the fibroblast cells from 3-month-old(♂) and 10-year-old(♀) pigs were used as donor cells for somatic cell nuclear transfer. The cloned embryos were transplanted into 16 surrogate sows and 25 cloned pigs were obtained. The growth and development indexes of these cloned pigs were all normal. After sexual maturity, the male and female cloned pigs mated naturally and 39 live piglets (F1) were obtained. This study successfully cloned Wujin pigs by somatic cell nuclear transfer technology, and cloned Wujin pigs had normal growth and developmental ability and reproductive performance. The study result provides a new way for the conservation of local pig breeds.

The aim of this study was to construct shRNA recombinant vector interfering insulin-like growth factor-II (IGF-II), and study the effects of IGF-II on the yak sertoli cells. The shRNA oligonucleotides targeting yak IGF-II were designed and synthesized and it was cloned into pLKO.1 plasmid vector, after transfected into yak sertoli cells, a stable cell line was obtained by puromycin selection. The relative expressions of IGF-II mRNA and protein were identified by qRT-PCR and Western blotting, respectively. The proliferation and apoptosis of sertoli cells were analyzed by proliferation measurement system. The results showed that the pLKO.1-shRNA vector interfering IGF-II was successfully constructed, which was stably expressed in sertoli cells and could interfere the expression of yak IGF-II effectively. Compared with the control group, the interference efficiency of pLKO.1-shRNA2 was the most significant， IGF-II expression down-regulated to 19.1% (P<0.05), and could reduce the expression of endogenous IGF-II protein by 76.3% (P<0.05). The stable cell line obtained by transfection and screening of pLKO.1-shRNA2 plasmid had significant lower activity of cell division and proliferation than that of control group after 48 h of culture (P<0.05). The results of qRT-PCR showed that the expressions of IGF-I and IGF-IIR in sertoli cells were significantly up-regulated (P<0.05), and the expressions of IGF-IR and Bcl-2 were significantly down-regulated (P<0.05) after interfering IGF-II. In conclusion, the interference plasmids of IGF-II were successfully constructed. After interference plasmid was transfected into yak sertoli cells, it could effectively inhibit the expression of IGF-II, change the expression pattern of IGF-IR and Bcl-2, and potentially affect the division and proliferation of yak sertoli cells, however, the specific regulation mechanism requires further studied.

The purpose of this study was to investigate the cecal microbial community composition and diversity of grazing Tibetan pigs, captive Tibetan pigs and lean type pigs (DLY pigs) in Tibetan plateau, in order to partially revealed the specificity of the intestinal microbial community of Tibetan pigs. Pigs with the same age were chosen, including 5 grazing Tibetan pigs, 5 captive Tibetan pigs and 5 DLY pigs. The grazing Tibetan pigs were free range husbandry by local herdsman. The captive Tibetan pigs and DLY pigs were reared in pens and fed with same diets. At the age of 160 days, all the pigs were slaughtered by anterior vena cava bloodletting. Cecal chyme was collected and rapidly frozen with liquid nitrogen. The V3-V4 regions of the 16S rRNA genes were amplified and sequenced using high-throughput sequencing techniques. The results showed that a total of 659 904 valid sequences were obtained from the V3-V4 region of 16S rRNA in 15 samples, including grazing Tibetan pigs 213 031, captive Tibetan pigs 219 417 and DLY pigs 227 456. Interestingly, the cecal OTUs quatity, Chao1 index, ACE index and Shannon index of the grazing Tibetan pigs were significantly higher than those of the other pigs (P<0.05), but there was no significant difference between the captive Tibetan pigs and DLY pigs (P>0.05). The microbiota of 3 types of pigs were divided into 13 phyla and 56 genera, and Firmicutes has the highest relative abundance in all pigs. The relative abundance of Actinobacteria and Verrucomicrobia were significantly higher in grazing Tibetan pigs than those of the others (P<0.05), but Bacteroidetes was remarkably higher in captive Tibetan pigs (P<0.05). The relative abundance of 11 genera in grazing Tibetan pigs was significantly higher than captive Tibetan pigs and DLY pigs (P<0.05). These results indicated that the cecal microbial community structure and diversity of grazing Tibetan pigs were unique, which could provide reference for further developing the Tibetan pig resources.

This study was conducted to investigate the effect of Lactobacillus reuteri on the expression of miRNAs in the jejunum of piglets. Six litters of healthy 1-day-old York×Rongchang piglets were selected and randomly divided into the two group，3 litters per group(n=27). The two groups were control group (daily orally adminitrated with 0.1% sterile peptone solution per piglet)，and the LR group (daily orally administrated with 1.0×10¹⁰ CFU of L. reuteri per piglet)， respectively. At 10 and 20 days of age, six piglets per group were selected for slaughter, respectively, and the jejunum samples were collected. After RNA extraction, the library was constructed and the miRNA expression profile of the jejunum was detected by Solexa high-throughput sequencing technology. Bioinformatics technology was used to conduct target genes prediction and functional analysis. The results showed that: 1) A total of 8 libraries were constructed and a total of 187 103 840 clean reads were obtained, of which the 22 nt length sequence accounted for the largest proportion; 2) Three hundred and fifty-four and 352 known miRNAs were respectively identified at 10 and 20 days of age, of which 329 miRNAs were co-expressed; 3) Differentially expressed (DE) miRNAs in the jejunum were found after L. reuteri administration, 7 and 9 DE miRNAs were obtained at 10 and 20 days of age，respectively. Notably, ssc-miR-218 was expressed differentially at two ages. 4) KEGG pathway analysis and target gene prediction on the differentially expressed miRNAs revealed that 10 221 target genes were enriched in 293 biological pathways, of which 66 were significantly enriched (P<0.05), including the MAPK signaling pathway and PI3K-Akt signaling pathway, which were known to regulate intestinal development and immune. 5) Six miRNAs were selected to validate the sequencing results by qRT-PCR, the qRT-PCR expression results corresponded well with those from the sequencing. The results indicates that L. reuteri may regulate intestinal health related pathways by influencing the expression of jejunal miRNAs.

The aim of this study was to investigate the effects of probiotic interaction on growth performance, intestinal nutrient digestion and absorption, and related sugar transporters in broiler. Two hundred 1-day-old male Arbor Acres broilers were selected, and then randomly divided into 4 groups with 5 replicates per group and 10 for each replicate. Lactobacillus casei, Lactobacillus acidophilus and Bifidobacterium lactis were mixed in proportion, and then fermented in milk. The chicks in the control group received normal drinking water, the chicks in probiotic group Ⅰ (mixed ratio of 2:1:1), group Ⅱ (mixed ratio of 1:2:1), and group III (mixed ratio of 1:1:2) received drinking water supplemented with 1% compound probiotics. All chickens were fed with normal diet for 42 days, every 21 days as a stage. The results showed that: 1) Compared to control group, the ADG(P<0.05) and feed conversion ratio (P>0.05) of broilers in all probiotic groups were improved. Furthermore, the production performance of broilers in probiotic group Ⅰ was higher than those of probiotic group Ⅱ and Ⅲ. 2) Compared to control group, specific activities of amylase, lipase, trypsin in jejunum of probiotic groups increased significantly (P<0.05). 3) In all probiotic groups, the intestinal villus height increased significantly (P<0.05), crypt depth decreased significantly (P<0.05), and the expression of glucose transporter 2 (GLUT2), particularly in probiotic group Ⅰ and Ⅲ, was significantly upregulated (P<0.05). In conclusion, probiotics interaction can enhance digestive enzyme activity, improve the structure of digestive tract, upregulate the expression of nutrient transporter, improve the nutrient digestibility and energy absorption efficiency, and further improve production performance of broiler. When the mixing ratio of L. casei, L. acidophilus and B. lactis was 2:1:1, the compound probiotics had a better effect on broilers.

In this study, in order to explore the relationship between intestinal microbiota and sheep growth and development, the differences between the intestinal microbiota of Altay sheep with different growth rates under the same feeding conditions and changes in intestinal microbiota species composition after affecting growth rate through diet were explored. Among the 300 lambs at 6-month-old, fast-growing and slow-growing male Altay sheep were screened according to the top 10% and the bottom 10% of daily weight gain, and were defined as high body weight group and low body weight group， respectively. Twelve animals were randomly selected from the high body weight group and they were divided into a high body weight restriction feed group (HR, n=6) and a high body weight control group (HC, n=6). Twelve sheep were randomly selected from the low body weight group and they were divided into a low body weight add feed group (LA, n=6) and a low body weight control group (LC, n=6). After 7 days of pre-feeding, the HR, HC, LC, and LA groups were fed diets with 75%, 100%, 100%, and 125% of energy intakes, respectively. And feeding trials were continued for 30 days. At the end of the feeding period, the animals were weighed and slaughtered to collect intestinal contents from the same site, DNA was extracted from these samples, and detection was performed using the Illumina Miseq sequencing platform. The results showed that under the same feeding standard, the composition of intestinal microbiota of Altay sheep with different growth rates was different. The abundance of Firmicutes, Actinobacteria, Saccharibacteria, Verrucomicrobia and Bifidobacteriaceae in the HC group was higher than that in the LC group, and the abundance of Proteobacteria and Paeniclostridium in the LC group was higher than that in the HC group. In the experiment of regulating the growth rate through diets, the low-energy diet significantly reduced body weight gain (P<0.05) of HR group; and the high-energy diet significantly increased body weight gain (P<0.05) of LA group. Regardless of promoting or inhibiting the growth rate, the abundance of intestinal Firmicutes and Bacteroidetes increased, and high-abundance Saccharibacteria always existed in the group with faster growth rate, while high-abundance Paeniclostridium always existed in the group with slower growth rate. A comprehensive analysis of the results of the two parts of the experiment showed that the abundance changes of Firmicutes and Bacteroidetes in the Altay sheep may be related to the diet and growth rate. The abundance change of Saccharibacteria is positively correlated with growth rate, and the abundance change of Paeniclostridium is negatively correlated with growth rate.

The purpose of this experiment was to study the effects of Gymnadenia conopsea polysaccharide (GCP) extract on growth performance, meat quality and antioxidant function house-fed mutton sheep under oxidative stress, so as to provide experimental basis for extensive research and development of Gymnadenia conopsea and its extracts. Twenty 6-month-old healthy male Small-Tailed Han sheep with body weight of (35.0 ±4.0) kg were randomly divided into two groups. The experimental group (n=10) and control group(n=10). The whole experiment lasted for 42 d and was divided into 3 periods: the first 15 days were the blank period; the middle 15 days were the treatment period; the last 12 days were the observation period ofter modeling. From the first day of the treatment period, the control group was fed with basal diet, and the experimental group was fed pellet basal diet containing 30 mg·kg^-1 GCP extract. On the 15th day of the treatment period, the sheep in each group were intraperitoneally injected with 10 mg·kg^-1 Diquat solution according to sheep body weight(BW), and then fed for another 12 days. On the 1 st, 15th, 30th, 33rd, 36th, 39th and 42nd morning of the whole experimental period, fasting weight and blood samples were collected before feeding. The serum was separated and the antioxidant indexes (SOD, GSH-Px, T-AOC, MDA) in serum were determined. On the 42nd day of the whole experimental period, 5 sheep randomly selected from each group were slaughtered and meat samples were collected to determine the meat quality. The results showed that the addition of GCP extract to the diet of the sheep could increase the average daily feed intake (P<0.05). When the body was subjected to oxidative stress, the indexes of daily gain and antioxidant enzymes recovered rapidly on experimental group, the mutton drip loss decreased significantly, and the mutton sheer force increased significantly(P<0.05). The results indicated that GCP extract could improve the growth performance of mutton sheep, and alleviated the decline of growth performance and antioxidant performance caused by oxidative stress, and had a certain effect of antioxidant stress.

The purpose of this study was to investigate the effect of steam explosion pretreatments, compound bacteria fermentation and their combined treatment on the degradation of fiber in Caragana. In flowering stage (April), fructicative stage (July) and dormancy stage (November), the whole plant of Caragana was collected and its general nutrients were determined. According to the research content, the experiment was divided into 3 parts: 1)Degradation of fiber components in Caragana powder was studied under 3 different steam explosion pressures (0.8、1.1 and 1.4 MPa). 2)The effect of compound bacteria fermentation on the degradation of Caragana powder fiber component was determined. 3) The effect of steam explosion + compound bacteria fermentation on the degradation of Caragana powder fiber component was studied. The 3 growth periods of Caragana powder in each experiment were regarded as fixed factors. All treatments were compared with untreated Caragana powder in the same period, and each treatment was repeated 3 times. The neutral detergent fiber, acid detergent fiber, lignin, hemicellulose and cellulose, and the activity of carboxymethyl cellulase and xylanase were detected in each treatment. The results showed that: 1) According to the nutrient component, Caragana had the highest feeding value at the fructicative stage. 2) Compared with the control group, the content of neutral detergent fiber, acid detergent fiber, lignin (only in flowering stage) and cellulose increased first and then decreased (P<0.05), Hemicellulose decreased linearly (P<0.01), and the content of neutral detergent solute decreased first and then increased (P<0.05) as steam explosion pressureincreasedat flowering and fructicative stage. 3) Compared with the same period before fermentation, the content of neutral detergent fiber (P<0.01), acid detergent fiber (P<0.01) and cellulose (P<0.01) decreased significantly and neutral detergent solute (P<0.01) increased significantly after fermentation. The hemicellulose decreased significantly at flowering and dormancy stage (P<0.01). 4) After steam explosion pretreatment and fermentation, the content of neutral detergent fiber (P<0.001), acid detergent fiber (P<0.05) and hemicellulose (P<0.001) decreased significantly, the content of cellulose decreased significantly at flowering and fructicative stage (P<0.01), and the content of neutral detergent solute (P<0.001) increased significantly with the increase of steam explosion pressure at the flowering stage and dormancy stage, and the content of lignin (P<0.05) decreased first and then increased at flowering and fructicative stage, and decreased significantly at dormancy stage (P<0.01) with the increase of steam explosion pressure, compared with the same period without steam explosion pretreatment. 5) After steam explosion pretreatment and fermentation, carboxymethyl cellulase activity (P<0.001) first decreased and then increased at the flowering anddormancy stage, while carboxymethyl cellulase activity (P<0.001) significantly increased in the fructicative stage with the increase of steam explosion pressure; the xylanase activity (P<0.001) increased linearly with the increase of steam explosion pressure in the three periods. The results indicated that the fiber content of Caragana at different harvest time could be significantly reduced by steam explosion, compound bacteria and their combined treatment, among which the effect of combined treatment was the best. This study provides scientific basis and data support for the effective evaluation of the nutritional value of Caragana and its application in animal husbandry.

The objective of this study was to explore the physiological mechanism of Apelin-13 regulating blood lipid metabolism in mice. Ninety-six mice with similar body weight were randomly divided into 4 treatments, with 4 replicates each. In a 14-day trial period, the mice of the control group and 3 treatment groups were injected subcutaneously with 100 μL 0.9% normal saline, 10, 20 and 50 μg·kg^-1 Apelin-13 each day, respectively. The growth performance, serum Apelin-13 and blood lipids contents were measured. The transcriptomic profiling of liver tissues was sequenced using Illumina Novaseq technology. The results showed that compared to control group, the serum Apelin-13 and high-density lipoprotein cholesterol contents were increased extremely significantly (P<0.01) and triglycerides contents were decreased significantly (P<0.05) in treatment groups. A total of 340 up-regulated and 350 down-regulation differentially expressed genes (DEGs) were identified among treatment groups and control group. The most significant enriched signaling pathway was retinol metabolism, in which cytochrome P450 (Cyp) and uridine diphosphate glucuronidase (Ugt) superfamily genes were mostly up-regulated. The results of this study indicated that Apelin-13 might regulate blood lipid metabolism in mice by regulating the expression of Cyp and Ugt superfamily genes in the retinol metabolism signaling pathway.

The differentially expressed proteins between sporulated oocyst and unsporulated oocyst of Cystoisospora suis strains were screened and compared. In this study, the C. suis infection model of piglets were established and a quantitative proteomic analysis between sporulated oocyst and unsporulated oocyst of C. suis were conducted by iTRAQ labeling and LC-MS/MS. Part of the iTRAQ results were validated by qRT-PCR. Total 174 proteins were identified, among which 40 proteins were differentially expressed, 38 proteins were upregulated and 2 proteins were downregulated. The differentially expressed proteins were mainly involved in metabolism, antibiotics biosynthesis, secondary metabolites biosynthesis and glycolysis/gluconeogenesis pathways. The results of this study provide reference for elucidating the developmental mechanism of C. suis sporulated oocyst and facilitated discovering a new biomarker for C. suis.

The present study aimed to develop an ELISA for detecting antibodies of Haemophilus parasuis by optimization of reaction conditions, determination of its cutoff value and evaluation of its application with autotransporter passenger domain (Apd) as the coating antigen. Firstly, the porcine sera from immunization and challenge experiments were used as HPS-positive and -negative control sera to optimize the Apd-ELISA reaction parameters and conditions. Next, the cutoff value was determined based on testing of the serum samples with confirmed background, and then the blocking solutions and the packing methods of plates were selected. Finally, Apd-ELISA was used to detect clinical serum samples and then compared with the Biocheck OppA-ELISA kit from Netherland. With the coating antigen at 0.5 μg·mL^-1, the tested sera being diluted at 1:200 and incubation for 45 min and HRP-labeled goat anti-pig second antibody being diluted at 1:15 000, Apd-ELISA displayed the improved discriminative capability and the reduced background signal. The cutoff value was determined to be 0.33 by calculation formula (S-N)/(P-N) and the OD_{630 nm} value of the 27 positive porcine sera and 40 negative porcine sera with the confirmed background. The blocking solution K and the vaccum package can preserve ELISA plates for 4 days at 50 ℃ (comparable to 22 months at 4 ℃). Detection of 1 179 clinical sera with Apd-ELISA indicated that the positive rates were 83.76%, 61.09% and 27.48% in sows, fattening pigs and piglets, respectively. Compared with the OppA-ELISA kit from Biocheck (OppA-ELISA), Apd-ELISA showed 100% identity to the results of OppA-ELISA kit in clinically negative sera and experimentally positive sera from the pigs immunized with the inactivated vaccine, but 4.76%(6/126)identity to the results in the sera from clinically positive sera. However, Apd-ELISA displayed 73.02%(92/126)identity to the results of OppA-Western blot in the sera from clinically and experimentally infected pigs. The present study obtained the Apd-ELISA kit that can effectively distinguish HPS-positive and -negative sera and can be stably preserved, which would be applied for antibody monitoring among pigs immunized with HPS-inactivated vaccine and subjected to infection of H. parasuis.

The purpose of this study was to construct a recombinant Lactobacillus reuteri (L. reuteri) expressing the cap protein of porcine circovirus type 2 (PCV2) and evaluate its effect on immune response in mice. The cap protein gene of PCV2b strain isolated and stored in the laboratory was amplified by PCR. A recombinant strain pPG-T7 g10-PPT-cap / L. reuteri expressing the cap protein was constructed using L. reuteri of pig origin as the host strain and explored the immune effect of BALB/c mice with recombinant bacteria orogastrically. Indirect ELISA was used to determine the level of antigen-specific IgG antibodies in the serum of mice after immunization, the levels of antigen-specific sIgA antibodies in stool, nasal wash, reproductive tract wash, and intestinal mucus, and the levels of various cytokines in mouse serum; MTT method was used to detect mouse spleen lymphocyte proliferation levels; flow cytometry (FCM) was used to detect the levels of CD4⁺ T cells and CD8⁺ T cells in mouse spleen lymphocytes; fluorescence quantitative PCR was used to detect the viral load of organs in challenged mice after immunization. The results showed that the serum levels of IgG antibodies in the mice of the oral immune recombinant strain group (OIG) were significantly higher than those in the control group (P<0.01); the levels of sIgA antibodies in the stool, nasal wash, reproductive tract wash, and intestinal mucus of the mice in OIG were significantly higher than those in the control group (P<0.01); Compared with the control group, the levels of cytokines in the serum of OIG were as follows: The levels of IFN-γ, IL-2, IL-4, IL-12 increased, the levels of IL-10 decreased, and the levels of IFN-α did not change significantly; Incubation of PCV2 and mouse spleen lymphocytes in vitro showed that the proliferation stimulating index of spleen lymphocytes in OIG was significantly higher than that of the control group (P<0.01); FCM results showed that CD4⁺ T cells and CD8⁺ T cells were higher than those of the control group; the results of fluorescent quantitative PCR showed that compared with the control group, the viral load in the OIG was significantly lower than that of the control group. In summary, the recombinant L. reuteri expressing the PCV2 cap protein were successfully constructed, and the constructed recombinant L. reuteri can stimulate mice to produce humoral and cellular immune responses after oragastrical immunization, and can exert a certain immune protection effect.

Vaccination is the key strategy to prevent and control classical swine fever in China. Detection of vaccine-induced antibody by ELISA is the main method to assess the efficacy of vaccination. In order to evaluate the ability of individual laboratories for antibody detection against classical swine fever virus, Certification and Accreditation of the People's Republic of China organized the national proficiency testing program “Diagnostic Techniques for Classical Swine Fever” (CNCA-19-A01). The National/OIE Reference Laboratory for Classical Swine Fever (NRLCSF) that affiliated to China Institute of Veterinary Drug Control is responsible for the implementation of the program. Positive and negative samples were prepared and numbered randomly. Each participant received 4 different samples, and then the samples were tested according to their selected ELISA methods. All participants should submit their test results and the original reports to NRLCSF. If the results is unsatisfied, the participant will have another chance to retest. If the result is still unsatisfied, the participant will be considered unqualified for CSFV antibody detection. One hundred and three institutions participated this program. The accurate rate is 92.23% (95/103) for the first round of test, and 100% for the first and second tests. This result demonstrated that all participate laboratories provide competence for the detection of CSFV antibody and they could meet the requirement for CSFV diagnosis and surveillance. This proficiency testing program could help participants to investigate the reason for disagreement and implement their deficiencies.

For the sake of verifying the immunogenicity of candidate epitope-polypeptide, the B and T cell epitopes of S1 protein of avian infectious bronchitis virus (IBV) H120 strain were predicted and the corresponding epitope-polypeptides were synthesized， and then were used to immunize mice, the immune effect was analyzed. Five epitope-polypeptides against S1 protein of IBV H120 strain were selected by epitope prediction software and acquired by chemical synthesis, then were immunized to mice. The specific antibodies, neutralizing antibodies and T lymphocyte subsets induced by each epitope-polypeptides were analyzed by indirect ELISA, neutralization test and flow cytometry. The ELISA results showed that the five epitope-polypeptides had good reactivity. The antibody titers of antisera induced by the five epitope-polypeptides sorted from high to low as follows: Pep76-106, Pep240-257, Pep511-537, Pep403-421, Pep135-172. The neutralization test results showed that the neutralization titers of antisera induced by the five epitope-polypeptides groups in mice were higher than that of the blank control group, and the order of neutralization titers was Pep240-257 = Pep403-421 = Pep511-537 > Pep76-106 = Pep135-172. The flow cytometry results showed that the percentages of CD3⁺, CD4⁺CD8^- and CD8⁺CD4^- T lymphocytes in all the five epitope-polypeptides groups were significantly higher (P<0.01) than those in the blank control group. The number of the CD3⁺ and CD4⁺CD8^- T lymphocytes sorted from large to small as follows: Pep403-421, Pep240-257, Pep76-106, Pep511-537 and Pep135-172. The number of the CD8⁺CD4^- T lymphocytes sorted from large to small as follows: Pep403-421, Pep76-106, Pep511-537, Pep240-257 and Pep135-172. In conclusion, Pep240-257, Pep76-106 and Pep403-421 could induce humoral immunity among the five epitope-polypeptides, while Pep403-421 could induce cellular immunity. Thus, peptide of Pep403-421 could induce cellular immunity and humoral immunity. This study laid a foundation for further understanding the immunological characteristics of the S1 protein and the development of diagnostic reagents and effective epitope vaccines.

To investigate the role of FABP4 on regulating fatty acid metabolism and autophagy in macrophage infected with BCG, small interfering RNA for FABP4 were transfected into RAW264.7 cells alone or combined with BCG infection. The immunoblot and immunofluorescence were used to detect the expression of FABP4, the accumulation of lipid droplets, the expression of fatty acid β-oxidation and autophagy related factors. The results showed that the infection of BCG increased the expression of FABP4 and accompanied with the accumulation of lipid droplets in RAW264.7. Moreover, the knockdown of FABP4 decreased lipid droplets but promoted the expression of carnitine palmitoyltransferase 1A (CPT1A) (P <0.05) and the production of ATP (P <0.05). Meanwhile, the knockdown of FABP4 suppressed the expression of AMPK, p-ULK1, ATG5, ATG7, ATG12 and LC3B which are autophagy related factors (P<0.001). Our results indicated that the knockdown of FABP4 promoted fatty acid oxidation, decreased fatty acid level and suppressed autophagy via AMPK signal pathway in BCG infected RAW264.7 cells.

When mammalian cells are stimulated by stress, multiple dense granular structures form in cells. These structures are called stress granules (SGs). G3BP1 is an important component and marker protein of stress granules. To dynamically monitor the formation of SG, the HeLa cell line stably expressing GFP-G3BP1 was constructed. The G3BP1 gene was amplified and cloned into a lentiviral vector to obtain a recombinant plasmid Lenti-GFP-G3BP1. The three-plasmid lentiviral packaging system was used to package the lentiviral particles expressing GFP-G3BP1, which were used to infect HeLa cells. The positive cells were initially selected by puromycin, and further selected by limited dilution to get the monoclonal cells stably expressing GFP-G3BP1. The function and expression of GFP-G3BP1 in the cell line were detected by Western blot and immunofluorescence assays. The obvious cytoplasm-specific fluorescence of G3BP1 was observed. Western blot results show a GFP-G3BP1 specific band, indicating that the HeLa cell line stably expressing GFP-G3BP1 was constructed successfully. Finally, the formation of SG in response to ARS/heatshock/Newcastle disease virus treatment were dynamically monitored. The results showed the gradual accumulation of SGs in response to these treatments. The specific cell line and method we established have laid the foundation for subsequent research on SG and Newcastle disease virus.

The aim of this study was to investigate the antimicrobial resistance, virulence genes of Campylobacter spp. isolated from chicken and swine farms in Jiangsu Province. Campylobacter spp. were isolated and identified from two hundred and fifty fecal samples collected from twenty-five animal farms in Jiangsu Province. Campylobacter strains were tested for the antimicrobial susceptibility against to 9 kinds of antimicrobial agents by using an agar dilution method. Eight virulence genes (cdtB, cadF, htrB, clpP, csrA, wlaN, cstⅡ, and cgtB) were detected by polymerase chain reaction. Ninety-three Campylobacter strains were isolated and identified from 250 samples, including 45 C. jejuni strains and 48 C. coli strains. The highest percentage of antimicrobial resistance was found for nalidixic acid (80.0%), tetracycline (71.1%) and ciprofloxacin (66.7%) in C. jejuni isolates. The C. coli isolates were most frequently resistant to erythromycin (87.5%), nalidixic acid (79.2%) and azithromycin (72.9%). Moreover, the multi-drug resistance (resistance to three or more antimicrobial classes) was common among Campylobacter isolates with a rate of 67.7%. On the other hand, the 93 Campylobacter strains showed a wide variation for the presence of the 8 virulence genes, cdtB and cadF were positive for all isolates，while htrB, clpP, csrA, wlaN, cstⅡ and cgtB was 97.8%, 76.3%, 18.3%, 5.4%, 2.2%, and 0%, respectively. The results indicated that the multiple drug resistance of Campylobacter strains from animal origin was relatively serious. In addition, the virulence-associated genes were detected widely among Campylobacter strains.

The purpose of this study was to investigate the variation of differential metabolites in mammary carcinomas bearing cats and healthy cats, and to explore the relationship between differential metabolites and the occurrence of feline mammary carcinomas. In this study, 6 feline mammary carcinomas serum samples were selected as test (T) group. Meanwhile, 6 healthy cats with similar age and same breed were selected as control (C) group. Serum samples were detected by ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (LC-MS). Differential metabolites were screened by principal component analysis (PCA), orthogonal projections to latent structures-discriminant analysis (OPLS-DA) and Student's t-test. Then the hierarchical clustering analysis (HCA) was carried out for the screened differential metabolites. The KEGG annotation and the metabolic pathway of differential metabolites were analyzed. The results showed that a total of 159 differential metabolites were identified in the 2 groups. Compared with C group, 49 differential metabolites were down-regulated and 110 differential metabolites were up-regulated in T group. Finally, a total of 5 differential metabolites which closely related to feline mammary carcinomas were selected. Ergothioneine (EGT) and creatine in T group were down-regulated, while indolelactic acid (IAA), choline and uric acid were up-regulated compared to C group. These differential metabolites indicated that during the development of feline mammary carcinomas, the body changes involved multiple metabolic pathways, such as glycine, serine and threonine metabolism, arginine and proline metabolism, histidine metabolism, tryptophan metabolism, purine metabolism and glycerophospholipid metabolism. This study provides a new idea for further research of the pathogenesis of feline mammary carcinomas.

In order to explore the species of endophytic fungi from a poisonous plant, Delphinium grandiflorum L. In this experiment, surface disinfection was used for separation, morphological identification was used, and phylogenetic tree was constructed using ITS sequence to determine the species of endophytic fungi. The results showed that 22 species of endophytic fungi were isolated from D. grandiflorum L., they were mainly divided into two groups according to their phylogenetic relationship, belonging to 6 classes, 6 orders, 6 families, and 7 genera. Among them, Alternaria sp. was the dominant species (accounting for 31.82% of the isolated strains, mainly distributed in flowers and leaves), endophytic fungi are most commonly distributed in flowers (accounting for 40.91% of the isolates), followed by leaves (31.82%), roots (22.73%) and stems (4.55%). The results indicated that the distribution of endophytic fungi in D. grandiflorum L. is different in different tissues.

The objective of this study was to explore the epidemic situation and pathogenic characteristics of swine influenza virus (SIV) in Shandong Province. In the spring of 2019, 130 swine nasal swab samples were collected from a slaughterhouse in Tai'an city, Shandong Province for virus isolation and identification. The whole genome of isolated virus was sequenced and analyzed. Meanwhile, 1 527 swine serum samples were collected from swine farms in 8 regions of Shandong province and their anti-SIV antibody were detected by HI assay using standard avian H9N2 antigen. The results showed that a H9N2 subtype influenza virus strain was isolated and named as A/swine/Shandong/TA009/2019(H9N2). The homology analysis showed that the isolated virus had close genetic relationship with A/environment-air/Kunshan/NIOSH-BL20/2018(H9N2) and A/environment-air/Kunshan/NIOSH-BL25/2018(H9N2), and the nucleotide homology of the gene fragments were above 99.5%. Phylogenetic analysis results demonstrated that HA and NA genes of the isolated virus belong to the Y280-like lineage, PB2 and M genes belong to the G1-like lineage, and PB1, PA, NP and NS genes belong to the SH/F98-like lineage. The cleavage site in HA protein is “PSRSSR/GL”, which was in accordance with the molecular biological characteristics of low pathogenic avian influenza virus.The position 216 of HA protein is L, and it has the ability to bind human-derived sialic acid α 2,6-Gal. The results of HI showed that 9 among 1 527 serum samples were positive with a positive rate of 0.59%. The isolated virus was swine-derived H9N2 virus, and serological investigations revealed that H9N2 subtype virus infection was present in swine herds in Shandong Province. The results of this study suggest that continuous surveillance of the SIV epidemiological situation and its pathogenic characteristics should be strengthened.

The interaction between JEV and the host at the miRNAs level was preliminarily explored by studying the miRNAs expression profiles of primary neurons in mice infected with JEV. Total RNA of JEV-infected and uninfected primary neurons of the suckling mice was extracted individually by Trizol and then analyzed miRNA expression profiles by high-throughput sequencing analysis. Significantly differentially expressed miRNAs were selected for verification by real-time quantitative PCR. Through bioinformatics analysis, 26 miRNAs with significant expression differences were screened out, among which 18 miRNAs were up-regulated and 8 miRNAs were down-regulated. The results of quantitative real-time PCR of the JEV-E gene indicated that the expressions of mir-21a-3p mir-223-5p mir-147-3p mir-155-5p and mir-146a-5p could promote the expression of JEV-E gene in neurons, while the expression of mir-301a was just on the contrast. The expression of miRNAs in primary neurons could affect the replication of JEV. This study provided the theoretical basis and direction for further studies on the regulatory function of miRNAs in the mechanism of neural dysfunction induced by JEV.

To further identify a new strain of Trichomonad isolated from the feces of a cat with chronic diarrhea, isolation and cultivation， morphological observation and gene homology analysis of 18S rRNA of the Trichomonad were accomplished in this study. Morphological observation showed that the Trichomonas was shaped pear or spindle, with 3 anterior flagella and 1 posterior flagella. The results of phylogenetic tree construction showed that Trichomonas isolated in this study is Trichomonas foetus, which is in the same evolutionary branch and is closely related to the sequence MH400076.1 in GenBank with 99% sequence similarity. Based on the above identification results, this Trichomonas isolated from cats with long-term chronic diarrhea is T. foetus.