Endometrial receptivity and decidualization are crucial parts of embryo implantation, which influenced the successful pregnancy and reproductive performance of mammalian. Therefore, it is of great significance to explore the regulatory mechanism of uterine physiological function to improve reproduction efficiency of female animals. Endometrial receptivity is an ability of the endometrium to allow blastocysts to localize, adhere, invade, and eventually implant. To transit into receptive state, the endometrium needs to undergo a series morphologically and functionally remodeling, during which endometrial stromal cells proliferate and differentiate into decidual cells, the process is called decidualization. Non-coding RNAs (ncRNAs) can affect a variety of physiological processes, including involvement in the regulation of endometrial function, and ceRNA network theories of lncRNA-miRNA-mRNA and circRNA-miRNA-mRNA are also important molecular mechanisms. In this review, the current research progresses of miRNA, lncRNA and circRNA in regulating mechanism of endometrial receptivity and decidualization were discussed, with a view to provide theoretical guidance and new ideas for further revealing mechanism of ncRNAs on the physiological function of mammalian uterus.

Recent years, techniques for in vitro embryo production (IVP) have advanced significantly, especially the ovum pick-up (OPU) technology grows widely. OPU is a new technology developed in the 1990 s that can obtain oocytes from excellent breeding heifers, which has a broad application prospect in the research of the mechanism of animal embryo production and development and in the clinical practice of human assisted reproductive technology. It can improve the resource protection efficiency of breeding females and precious wild animals, and the genetic potential of excellent females, and provide the necessary supporting technology for the development of cattle biotechnology. The quality of oocytes is the key to the success of the IVP system, and the in vitro maturation (IVM) process of the oocytes is critical for successful fertilization and embryo development. Oocyte IVM completes the cytoplasmic and nuclear maturation of the oocyte, preparing all necessary components for development to the activation of the embryonic genome. Developmental ability of oocytes matured in vitro is lower than that of oocytes matured in vivo, non-synchronization of oocyte meiosis and nucleocytoplasmic maturation, oxidative stress damage, et al are important for successful fertilization and embryo development. Therefore, it is necessary to further improve the in vitro maturation ability of oocytes to improve the bovine IVP system. In this paper, the problems of in vitro maturation of oocytes such as non-synchronization of nucleocytoplasmic maturation and oxidative stress were summarized, as well as the improvement effects of C-type natriuretic peptide, melatonin, FLI and other substances on oocytes IVM were discussed in order to improve the bovine IVP system.

The reproductive performance of sheep is a very important economic trait. The number of lambs is directly related to the number of ovulation each time, and the growth and development of ovum is influenced by the interaction of ovarian endocrine and paracrine. FecB gene is the first multifetal major gene discovered in sheep. The BMP signaling pathway in the ovary of sheep carrying this gene is damaged and the activity is reduced, and some members of TGF-β family are inactivated, which inhibits the proliferation of GCs and improves the sensitivity of FSH, effectively improves ovulation rate and lambing rate of sheep, which is conducive to expanding the breeding scale and improving economic benefits. In this paper, the discovery of FecB gene, its action mechanism, its effect on follicular development, reproductive ability and offspring growth and development, as well as its application in breeding were reviewed, in order to provide reference for the mechanism research and breeding work of improving sheep reproductive performance.

The prevalence of numerous genetic subtypes strains of bovine viral diarrhea virus (BVDV) and the expansion of its host spectrum interfere with the prevention and control of BVDV. European countries have adopted the measure of comprehensive detection and culling of BVDV-positive cattle and achieved a certain prevention and control effect, but considering the actual production situation in China, vaccination is still the optimal strategy. An in-depth understanding of the ‘game’ between BVDV proteins and the host immune system during virus infection will help researchers to effectively avoid the potential interference of antigen candidate proteins on immune effects in the term of vaccine development. In addition, vigorously developing low-cost, high-efficiency new-type vaccines (such as epitope peptide vaccine and virus-like particle vaccine) can enhance the biosafety of BVDV vaccines based on integrating viral immunodominant antigenic epitopes, and meet the requirements for the prevention and control of BVDV with vaccines. Thus, in order to provide proof-of-concept towards the development of BVDV vaccine and BVDV prevention and control, this paper reviews the researches on the immunological function and characteristics of BVDV proteins and the current situation of BVDV vaccine development.

Salmonella is a zoonotic pathogen. It has a significant impact on human and animal health, and has caused significant economic losses to animal husbandry. Therefore, it is urgent to explore its pathogenic mechanism. Lipopolysaccharide, located in the outer membrane of Salmonella, is an important virulence factor. The modification of its structure can affect the immune escape ability, resistance and virulence of Salmonella. Based on this phenomenon, the mutant strains obtained by artificially modifying LPS can be used in the prevention and control of salmonellosis. In addition, low-toxicity derivative of LPS can be used to be vaccine adjuvants due to its useful immunostimulatory properties. Based on the above, the structural modification and its effects, biosynthesis and transport, and the application of Salmonella lipopolysaccharides are reviewed in this paper. Grasping the progress of this research is conducive to the in-depth study of the pathogenic mechanism of Salmonella,as well as the prevention and control of disease.

The purpose was to explore candidate genes related to the eye muscle area, predicted lean meat percentage and average backfat thickness of Large White pigs by using the weighted single-step genome wide association study and making full use of the phenotype, pedigree and genotype information of the population. The phenotypic record data of eye muscle area, predicted lean meat percentage and average backfat thickness of 21 754 Large White pigs were collected, including 1 259 individuals with genotype data. Through analysis of variance and the weighted single-step genome wide association study, quantitative trait loci (QTL) that are significantly related to traits were determined. The gene annotation, gene ontology (GO) and kyoto Encyclopedia of genes and genes (KEGG) pathway enrichment analysis were carried out. The results showed that the heritability of eye muscle area, predicted lean meat percentage and average backfat thickness in Yorkshire pigs were 0.450 6±0.017 3, 0.496 8±0.017 4 and 0.475 8±0.017 2, respectively. Using the weighted single-step genome wide association study, 10 candidate QTL regions significantly associated with eye muscle area, 8 candidate QTL regions significantly associated with predicted lean meat percentage, and 12 candidate QTL regions significantly associated with average backfat thickness were localized. Follow-up gene annotation, GO function and KEGG pathway enrichment analysis revealed 36 candidate genes significantly associated with eye muscle area, with a total of 7 terms enriched; 29 candidate genes significantly associated with predicted lean meat percentage, with a total of 2 terms enriched; and 41 candidate genes significantly associated with average backfat thickness, with a total of 11 terms enriched. Analysis of the genes contained in these terms showed that some of these candidate genes were associated with growth and development, muscle fat and bone development. For example, ADAL gene affects adipocyte differentiation and other activities; FGF9 gene is reported to be involved in bone development and other processes. In this study, the important candidate genes related to growth traits of Yorkshire pigs were located through the weighted single-step genome wide association study. The results extended the related research of growth traits of Yorkshire pigs, and provide important molecular markers for genetic improvement of growth traits in the future.

This study aimed to explore the correlation among growth traits in Ujumqin sheep and the optimal regression equation of body size to body weight was established, and analyze the effect of TRHDE gene polymorphism on the growth traits of Ujumqin sheep. In this study, 336 well-developed Ujumqin sheep (176 males and 160 females) were selected as research objects, and growth traits were collected at 4-month-old and 6-month-old. Genotypes of SNPs in the TRHDE gene were detected using the sheep Illumina OvineSNP600 BeadChip in a population of 336 Ujumqin sheep. SPSS 26.0 software was used for correlation and regression analysis among growth traits and association analysis between TRHDE gene polymorphism and growth traits. The results showed that the optimal regression equations of 4-month-old and 6-month-old male and female Ujumqin sheep were as follows:Y₁=-53.66+0.77X₁+0.39X₂, Y₂=-47.83+0.63X₁+0.45X₂, Y₃=-47.01+0.42X₁+0.13X₂+2.82X₃+0.19X₄+0.28X₅, Y₄=-61.32+0.25X₁+0.40X₂+3.15X₃+0.39X₄. The results of the association of TRHDE gene SNPs with growth traits in Ujumqin sheep showed that rs404402551A>G locus was significantly or extremely significantly associated with body weight and chest circumference at 4-month-old and body weight, chest circumference and chest width at 6-month-old (P<0.05; P<0.01). The rs415608385C>A locus was significantly or extremely significantly associated with body weight at 4-month-old and body weight, body height, body length and chest width at 6-month-old (P<0.05; P<0.01). The rs426657887G>A locus was not significantly associated with each growth trait in Ujumqin sheep (P>0.05). And the rs404402551A>G and rs426657887G>A loci were in strong linkage disequilibrium in the Ujumqin sheep. In conclusion, the greater potential for selection of body weight in Ujumqin sheep and chest circumference can be used as an important indicator for assessing body weight in Ujumqin sheep. The rs404402551A>G and rs415608385C>A loci of the TRHDE gene can be used as potential molecular markers for growth and development in Ujumqin sheep, which can provide reference for molecular marker-assisted selection for breeding in Ujumqin sheep.

The aim of this study was to investigate the regulatory effects of programmed cell death 4 (PDCD4) on the apoptosis of mammary epithelial cells (GMECs) and milk composition in goat. In this study, the dual luciferase reporter assay was used to verify the targeting relationship between miR-21-5p and PDCD4, and the regulatory effects of miR-21-5p on PDCD4 was detected by RT-qPCR and Western blot. The PDCD4 small interfering RNA (si-PDCD4) and PDCD4 overexpression vector (pc3.1-PDCD4) were transfected into GMECs to explore the effect of PDCD4. EdU and CCK-8 tests were used to detect the effect of PDCD4 on the proliferation of GMECs, flow cytometry was chosen to detect its effect on apoptosis of GMECs. After transfection, the ELISA kit was used to detect the secretion of β-casein and triglyceride (TG) in GMECs. Finally, the Western blot tests was used to detect the protein expression of PI3K, mTOR, S6K, ERK, Bcl-2, Bax in GMECs. The results of the dual luciferase reporter assay showed that PDCD4 was one of the target genes of miR-21-5p. The results of the EdU, CCK-8 and flow cytometry tests showed that the activity of GMECs was significantly decreased after overexpression of PDCD4 (P<0.01), indicated that PDCD4 inhibited the proliferation of GMECs and promoted the apoptosis of GMECs (P<0.01); the activity of GMECs was significantly increased after interfering PDCD4 (P<0.01), the pro-apoptotic level of GMECs was significantly reduced (P<0.01). The results of the ELISA kit test showed that the secretion of β-casein and TG in GMECs was significantly decreased after overexpression of PDCD4 (P<0.01), and they were significantly increased after interfering PDCD4 (P<0.01). The results of the Western blot test showed that, compared with the control group, the protein expressions of phosphorylated PI3K, mTOR, S6K and ERK were significantly decreased after overexpression PDCD4 (P<0.05), the protein expression of Bax was significantly up-regulated (P<0.01), and the protein expression of Bcl-2 was significantly down-regulated (P<0.01). After interfering PDCD4, the protein expression of phosphorylated PI3K, mTOR, S6K and ERK were significantly increased (P<0.05), the protein expression of Bax was significantly decreased (P<0.05), and the expression of Bcl-2 was significantly up-regulated (P<0.05). This study indicates that PDCD4 promotes the apoptosis of dairy goat mammary epithelial cells by activating the ERK/Bcl-2/Bax signaling pathway, and decreases the secretion of β-casein and TG in dairy goat mammary epithelial cells by inhibiting PI3K/mTOR/S6K signaling pathway. And miR-21-5p targets PDCD4 and can regulate its expression in GMECs.

In order to elucidate the expression pattern of AGRP gene in goat skin and its action mechanism on melanin production. The tissue expression level of AGRP gene in different tissues of Youzhou dark goats and skin tissue of 3 Banjiao goats were detected by qRT-PCR. And the effects of exogenous AGRP protein at different concentrations on melanocytes were analyzed by in vitro experiments. The cell viability was detected by EdU method, and cAMP activity was detected by cyclic adenosine monophosphate kit, as well as its effects on the content of eumelanin and the mRNA expression of melanin-related genes MC1R, TYR, TYRP1 and DCT. This results showed that AGRP gene was expressed in different tissues of Youzhou dark goats and its expression in the pituitary gland was the highest (P<0.05). Meanwhile, the expression level of AGRP in the skin of Banjiao goat and Youzhou dark goat were significantly different (P<0.05). The addition of different concentrations of AGRP protein had no significant effect on the melanin production and proliferation of melanocytes (P>0.05). cAMP activity in the melanin production pathway significantly decreased (P<0.05) with the increasing of AGRP concentration. The 200 nmol·L^-1 AGRP significantly inhibited the expression of MC1R mRNA and enhanced the mRNA expression of TYR and TYRP1 (P<0.05). The addition of exogenous α-MSH at different concentrations could significantly (P<0.05) salvage the effects of AGRP on function of melanocyte. AGRP gene is closely related to that phenotype of Youzhou dark goat skin. Determining the biological function of AGRP in melanin production can lay the foundation for further revealing the mechanism of AGRP on melanin deposition in skin of Youzhou dark goat.

A total of 1 937 body weight and body size traits data of 564 Jinnan cattle (279 bulls and 285 cows) from 2011 to 2021 were collected from the National Jinnan Cattle Genetic Resources Gene Protection Center in Yuncheng, Shanxi Province. Genetic parameters of body weight and body size traits of Jinnan cattle at birth, 6, 12, 18 and 24 months of age were estimated. Using REML algorithm in ASREML software and multi-trait animal model, with birth year, birth season and sex as fixed effects, the heritability, genetic correlation and phenotypic correlation of body weight and body size traits in 5 different growth stages of Jinnan cattle were estimated. The results showed that birth year had significant effects on the body weight and body size traits at different growth stages of Jinnan cattle(P<0.01). Birth season and sex had little effect on early growth and development of Jinnan cattle, but had great effect on late growth and development. The estimated heritability of body weight from birth to 24 months of age ranged from 0.22 to 0.35, the estimated heritability of body height ranged from 0.18 to 0.31, the estimated heritability of stature ranged from 0.17 to 0.43, the estimated heritability of body length ranged from 0.10 to 0.24, and the estimated heritability of chest girth ranged from 0.22 to 0.29. The estimated values of abdomen circumference heritability ranged from 0.25 to 0.33, and the estimated values of shin circumference heritability ranged from 0.16 to 0.56. The body weight and body size traits of Jinnan cattle at different growth stages showed strong positive genetic and phenotypic correlations. The genetic and phenotypic correlations ranged from 0.37 to 0.97 and 0.47 to 0.96, respectively. The genetic parameters of growth and development traits of Jinnan cattle were systematically evaluated and analyzed in this experiment. It was found that the body weight and body size at different growth stages of Jinnan cattle belonged to medium and high heritability traits, and the genetic correlation and phenotypic correlation of these traits showed strong positive correlation. Among them, weight, stature and shin circumference at 18 months of age could be considered as the target traits for constructing selection index of Jinnan cattle due to their high heritability, strong genetic correlation and phenotypic correlation. The results of this experiment can provide a theoretical basis for the scientific conservation of Jinnan cattle and the development and utilization of germplasm resources.

This study aimed to explore the role and regulatory mechanism of lncRNA-LRTN4RL1- AS/miR-27a-3p/PPARγ axis in milk fat synthesis of dairy cows. Bovine mammary epithelial cells (BMECs) were used as experimental objects. Three biological repeats were carried out for the treatment group and the control group. Real time quantitative PCR (RT-qPCR), Western blot (WB), Oil Red O staining, triglyceride detection and double luciferase report were used to detect the effect of lncRNA-LRTN4RL1-AS on the expression of triglyceride accumulation and lipid synthesis related genes in BMECs. LRTN4RL1-AS-miR-27a-3p-PPARγ had targeting relationship of competing endogenous RNAs(ceRNA) was verified. The results showed that LRTN4RL1-AS was mainly expressed in the cytoplasm. After interfering with LRTN4RL1-AS, the lipid droplet formation and triglyceride accumulation of BMECs were significantly inhibited (P<0.05), and the expression of lipid synthesis related genes CEBPα (P<0.01), CEBPβ (P<0.01) and PPARγ were significantly down-regulated (P<0.05), and the expression of lipid catabolism gene HSL was significantly up-regulated (P<0.05). Double luciferase report assay showed that the luciferase activity in mimics-miR-27a-3p in LRTN4RL1-AS mutant group was significantly higher than that in LRTN4RL1-AS wild type group (P<0.01). It was confirmed there was a targeted binding relationship between LRTN4RL1-AS and bta-miR-27a-3p. Cotransfection experiment result showed that inhibitor-miR-27a-3p reversed the inhibition effect of interference LRTN4RL1-AS on milk fat synthesis (P<0.05), and reduced the inhibition effect of si-LRTN4RL1-AS on PPARγ gene expression. The results show that LRTN4RL1-AS regulates milk fat synthesis of BMECs by competitively binding miR-27a-3p to regulate the expression of PPARγ.

This study aimed to investigate the regulation and mechanism of Mtmr3 (myotubularin related protein 3) on proliferation and differentiation of C2C12 cells. In this study, mouse myoblast cell line (C2C12) was used as experimental material. qRT-PCR and immunofluorescence staining were used to detect the mRNA expression level of Mtmr3 gene in myoblast growth and differentiation stages, and the distribution morphology on day 6 of differentiation. The small interfering RNA (siRNA) of Mtmr3 was synthesized and divided into siRNA NC control group and Mtmr3 siRNA treatment group (n=3). EdU, CCK-8, qRT-PCR and Western blot were used to detect the influence of Mtmr3 siRNA on myoblast proliferation and differentiation, and the relevant mechanisms of regulating myoblast proliferation and differentiation were studied through signal pathways. The results showed that the mRNA expression level of Mtmr3 decreased in the growth stage, but increased gradually in the differentiation stage, and the immunofluorescence staining of Mtmr3 showed a myotube shape. After interfering with Mtmr3, mRNA and protein expression levels of Mtmr3 gene were significantly decreased (P<0.01). In proliferation test, the percentage of EdU positive cells in total cells and cell viability were significantly increased after transfection of Mtmr3 siRNA (P<0.01). After the interference of Mtmr3 expression, the mRNA and protein expression levels of Pcna and Cdk4 were significantly increased (P<0.01), and the mRNA expression level of Ccnd gene was significantly increased (P<0.01). In the differentiation test, the expression level of differentiation marker gene Myhc protein was significantly inhibited after transfection of Mtmr3 siRNA (P<0.01), and the mRNA expression levels of Mtmr3, Myog and Myhc genes were significantly inhibited at the 4th and 6th day of cell differentiation (P<0.01). After interfering Mtmr3 expression in proliferative stage and differentiation stage, phosphorylated protein expression levels of Mtor and P70s6k were significantly increased and significantly decreased, respectively (P<0.01).The results of CCK-8 showed that Mtmr3 siRNA could counteract the inhibitory effect of Rapamycin (Rapa), a specific inhibitor of Mtor pathway, and promote myoblast proliferation compared with the control group (P<0.01); The mRNA expression levels of Mtor, P70s6k, Myog and Myhc genes were significantly decreased in myoblasts treated with Rapa during cell differentiation (P<0.01). In summary, the expression level of Mtmr3 in myoblast growth stage showed a downward trend, while that in differentiation stage showed a gradual upward trend, and the immunofluorescence staining of Mtmr3 showed a myotube shape. Interfering with Mtmr3 expression promotes myoblast proliferation by activating the Mtor pathway, and inhibits myoblast differentiation by inactivating the Mtor pathway.

This study aimed to analyze the expression of Toll-like receptor 7/8 (TLR7/8) in boar sperm and reproductive organs, and to explore whether boar X sperm and Y sperm could be separated by their ligand treatment. In this study, the mRNA expression levels of TLR7 and TLR8 genes in testis, caupt, corpus, caudu and sperm of 3 healthy adult boars were analyzed by qRT-PCR. The protein expression pattern of TLR7 and TLR8 in testis and epididymis of boar was investigated by immunohistochemistry. Their expression in sperm of different species (healthy adult mice, bulls and boars) was analyzed by cellular immunofluorescence. Finally, the effects of TLR7 and TLR8 on sperm motility and X/Y sperm separation were investigated by co-incubating their ligand R848 with boar sperm. The results showed that TLR7/8 mRNAs were expressed in boar testis, caupt, corpus, caudu and sperm. TLR7/8 proteins were mainly expressed in germ cells in testis and in columnar cell microvilli in epididymis. TLR7/8 proteins were only expressed in the tail of mouse and bull X sperm, but not in Y sperm. However, TLR7/8 proteins were expressed in both boar X and Y sperms with no significant difference in expression pattern. And TLR7 protein was mainly located in the acrosome of boar sperm head, while TLR8 protein was mainly located in the tail of boar sperm. Compared with the control group, the sperm motility of the treated group with TLR7/8 ligand R848 was reduced, but the sex ratio of the upper sperms was not significantly different (P>0.05). In conclusion, there are no significant difference in the expression of TLR7/8 between boar X sperm and Y sperm, but their expression patterns are different from those of mouse and bull sperm, so TLR7/8 may not be suitable for the separation of boar X sperm and Y sperm.

The purpose of this experiment was to study the effects of PGF2α injection at different luteal stages on luteal tissue morphology, expression of PGF2α receptors and programmed necroptosis-associated genes in Hu sheep. Forty eight healthy ewes with similar body weight were used in this study, which were in good condition and have undergone estrus synchronization. They were randomly divided into 6 groups, namely the early-luteal phase experiment group and control group, the mid-luteal phase experiment group and control group, and the late-luteal phase experiment group and control group. The ewes in 3 experimental groups were injected with 1 mL PGF2α (0.1 mg) on day 6 (early-luteal phase), day 11 (mid-luteal phase) and day 16 (late-luteal phase), respectively. The ewes in 3 control groups were injected with 1 mL normal saline at the same time. The corpus luteum tissue was collected at 3 h after each injection for gene expression detection and HE staining for the detection of morphological changes of corpus luteum. The results showed that, after the injection of PGF2α, the morphology of corpus luteum changed, with inflammatory cell infiltration and apoptosis, especially in the mid-luteal phase, with a higher degree of degeneration. For ewes that were not injected with PGF2α, the mRNA expression level of FPA in the corpus luteum in the early-luteal phase was significantly higher in the early-luteal and mid-luteal phases compared to the late-luteal phase (P<0.05); the FPB expression level in the corpus luteum in the early-luteal phase was significantly higher than that in the late-luteal phase (P<0.05), and there was no significant difference from the mid-luteal phase (P>0.05); the expression level of TNFR1 in the mid-luteal phase was significantly higher than that in the early-luteal and late-luteal phases (P<0.05); The expression level of RIPK3 in the mid-luteal phase was significantly higher than that in the late-luteal phase (P<0.05), and both of them was no significant difference to the early-luteal phase (P>0.05); TNF-α, RIPK1 and MLKL expression levels did not significantly change throughout the luteal phase (P>0.05). In the early-luteal phase, injection of PGF2α significantly reduced the expression of FPA (P<0.05), and significantly increased the expression of FPB, RIPK1, MLKL (P<0.05), and had no significant effect on the expression of TNF-α, TNFR1, and RIPK3 (P>0.05).In the mid-luteal phase, injection of PGF2α could significantly up-regulate the expression of FPB, TNF-α, RIPK1 and MLKL (P<0.05), but had no significant effect on the expression of FPA, TNFR1 and RIPK3 (P>0.05); In the late-luteal phase, injection of PGF2α could significantly up-regulate the expression of RIPK1 (P<0.05)), had no effect on the expression of FPA, FPB, TNF-α, RIPK3 and MLKL (P>0.05). The mRNA expression level of FPA was positively correlated with RIPK3 and TNFR1 (P<0.05), and the mRNA expression level of FPB was positively correlated with MLKL and TNF-α (P<0.05). The results show that PGF2α may initiate necroptosis through the TNF-α/TNFR1 pathway after binding to its receptors FPA and FPB, and promote the degeneration of the corpus luteum. FPB plays a major role in mediating PGF2α-induced luteal degeneration and necroptosis, and PGF2α has a better effect on inducing luteal degeneration in the mid-luteal phase.

The aim of the present study was to clone goat BIRC5, and to reveal the role of BIRC5 gene in regulating the cycle and apoptosis of goat testis cells through the overexpression or interference of BIRC5. These data will be beneficial for exploring the role of BIRC5 gene in goat. The BIRC5 gene sequence was cloned by RT-PCR from spleen tissue of 3 three-day-old healthy Jianzhou male goats with 3 kg of body weight, and sequenced for further bioinformatics analysis using online softwares. The clone recovery product was ligated to a eukaryotic expression vector to construct pcDNA3.1(+)-BIRC5. Effective siRNA was designed and screened based on goat BIRC5 gene sequence. After pcDNA3.1(+)-BIRC5 and siRNA were respectively transfected into goat testicular cells, the expression of BIRC5 protein was detected by Western blot, and cell cycle and apoptosis were detected by flow cytometry. Meanwhile, the expression of apoptosis-related genes Bax, Caspase3, Caspase7, Bcl-2, p53, BCL2L11 and PARP1 were detected by RT-qPCR. A length of 506 bp BIRC5 gene sequence was cloned successfully, including 28 bp of 5'UTR, 429 bp of CDS region, and 49 bp of 3'UTR,encoding 142 amino acids. The phylogenetic tree showed that BIRC5 had the closest relative to Bos taurus. The overexpression of BIRC5 gene inhibited the apoptosis of cells, and the cells were arrested in G2/M+S phase; it also down-regulated the expression of Caspase7, p53, BCL2L11, Bcl-2 and Bax gene mRNA, but did not significantly change the mRNA expression of Caspase3 and PARP1. siRNA interference of BIRC5 gene promoted the apoptosis of cells, and the cells were arrested in G0/G1 phase; it also up-regulated the expression of Caspase3, Caspase7 and PARP1 gene mRNA, and down-regulated the expression of p53 and Bcl-2 gene mRNA, but did not significantly change the mRNA expression of Bax and BCL2L11. Goat BIRC5 gene could inhibit the apoptosis of testis cells and promote the arrest of goat testicular cells in G2/M+S phase. These data may lay a foundation for further study of BIRC5 gene function.

The purpose of this study was to explore the effect of TAS1R3 gene on the expression of autophagy-related genes in Congjiang Xiang pigs through mTOR. In this experiment, three 30-day-old healthy male Congjiang Xiang pigs were selected, testis tissue was collected and Leydig cells were cultured. The interference vector and empty vector were transfected into the cells, and their effects on the expression of TAS1R3 gene were detected by qRT-PCR. The effects of TAS1R3 gene interference on the expression of taste receptor family 1 receptor (TAS1R1), extracellular signal-regulated kinase 1 (ERK1), extracellular signal-regulated kinase 2 (ERK2), rapamycin target protein (mTOR), BECN1 and microtubule-associated protein 1 light chain 3 β (MAP1LC3B) were detected by qRT-PCR and Western blot. The results showed that when TAS1R3 gene was interfered, compared with shRNA-NC control group, the mRNA expression of TAS1R1, ERK1, ERK2 and mTOR genes decreased significantly(P<0.01), while the expression of autophagy factors BECN1 and MAP1LC3B increased significantly (P<0.01). The results of Western blot showed that when TAS1R3 gene was silenced, compared with shRNA-NC control group, the expression of T1R3, T1R1, p-mTOR, p-S6K1 and p-ERK12 decreased significantly(P<0.01), while the expression of autophagy protein Beclin 1 increased significantly(P<0.01). In this experiment, by transfecting TAS1R3 gene interference vector into Leydig cells, it was found that when TAS1R3 gene was interfered, it could affect the expression of mTOR gene, and then regulate the expression of downstream autophagy factors, suggesting that TAS1R3 gene is closely related to autophagy of Leydig cells, which provides a reference for further study on the regulation mechanism of TAS1R3 gene affecting the reproduction of Xiang pigs by regulating autophagy.

As an anti-nutrient, phytic acid was widely presented in plant feed materials, and it was often complexed with Cu²⁺, Zn²⁺, Fe²⁺, Ca²⁺, Mg²⁺ and other ions to reduce the bioavailability of mineral elements in livestock and poultry. Phytase could effectively degrade phytic acid, thereby releasing the complexed metal elements, thus improving the utilization of Cu, Zn, etc., and reducing the excretion. The purpose of this experiment was to determine the effect of phytase supplementation on the production performance of broilers and the emission of Cu, Zn and other elements in broiler chickens under reduced dietary Cu levels. A total of 480 healthy one-day-old broilers were randomly divided into 3 groups with 8 replicates in each group. The control group was fed a standard diet (8 mg·kg^-1 Cu without phytase), and the dietary Cu levels of the two experimental groups were 4 mg·kg^-1(50% Cu) and 0 mg·kg^-1 (0% Cu), and 1 200 U·kg^-1 phytase was supplemented to the diets, respectively. The production performance of broilers and the content of Cu, Zn and other elements in blood and bones were determined. Metabolic tests were carried out at 4 and 7 weeks to determine the apparent utilization of Cu and Zn and their excretion in feces. The results showed that phytase supplementation had no significant effect on the production performance of broilers at all stages when the dietary copper level was reduced (P>0.05), but had a promoting effect on the 21 d broiler tibia index (P=0.07), and the Mn content of the tibia in 0% Cu group was significantly higher than that in the CON group (P<0.05) at 21 d. In the 4th week, the apparent bioavailability of Ca, P, Cu, Zn, and Mn in the two Cu decreasing groups were significantly increased (P<0.05), and the apparent metabolic rates of Cu, Ca, and P increased in a dose-dependent manner. At the 7th week, the apparent metabolic rates of Cu and Zn in the 50% Cu group were significantly higher than those in the CON group (P<0.05). Compared with the CON group, the excretion of Cu, Zn and Mn in 0% Cu and 50% Cu groups were significantly decreased at the 4th week (P<0.05), and the excretion of Cu at the 7th week was significantly decreased (P<0.05). In conclusion, the supplementation of 1 200 U·kg^-1 phytase in the diet had no obvious effect on the production performance of broilers when the diet Cu level was reduced to 50% or 0% of the standard, but it could increase the apparent utilization rate of dietary Cu, Zn and reduce the excretion of fecal Cu and Zn.

The aim of this experiment was to study the effects of different crude protein levels of artificial pigeon milk on growth performance, serum antioxidant and intestinal development of the suckling pigeons in order to determine a reasonable crude protein level of artificial pigeon milk. One hundred and ninety-two 7-day-old suckling pigeons were randomly divided into 4 groups with 8 replicates and 6 suckling pigeons in each group. They were fed artificial pigeon milk with crude protein levels of 16.5%, 18%, 19.5% and 21% (CP16.5%, CP18%, CP19.5% and CP21% groups), respectively, for 18 days. The results were showed as follows:1) There were no significant differences in initial body weight, final body weight, average daily gain (ADG), average daily feed intake (ADFI) and feed/gain ratio (F/G) among the groups of suckling pigeons(P>0.05). 2) The carcass rate of suckling pigeons in CP21% group was the highest and significantly higher than those of CP18% and CP19.5% groups (P<0.05); The half-evisceration rate in CP16.5% group was the highest and significantly higher than that in CP19.5% group (P<0.05); There was no significant difference in the evisceration rate among all groups (P>0.05); The breast muscle rate in CP21% group was the highest and significantly higher than that of CP18% group (P<0.05). 3) The apparent metabolic rates of crude protein, crude fat and dry matter in the CP16.5% group were the highest, but only the apparent metabolic rates of crude protein and dry matter in the CP16.5% group were significantly higher than those of the other groups (P<0.05), and there was no significant difference in the apparent metabolic rates of crude fat among all groups (P>0.05). 4) The spleen index was significantly higher in the CP18% group than other groups (P<0.05); The kidney index in the CP16.5% group was significantly lower than other groups (P<0.05); The gizzard index in the CP16.5% group was the highest and significantly higher than other groups (P<0.05); The bursal index in the CP16.5% group was the highest and significantly higher than the CP21% group (P<0.05); The heart, liver and glandular stomach indices of the suckling pigeons had no significant difference among all groups (P>0.05). 5) The height of the duodenal villi in the CP21% group was significantly lower than those of the other groups (P<0.05), the depth of the duodenal crypt had no significant difference among all groups (P>0.05), and the ratio of the duodenal villi height to crypt depth (V/C) ratio in the CP21% group was significantly lower than those of the CP18% and CP19.5% groups (P<0.05). The jejunum and ileum morphology index had no significant difference among groups (P>0.05). 6) Serum malondialdehyde in the CP16.5% group was significantly lower than the CP21% group (P<0.05), while serum total antioxidant capacity, catalase and superoxide dismutase levels among all groups had no significant difference (P>0.05). In conclusion, the crude protein level of artificial pigeon milk at 16.5% to 21% could maintain the normal growth performance of pigeons, and with the increase of crude protein level of artificial pigeon milk, the carcass rate and breast muscle rate of pigeons increased, but the serum antioxidant capacity decreased. The highest crude protein apparent metabolic rate was found at 16.5% crude protein level of artificial pigeon milk, and it could meet the nutritional needs of growing pigeons. Therefore, the crude protein level of artificial pigeon milk is recommended to be 16.5% under the conditions of this experiment.

The purpose of this study was to explore the effects of cocktail composed of four pathogenic phages on growth performance, blood routine, serum biochemical index, antioxidant capacity, inflammatory cytokines, immunoglobulin and fecal microbiota of weaned piglets, so as to evaluate the safety of preparation of phage in vivo. Twelve healthy 21-day old weaned piglets with similar body weight were randomly divided into control group (CON) and phage group (Phage) with 6 piglets per group. The phage group was given 10 mL phage cocktail by gavage for 20 consecutive days (the total amount of each phage was 5×10⁸ PFU), and the control group was gavaged equal volume of SM buffer. Blood and stool samples were collected at day 11 and 21. The results showed that:1) there was no significant difference in growth performance between the two groups. 2) There was no significant difference in blood routine indexes between the two groups on the 11th and 21th day (P>0.05). The albumin content in serum biochemical indexes of piglets in phage group increased significantly on the 11th day (P<0.05), but returned to the level of control group on the 21th day. No significant difference was present in other serum biochemical indexes between the two groups on the 11th and 21th day (P>0.05). 3) Compared with the control group, the enzymatic activity of CAT in serum of piglets in phage group had an upward trend (P=0.066), and no significant difference was found in other antioxidant indexes between the two groups (P>0.05). 4) At day 11, the serum IFN-γ, TNF-α and IL-1β levels of the piglets in the phage group were significantly increased, (P<0.05), and the IL-10 level was significantly decreased (P<0.05). At day 21, the levels of IFN-γ, TNF-α, IL-17 and TGF-β in the phage group were significantly increased (P<0.05), and the IL-4 and IL-10 were significantly decreased (P<0.05). 5) Compared with the control group, the levels of IgA, IgG and IgM in serum of piglets in phage group at 21 days were significantly increased (P<0.01). 6) There was no significant difference in α diversity indexes of fecal microbiota between the two groups at day 11 (P>0.05). On day 21, compared with the control group, the Shannon index in phage group was significantly increased (P<0.05). β-diversity analysis showed no significant difference in microbial community composition between the two groups at day11 and 21. The results of differential flora analysis showed that there were no bacterial families and genera with significant differences in relative abundance between the two groups on the 11th day, while on the 21th day, compared with the control group, the relative abundance of Oscillospiraceae in the phage group was significant increased (P<0.05), and this difference at the genus level was manifested as a significant increase in the relative abundance of Ruminococcus_gauvreauii_group (P<0.05). In conclusion, continuous gavage of the phage cocktail for 20 days had no significant effect on the growth performance and routine blood parameters of piglets; It has a positive effect on antioxidant function and significantly improves the α diversity of bacterial community. At the same time, the serum levels of proinflammatory cytokines and immunoglobulin were significantly increased.

Porcine circovirus type 3 (PCV3) is a new genotype of porcine circovirus firstly reported cause reproductive in sows in the United States in 2016, pathogenesis is not fully known. In order to study the pathogenicity of PCV3 in weaned piglets, a PCV3 strain was isolated using PK-15 cells from clinical tissue samples in this study and was named as PCV3/CH/HB/XY/2018, which belonged to PCV3a-IM genotype indicated by whole genome sequencing (WGS). Groups of five 28-day old weaned piglets were infected by both nasal inoculation and intramuscular injection with PCV3 positive tissue homogenate and the 8^th generation virus culture, respectively, meanwhile, a blank control group was set up. The results of infection test in piglets were evaluated in terms of clinical manifestation, body temperature, weight gain, viremia, tissue distribution and pathological changes. The results showed that after infection with PCV3 positive tissue homogenate or virus culture, the piglets in both groups showed high body temperature, emaciation, dermatitis and slow growth, and viremia lasted 14 days but disappear before 21 days post infection. Meanwhile, PCV3 was mainly found in tonsils and lymph nodes, and the highest viral load of PCV3 was found in spleen, lung and tonsils, the pathological changes in the lungs were characterized by interstitial pneumonia, lymphoid nodular hyperplasia and eosinophilia. This study proved that PCV3/CH/HB/XY/2018 was pathogenic to weaned piglets, and the isolation of this virus laid a foundation for the PCV3's pathogenesis mechanism research of PCV3-associate disease.

To investigate the prevalence and genomic characterization of porcine reproductive and respiratory syndrome virus (PRRSV) in Fujian province and provid a theoretical basis for the prevention and control of PRRS, the complete genome of 32 isolates from Fujian province from 2017 to 2021 were analyzed. Tissues and serum were collected from suspected of being infected with PRRSV in Fujian during 2017-2021 were used for virus isolation and full-length genomics sequences amplification by RT-PCR. Genome analyses were performed using the DNASTAR 7.0 package. Phylogenetic trees were constructed by the MEGA 7.0 software using the neighbor-joining method, the genomic recombination of PRRSV were analyzed by using RDP 4.10 and Simplet SimPlot 3.51 softwares. Thirty-two PRRSV-2 strains were successfully isolated. The full length of 32 isolates were determined to be 14938-15439 bp, excluding the poly (A) tail and shared 81.4%-99% identity with PRRSV-2 representative strains and only 59.8%-60.6% with LV. Genotyped of 32 PRRSVs were tested by genetic evolutionary tree constructed by the whole genome and ORF5 gene, and the results showed 75% agreement. Genomic sequence analysis showed Nsp2 and ORF5 are the most variable protein in PRRSV genome, Nsp2 protein has different degrees of amino acid deletion (1-155 aa) and GP5 had high variability in the primary neutralizing epitope (PNE). Recombination analyses revealed a high recombination probability (27/32) and complex recombination patterns were found in the 32 PRRSV strains, including:L1+L8, L1+L3, L8+L5, L8+L3, L1 (Sublineage1.8+Sublineage1.5)+L8, L1+L5+L8, L1+L3+L8, L1+L3+L5, L1+L3+L5+L8. Additionally, PRRSV genomic recombination hotspots mainly distributed in Nsp1, Nsp2, Nsp9 and ORF3 genes. In summary, the epidemiological and evolutionary characteristics of PRRSV indicating the circulating strains in Fujian Province are recombinant strains with NADC30-like PRRSV as the main parent. Genotypes of PRRSV should be based on the results of the full-length genome.

Porcine deltacoronavirus (PDCoV) can cause diarrhea, vomiting and even lead to death of newborn piglets, which has serious harm to pig breeding. Phenylalanine dipeptide and its derivatives can be self-assembled into nanometer supramolecular assemblies, which have the function of loading and delivering drugs. This study was conducted to explore whether bridging diphenylalanine dipeptide can enhance the immunogenicity of PDCoV. Firstly, its cytotoxicity was evaluated. Then, it was used as adjuvant (0.1 mg·mL^-1) and mixed with equal volume of inactivate PDCoV (PDCoV HNZK-02, TCID₅₀=10⁸·0.1 mL^-1) to prepare inactivated vaccine. Mice (200 μL per mouse) were immunized with inactivated vaccine, and the specific IgG antibody was detected by ELISA method, the SI index of lymphocyte proliferation was detected by CCK-8 kit, and the activity of specific cytotoxic T lymphocytes (CTL) was detected by lactate dehydrogenase (LDH). After 5 weeks of immunization, mice were challenged with PDCoV (500 μL per mouse) by intragastric administration, and the viral load of ileum and other tissues was detected 3 days later. The results showed that bridged diphenylalanine dipeptide had no obvious toxic effect on porcine testicular cells. Mice immunized with inactivated PDCoV with bridging diphenylalanine dipeptide could produce high level of IgG, SI and CTL were higher than those of the inactivated PDCoV group, and the intestinal tissue viral load was significantly lower than that of the inactivated PDCoV group. Bridged diphenylalanine dipeptide showed a good adjuvant effect and has the potential to become a new safe and effective adjuvant.

Biomineralization technology can enhance the heat resistance of foot-and-mouth disease (FMD) virus-like particles (VLPs), but there is no experimental data to support the feasibility of mineralized FMD VLPs for clinical use. The aim of this study was to evaluate the immunogenicity of the mineralized FMD VLPs vaccine in cattle and pigs. The pigs were immunized with VLPs and mineralized VLPs, and the specific antibody titers in the serum of the immunized pigs were detected by liquid-phase blocking ELISA (LPBE). The specific IFN-γ secreted by peripheral blood lymphocytes (PBLs) after stimulation in vitro was detected by enzyme-linked immunospot assay (ELISpot). Cattle were immunized with mineralized type A VLPs, mineralized type O VLPs, and mineralized type A and type O bivalent VLPs, respectively. The serum specific antibody, neutralizing antibody, IL-4 and IFN-γ contents were detected by LPBE, neutralization test and sandwich ELISA. The results showed that there was no significant difference in serum antibody level between VLPs and mineralized VLPs immunized pigs.Compared with VLPs, the level of IFN-γ secreted by PBLs of mineralized VLPs immunized pigs was higher than that of VLPs immunized pigs (P<0.05). The serum of cattle immunized with the three vaccines contained more IL-4 and IFN-γ (P<0.05), and there were no significant differences in the titers of serum specific antibodies and neutralizing antibodies between the mineralized bivalent vaccine and the corresponding mineralized monovalent vaccine. In conclusion, mineralized VLPs have good immunogenicity for both cattle and pigs, mineralized VLPs of different serotypes can be used in combination, and mineralized shells can enhance the ability of VLPs to induce cellular immune response.

The study aimed to explore the efficacy of the different emulsification methods and stability of foot-and-mouth disease (FMD) vaccine adjuvanted with cultivated Artemisia rupestris L. crude polysaccharides (CARCP). ICR mice were subcutaneously immunized with FMD-Ag (inactivated foot-and-mouth disease viral antigen) adjuvanted with ISA-206 or ISA-206+CARCP prepared by stirred emulsification and ultrasonic emulsification. FMD-specific antibodies and T cell subsets from spleen were detected. After FMD-Ag+ISA-206 and FMD-Ag+ISA-206+CARCP by stirred emulsification were placed at 4℃ and 25℃ for 30 and 180 d, respectively, ICR mice were immunized with these vaccine formulations and tested humoral and cellular immunity for the analysis of stability. Results were as follows:Compared with commercial FMD vaccine, the effects of different FMD vaccine formulations prepared by stirred emulsification and ultrasonic emulsification had no significant difference on FMD-specific IgG and CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells (P>0.05). After placing at 4℃ for 30 and 180 d, compared with commercial FMD vaccine, there was no significant difference in IgG levels and CD4⁺, CD8⁺, CD4⁺CD44⁺, CD8⁺CD44⁺T cells in FMD-Ag+ISA-206 and FMD-Ag+ISA-206+CARCP group (P>0.05). After placing at 25℃ for 30 and 180 d, compared with commercial FMD vaccine, the levels of antibodies and T-cell subsets in FMD-Ag+ISA-206 group were significantly reduced (P<0.05), while there was no significant difference between antibody levels and T-cell subsets in FMD-Ag+ISA-206+CARCP group (P>0.05). Stirred and ultrasonic emulsification have similar immune effects on FMD vaccine. Under different storage conditions, CARCP enhanced the stability of FMD vaccine. These data provided some experimental thoughts for CARCP polysaccharide adjuvant in the development of FMD vaccine adjuvant.

Goose astrovirus (GAstV), as an emerging pathogen causing visceral gout and high mortality in goslings and result in huge economic losses. Thus, it is very important to develop credible detection methods to prevent this diseases wide spread in the absence of effective treatment measure. In order to establish a rapid, specific and sensitive method for detection of genotype GAstV Ⅰ and GAstV Ⅱ viruses, a duplex TaqMan real-time quantitative reverse transcription PCR (RT-qPCR) assay was established. Two pairs of specific primers and two kinds of TaqMan probes labeled with different fluorescent groups according to the RdRp gene sequences were designed and synthesized. The results showed that the minimum detectable amounts of GAstV Ⅰ and GAstV Ⅱ were 43.3 and 6.49 DNA copies·μL^-1, respectively; the coefficient of variation of repeated tests was less than 0.5%; and the method had no cross reaction to viral nucleic acids such as DPV, GPV, GTMUV, GRV, AIV (H9N2), etc. In summary, the results above indicate that this duplex real-time PCR method can distinguish GAstV Ⅰ and GAstV Ⅱ with high sensitivity and specificity. It can be used for the clinical rapid differential diagnosis of GAstV Ⅰ and GAstV Ⅱ, and quantitative analysis of two genotypes of GAstV.

Diarrhea caused by different pathogens can lead to changes in the structural characteristics of intestinal flora of calves. In order to study the diversity of intestinal flora, 38 fresh faecal samples were collected from three large-scale cattle farms in western Inner Mongolia, including 30 diarrhoea faecal samples and 8 healthy faecal samples. The diarrhoea-associated pathogens were tested by PCR and then the samples were divided into three groups, the healthy group (HC), the bovine rotavirus (BRV)-infected diarrhoea group (DC_a) and the Escherichia-Shigella infection diarrhoea group (DC_b). Total DNA was extracted and the highly variable V3-V4 region of the bacterial 16S rDNA gene was amplified by PCR using universal primers, sequenced by the Illumina NovaSeq platform and the sequencing results were analyzed by bioinformatics. The results of α diversity index showed that there was a significant difference in intestinal microbial diversity between diarrhea and healthy calves, and the α diversity index of DC_b group was significantly lower than that of the other two groups(P<0.05). At phyla level, the dominant phyla in the three groups were Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria, but their relative abundance were significantly different. In DC_b group, Firmicutes were significantly decreased (P<0.05) and Actinobacteria were significantly increased (P<0.05). At the genus level, the dominant genera and relative abundance of the three groups were different. PICRUSt2 function prediction analysis showed that compared with HC group, DC_a group significantly decreased mannose-degradation, galactose degradation, sugar and vitamin biosynthesis and other related metabolic pathways. DC_b was significantly enriched in hexitol degradation and L-tryptophan biosynthesis. This study showed that the difference between healthy calves and diarrhoeic calves was significant (P<0.05) in the intestinal flora of the phylum Firmicutes and Actinomycetes; Not only the pathogenic bacteria in the phylum Aspergillus can cause diarrhoea in calves but also lead to an inflammatory response in the intestine; Family Rumexcoccus has multiple functions and it is difficult to infer functional differences between genera from amplicon sequencing data; It can also be concluded that diarrhoea is closely related to energy and nutrient metabolism and amino acid metabolism.

The detection rates of Pan drug resistant bacteria and multi drug resistant bacteria are increasing, and phages have good application prospects in preventing the transmission and infection of treatment resistant bacteria. The purpose of this study was to isolate mild bacteriophages with wide cleavage spectrum and enrich the Salmonella mild phage library. A temperate phage named Salmonella virus PEA2-3 was isolated and purified using mitomycin C induction, and some of its biological characteristics were determined and bioinformatics analysis was performed. TEM revealed that the phage head was approximately 61 nm wide and the tail was 93 nm long, and it belonged to the order Caudovirales, genus Rosemountvirus. The optimal multiplicity of infection was 0.01; The one-step growth curve showed an incubation period of 20 min and a lysis period of approximately 80 min; And was able to lyse Salmonella (38/72) and Escherichia coli (23/52) of different origins, which indicated it to be a lysogenic phage with a wide lysis spectrum, that had better application value. The genome sequencing results showed that its genome was 52 770 bp in length with a GC content of 45.99%, and the genome was annotated to a total of 73 CDS, 8 CDS of which were assigned to known functional proteins, and no antibiotic resistance genes or virulence genes was existed. The whole genome of phage PEA2-3 was submitted to the GenBank database of NCBI and obtained the sequence number of MW508890. The results suggest that this strain is a highly efficient and mild phage with a wide lytic spectrum that can lyse Escherichia coli and Salmonella simultaneously, providing a good application basis for the prevention and control of the spread of drug-resistant bacteria.

The aim of this experiment was to determine the characteristics and tissue distribution of the Ctype lectin 4(Tc-ctl-4)gene of Toxocara canis. The full-length gene of Toxocara canis C-type Lectin 4 (Tc-ctl-4) was amplified as template according to the genomic data of Tc-ctl-4(AF126830.1) and sequence analysis and multiple sequence alignment were performed. The prokaryotic expression vector Tc-ctl-4/pCold TF was constructed, the recombinant protein was purified and polyclonal antibody was prepared. The transcriptional level of Tc-ctl-4 and the tissue distribution of Tc-CTL-4 were detected by quantitative real-time PCR (qRT-PCR) and indirect immunofluorescence histochemistry. The results showed that the full-length sequence of Tc-ctl-4 gene was 842 bp, encoding 280 amino acids.A conserved Ctype lectin like domain (CTLD) was identified in multiple sequence alignments. SDS-PAGE showed that the recombinant protein Tc-CTL-4 was about 86 ku in size and expressed in soluble form. The protein was purified by Ni-NTA affinity chromatography, and high purity protein was obtained by elution with 500 mmol·L^-1 imidazole, which was used to immunize the New Zealand white rabbits to prepare polyclonal antibody. Indirect ELISA showed that the antibody titer was>1:160 000, indicating that the recombinant protein had good immunogenicity and SDS-PAGE showed that the purity of the purified antibody was higher than 95%. Western blot showed that the antibody could specifically bind to Tc-CTL-4, indicating that the antibody was highly specific. The qPCR results showed that the transcription level of Tc-ctl-4 was the highest in the uterus of female worms and the seminal vesicle of male worms. Indirect-fluorescence immunohistochemical assays were carried out to detect the tissue distribution of Tc-CTL-4, which showed predominant distribution of Tc-CTL-4 in the reproductive tissues of adult T. canis, such as in the uterus, oviduct of adult female T. canis and in the vas deferens of adult male T. canis, and body wall of T. canis. It is suggested that Tc-ctl-4 might play an important roles in the reproduction and immunity of T. canis.

This study aimed to determine the effects of PGD₂ members in the prostaglandin family, which are rarely reported in the reproductive field, on the endocrine function of luteal cells and its apoptosis-related gene expression in a single environment, and to analyze its mechanism in the luteal regression, providing a new theoretical basis for the comprehensive analysis of the biological role of prostaglandin family members. The ovarian tissues of the middle stage of the corpus luteum of the empty pregnant female goat were collected, isolated and purified by collagenase II digestion and pancreatic enzyme differential centrifugation to obtain goat corpus luteal cells. DMEM/F12 was used for in vitro culture to observe the growth status of luteal cells at different time of in vitro culture, and the luteal cells were identified by immunohistochemistry and cell morphological characteristics. PGD₂ was set to three different gradients to determine the dose-dependent relationship of its effect on the luteal cells. Finally, luteal cells were treated with the best dependent dose of PGD₂, the endocrine function changes of luteal cells were detected by ELISA, the apoptosis rate of luteal cells were measured by flow cytometry, and the expression level of mRNA/protein expression of apoptosis-related genes were detected by qRT-PCR/Western blot. The results showed that the goat primary luteal cells were successfully isolated and obtained after identification of the specific expression protein of synaptophysin (SYP). Real-time observation of cell morphology and ELISA detection showed that luteal cells had a full cytoplasm, compact shape, and clear morphology at 5 days of ex vivo culture, and the endocrine P₄ level of luteal cells was the highest. At this time, the cell growth curve results also confirmed that cell growth peaked at 5 days of ex vivo culture, and the cell growth curve was inverted "S" shaped. After 48 h of logarithmic peak cells seeding and PGD₂ treatment, it was found that the order of P₄ secretion levels of luteal cells in different dose groups was 2 μg group < 3 μg group < 1 μg group. At the same time, in the 2 μg dose treatment group, the concentration of P₄ in the medium decreased significantly compared with the control group (P<0.01). The expression of the steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) genes were significantly downregulated (P<0.05). In addition, the results of flow cytometry detection and Flow Jo software analysis showed that the apoptosis rate of luteal cells increased significantly (P<0.05). qRT-PCR and Western blot results confirmed that the anti-apoptotic factor B lymphoma-2 (BCL-2) mRNA and protein were significantly downregulated (P<0.05), and the pro-apoptotic factor cysteine aspartate-specific protease-3 (Caspase-3) mRNA and protein were significantly upregulated (P<0.05). The above results show that PGD₂ can not only inhibit the endocrine function level of luteal cells by downregulating the expression of StAR/3β-HSD gene, but also accelerate the apoptosis process of luteal cells by downregulating the expression of anti-apoptotic BCL-2 gene and upregulating the expression of pro-apoptosis Caspase-3 gene, and finally participate in the molecular regulation of luteal cells in the form of dual pathways. This lays a solid foundation for further improving the biological functional effects of prostaglandin family members and subsequently comprehensively exploring the molecular regulatory network mechanism of luteal maintenance and degradation in domestic animals.

The purpose of this study was to investigate the effect of porcine reproductive and respiratory syndrome virus infection on the flora in the lungs and intestines of piglets and the histological changes in the lungs and intestines. Fourteen weaned healthy piglets of 35 day old were randomly divided into infection group (n=7) and control group (n=7) after 7 days of adaptive feeding. The piglets in the infection group were inoculated with 2 mL 1×10⁵ TCID₅₀·mL^-1 PRRSV JTS virus solution, and the piglets in the control group were inoculated with 2 mL DMEM. Three piglets in the infection group died on 10, 12, and 19 days, respectively. The surviving piglets in the experimental group and the control group were euthanized on 21 days, and lung, intestinal tissue samples and intestinal contents were collected. Immunohistochemical staining and HE staining were used to observe the histopathological changes of lung and intestine, and high-throughput sequencing based on 16S rRNA Gene was used to analyze the flora structure of piglet lung and intestine. The results showed that the virus was distributed in the lungs and intestines of piglets in the infection group; Compared with the control group, there were significant inflammatory reactions in the lungs and intestines of the infected group. The analysis of microbial composition, structure and diversity showed that the indexes of Chao1 and ACE of piglets in the infection group increased in the lung and decreased in the intestine, while the indexes of Shannon and Simpon increased in the lung and decreased in most intestines. At the phylum level, the proportion of harmful microorganisms such as Proteus increased, while the proportion of beneficial microorganisms such as Firmicutes decreased; In the infection group, the relative proportion of pasteurebacteriaceae, duodenal lactobacilliaceae, ileal Enterobacteriaceae, cecum, colon and rectum decreased significantly, and the proportion of Pseudomonas in lung and duodenum increased significantly; In terms of genus level, the level of beneficial bacteria and lactic acid bacteria in the infection group decreased significantly, the β diversity proves that the clustering effect of small intestine and large intestine is consistent. It can be seen that PRRSV can cause lung lesions and intestinal inflammation in piglets, and affect the composition, abundance and function of lung and intestinal flora.

This study was conducted to research the molecular mechanism of inflammatory response of recombinant Candida krusei 14-3-3 protein (rCK14-3-3) treat on the mammary epithelial cells (MAC-T), so as to provide a theoretical basis for the systematic study of the mechanism of MAC-T damage induced by Candida krusei. The recombinant plasmid of Candida krusei BMH1 gene was constructed by prokaryotic expression and transformed into E. coli BL21 (DE3), rCK14-3-3 protein was identified by Western blot after induction and purification; the optimal concentration and time of rCK14-3-3 on MAC-T were screened by CCK-8 method; the expression levels of the Toll receptors, MAPK and NF-κB signaling pathway major proteins of MAC-T was determined by Western blot;the expression level of the inflammatory factors IL-1β, IFN-γ, IL-18, TNF-α, IL-6 and IL-12 were determined by ELISA in MAC-T. The recombinant expression vector pET-21a-BMH1 was successfully constructed, the rCK14-3-3 protein has biological activity after expression and purification. The purity of rCK14-3-3 protein reached more than 90% and the concentration was 0.2 mg·mL^-1. Compared with the blank control group, MAC-T was treated by rCK14-3-3 of 50 μg·mL^-1, the protein expression level of TLR2, TLR4, and MyD88 was very significantly increased at 1 and 3 h (P<0.01), at the time of 6 h, the TLR4 expression level was significantly reduced (P<0.05), no significant changes in TLR2 and MyD88 expression occurred; No significant change of p-JNK expression at 1 h, it was very significantly decreased at 3 and 6 h (P<0.01); The expression level of p-ERK was increased significantly at 1, 3 and 6 h (P<0.05 or P<0.01); The expression level of p-p38 was very significantly reduced at 1 h (P<0.01), and it increased very significantly at 3 and 6 h (P<0.01); The expression levels of p-IκB-α and p-p65 were all significantly increased at 1, 3, and 6 h (P<0.01); MAC-T was treated with 50 μg·mL^-1 of rCK14-3-3 showed a significant or extremely significant upregulation in IL-1β, IFN-γ, IL-18, and TNF-α after 1, 3 and 6 h (P <0.05, P<0.01), IL-6 increased significantly after 1 and 3 h (P <0.01), and decreased significantly after 6 h (P<0.05), IL-12 increased significantly after 1 h (P<0.01), and decreased significantly after 3 and 6 h (P<0.01). In the study, the rCK14-3-3 protein of biological activity was successfully expressed, which can induce an inflammatory response in MAC-T through the TLR2/4-MAPK/NF-κB signaling pathway, and rCK14-3-3 protein can be one of the important factors causing MAC-T inflammation.

Trichophyton rubrum (T. rubrum) is an important pathogen causing dermatophytosis. The dry twig and leaf of Platycladus orientalis (L.) Franco is called Cacumen Platycladi and recorded in the "Miao Medicine" for the treatment of tineas. The study aimed to investigate the main anti-T. rubrum components and mechanisms of Cacumen Platycladi, and to provide potential candidate anti-T. rubrum agents. In this study, the antifungal activity of Cacumen Platycladi main ingredient against T. rubrum was determined by minimum inhibitory concentration and spore germination rates. The mycelial morphology and spores ultrastructure of T. rubrum were observed by using electron microscopy; and the effects of the active substances of Cacumen Platycladi on the permeability, integrity, and ergosterol content of T. rubrum cell membrane were further examined, so as to elucidate the mechanism of T. rubrum inhibition by the active substances of Cacumen Platycladi. Finally, we used GC-MS and qRT-PCR to find its target of action against T. rubrum. The results showed that the petroleum ether fraction of Cacumen Platycladi exhibited the strongest anti-T. rubrum activity and in which α-pinene was proved to be the main ingredient with a MIC 32 μg·mL^-1. Alpha-pinene could induce membrane damage, higher significant cellular leakage (P<0.01) and ergosterol and 24(28)dehydroergosterol amount significant decreasing within 24 h (P<0.05); Cause highly significant reduction (P<0.01) of the relative expression of ERG3 gene and the "ERG3 inhibition" related mark sterol Ergosta-7,22-dien-3β-ol occured in sterols pattern. The results showed that α-pinene is the most potent anti-T. rubrum component in Cacumen Platycladi and ERG3 was its target.

This study aims to reveal the interaction mechanism between the pathogen and the midgut cells by analyzing the differential expressed proteome of the midgut after Apis mellifera worker infected with/without Vairimorpha ceranae(Nosema ceranae). The 5-day old workers were fed ad lib with sucrose solution containing 10⁴spore·μL^-1 for 48 h, then raised in the incubator under 30℃ for 18 days(23 days old). The results showed that the survival rate of workers in infected group was only 34.81%, which was significantly lower than that of the control, 71.85% (P<0.05). The midgut protein concentration was significantly decreased either (P<0.05), however, there was no significant difference in the daily consumption of sucrose solution per bee (P>0.05). The comparison of the midgut proteomes of the infected bee and the control by using tandem mass tags (TMT) showed that, 565 proteins were significantly differentially expressed, in which 301 proteins were up-regulated and 264 proteins were down-regulated in the infected midgut. The enrichment of different protein GO functional annotations showed that the cell process, energy metabolism, cell morphology, protein complexes, molecular binding, and catalytic activity in the infected midgut cells were significantly affected. Through the enrichment of the KEGG pathway, it was found that aminoacyl-tRNA biosynthesis, protein processing in the endoplasmic reticulum, and various types of N-glycan biosynthesis were significantly up-regulated, as well as some antioxidant proteins. The proteins enriched in amino acid metabolism and sucrose metabolic pathways were significantly down-regulated. It was revealed that the infection of V.ceranae at late stage significantly compromised the protein expression relatived to the digestion and immunity. These significant differential proteins provides a strong support for the subsequent screening and verification of interaction proteins between V. ceranae and A. mellifera.

Ocular and nasal swabs were collected from cats with upper respiratory tract infections in a cattery in Harbin, and the initial diagnosis was a mixed infection of feline herpesvirus-1 (FHV-1) and feline calicivirus (FCV) by PCR. CRFK cells were used to isolate viruses from the swabs of infected cats. Nucleic acid assay of the first two generations of cell cultures showed that FHV-1 and FCV were both positive, and only FCV was isolated from the third generation. FHV-1 was isolated by serum neutralization using the prepared mouse-derived polyclonal antibody of the isolated FCV, which named HRB2019 strain. HRB2019 was identified by morphological observation of viral particles, indirect immunofluorescence assay (IFA) and gene sequence analysis. And the growth kinetics in vitro of HRB2019 was studied. The results showed that HRB2019 could produce typical cytopathic effect on CRFK cells and viral titer of the fourth generation of virus culture reached 1×10^8.43TCID₅₀·mL^-1. Electron microscopy examination showed spherical virus particles with vesicle membrane, and its diameter was about 200 nm. IFA results showed that HRB2019 could bind to FHV-1 positive serum and appeared specific fluorescence. The gD gene of the isolate was amplified and the nucleic acid sequence analysis showed that the isolate was highly homologous with the domestic and foreign prevalent strains. The one-step growth curve of the virus showed that the virus began to replicate and proliferate from 12 h after inoculation, and the rapid proliferation period was from 12 to 36 h. The viral titers peaked at 48 to 72 h and began to decline at 84 h. In this study, FHV-1 was successfully isolated for the first time from samples infected with a mixture of FCV and FHV-1, which provided important data for epidemiological investigation and pathogenetic study of FHV-1 and provided methodological basis for isolation of growth-vulnerable strains in mixed infection.

This study was conducted to investigate the effect of enhancer of Zeste homolog 2 (EZH2) inhibitor GSK126 on the proliferation, migration, apoptosis and epithelial-mesenchymal transition (EMT) of canine mammary tumor cells. Canine mammary tumor cells (CHMm cells) in logarithmic growth phase were collected, and different concentrations of GSK126 (0, 10, 20, 40 μmol·L^-1) were used to culture CHMm cells. After 48 h, tetramethylazolium salt colorimetric method (MTT) and cell clone formation assay were detected for the proliferation ability of cells. Transwell assay was performed to evaluate the migration ability. The cell apoptosis was detected by Annexin V-FITC/PI flow cytometry, and the relative expression of apoptosis-related factors Bcl-2 and Bax, epithelial-mesenchymal markers E-cadherin, N-cadherin, Vimentin, EZH2 were determined by qRT-PCR and Western blot method. After treated with GSK126 for 48 h, the proliferation and migration of CHMm cells were inhibited in a concentration-dependent manner. The apoptosis rate of the GSK126 treatment group was significantly increased. The relative expression of pro-apoptotic factor Bax, epithelial marker E-cadherin mRNA and protein compared with control group was significantly increased as the concentration of GSK126 treatment group, in contrast, anti-apoptotic factor Bcl-2, mesenchymal marker N-cadherin, Vimentin and EZH2 mRNA and protein was significantly reduced. EZH2 inhibitor GSK126 may inhibit the proliferation, migration, EMT and induce apoptosis of canine mammary tumor cells, which provides a theoretical basis for the treatment of canine mammary tumor cells.

The purpose of this study was to investigate the protective effect and mechanism of coenzyme Q10 (CoQ10) on lipopolysaccharide (LPS)-induced acute lung injury in mice. Seventy-two healthy male C57BL/6 mice aged 8 weeks were selected and randomly divided into 6 groups:control group (CON group, 100 μL corn oil), CoQ10 intervention group (CHQ group, 50 mg·kg^-1 CoQ10), model group (LPS group, 100 μL corn oil + LPS), low-dose CoQ10 treatment group (LDQ group, 2 mg·kg^-1 CoQ10 + LPS), medium-dose CoQ10 treatment group (LMQ group, 10 mg·kg^-1 CoQ10 + LPS) and high-dose CoQ10 treatment group (LHQ group, 50 mg·kg^-1 CoQ10+LPS). The mice were continuously given different doses of CoQ10 or corn oil by gavage for 14 days, and intranasally instilled 50 μL 1 mg·mL^-1 LPS to establish an acute lung injury model. After 24 h, the mice were sacrificed, and the left lung was harvested, weighed and dried to calculate the wet-dry weight ratio (W/D) of lung tissue. The protein concentration of alveolar lavage fluid (BALF), malondialdehyde (MDA) content, reactive oxygen species (ROS) concentration, total superoxide dismutase (SOD) activity and reduced glutathione (GSH) level in lung tissue were detected. Hematoxylin and eosin (HE) staining was used to observe the pathological changes of lung tissues. TUNEL staining was used to detect the apoptosis rate of lung tissue. RT-PCR technology was used to detect the expression of mitochondrial apoptosis pathway-related genes. Western blot was used to detect the expression of MKP-1/Nrf2 pathway-related proteins in lung tissue. The results showed that compared with CON group, the W/D value and BALF protein concentration in the LPS group were significantly increased (P<0.01); lung tissue hemorrhage, alveolar wall thickening, inflammatory cell infiltration; MDA and ROS contents were significantly increased (P<0.01), SOD activity and GSH content were significantly decreased (P<0.01); TUNEL apoptosis rate and mitochondrial apoptosis pathway related gene expression were significantly increased (P<0.01); MKP-1 and Nrf2 protein expression were significantly decreased (P<0.01). Low-dose, medium-dose and high-dose CoQ10 significantly or extremely significantly reversed the changes of the above indexes induced by LPS (P<0.05, P<0.01), and high-dose CoQ10 was more effective (P<0.01). In addition, there was no significant difference in all indicators between the CHQ group and the CON group (P>0.05). The results suggest that CoQ10 can improve LPS-induced acute lung injury in mice by activating MKP-1/Nrf2 signaling pathway, enhancing anti-oxidative stress ability, reducing ROS production and inhibiting mitochondrial apoptosis.

The study aimed to investigate the protective effect of Limonin (LM) on kidney injury induced by high-fat diet in mice. Twenty-five male C57BL/6 mice weighed 21-23 g were selected and randomly divided into five groups (n=5) according to the test requirements:control group, LM group, high-fat group, high-fat + low-dose LM group (60 mg·(kg·d)^-1) and high-fat+high-dose LM group (120 mg·(kg·d)^-1). After 12 weeks the body weight of mice were detected. The automatic biochemical analyzer was used to measure triglycerides (TG), total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), creatinine (Cr), and urea nitrogen (BUN) in serum. The kits were used to measure TC, TG in the kidney. Hematoxylin-eosin (HE) and periodic acid-schiff (PAS) staining were performed to observe histological changes of kidney tissues. The protein expression of fatty acid synthase (FAS), sterol regulatory element binding protein 1 (SREBP1), adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) in the kidney were measured by immunohistochemistry. The protein expression of AMP-activated protein kinase (AMPK), phosphorylated AMPK (P-AMPK), FAS, SREBP1, ATGL, HSL and kidney injury molecule 1 (KIM1) was measured by immunoblot. The bound ability of LM、AICA Riboside (AICAR) and Dorsomorphin(Compound C) with AMPK was examined by molecular docking. The results showed that compared with high-fat group, LM intervention significantly reduced TG content in serum and kidney (P<0.05), improved histopathological kidney injury in mice fed with high-fat diet, promoted AMPK phosphorylation (P<0.05), significantly up-regulated the protein expression of ATGL and HSL (P<0.05), and inhibited the protein expression of SREBP1 and FAS (P<0.05). In addition, molecular docking showed that LM could bind tightly to AMPK proteins and had multiple sites identical to those of AICAR binding AMPK. The results suggest that LM attenuates kidney fat ectopic deposition and improves the high-fat diet-incluced kidney injury in mice by activating AMPK.

This experiment aims at investigating the effects of chronic ethanol poisoning on goat blood metabolites and its important metabolic route, screen prospective diagnostic biomarkers, and comprehend some of the etiology of nervous system disorders of the disease and early detection. Fifteen healthy 4-month-old local goats were randomly assigned to three groups:65% distiller's grains, 75% distiller's grains, basic diet control group. Three sets of blood samples were obtained every 15 days during the trial for the detection of liver and kidney, calcium, and protein metabolism indicators. The model of goat distiller's grains chronic poisoning was deemed successful when combined with clinical symptoms. Collect blood samples and screen differential metabolites utilizing ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, analyze metabolic enrichment pathways of differential metabolites, and evaluate the expression of key genes on the significant differential metabolic pathways. The metabolome results showed that chronic distiller's grain poisoning caused up-regulation of dopamine (DA) and 2-Furoylglycine (2-FG) and down-regulation of prostaglandin I₂ (PGI₂) in goats; synaptic vesicle cycle, dopaminergic synapses, Metabolic pathways such as ethanol toxicity, taurine, and hypotaurine metabolism, histidine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, tricarboxylic acid cycle, sphingolipid signaling pathway, and cAMP signaling pathway were Significant difference. Among them, the metabolic pathways of ethanol poisoning changed significantly, and the impact factor was >0.2. In the metabolic pathway of alcohol intoxication, the expressions of TH and DAT in group II were significantly decreased (P<0.05); the expressions of DDC and MAOA showed a decreasing trend (P>0.05). In summary, chronic lees intoxication can cause significant alterations in organic acids, amino acids, lipids, lipid-like molecules, amines, alcohols, fatty acids, and steroids metabolites in the blood of goats, and DA, 2-FG, and PGI₂ can be used as potential diagnostic biomarkers for chronic lees intoxication disease. The common pathway of differential metabolite enrichment is the ethanol intoxication metabolic pathway, in which DAT and TH mRNA expression is decreased, resulting in elevated DA concentrations in the organism, which in turn causes neurological dysfunction in intoxicated goats.

This study aimed to understand the molecular genetic variation and virulence characteristics of porcine pseudorabies virus (PRV) in China. The main glycoprotein gene sequences (gB, gC and gE) of PRV ZJ strain isolated from a pig farm inoculated with Bartha-K61 vaccine were determined. and its pathogenicity to 6-week-old mice was studied. Phylogenetic analysis results showed that this strain was highly homologous to epidemic strains in China after 2012, and had the genetic characteristics of the mutant strain. However, there was a unique change of S⁵¹⁰G in the amino acid position 510 of gE gene, which was different from the mutant strain. ZJ strain was highly pathogenic to BALB/c mice, and the challenged mice showed typical neurological symptoms. The mortality rate reached 100% on day 5 after the mice were infected with 100 LD₅₀. This study laid a foundation for further exploration of the variation characteristics and pathogenesis of this virus.