Germ cells are crucial for transmitting genetic material in living organisms and are vital for livestock breeding. The increasing demand for livestock products promoted the development of precision breeding techniques. Recent advancements in gene-editing have significantly improved the effect of genetic enhancement and disease treatment, highlighting the importance of integrating this technology into livestock germ cell research. The research on CRISPR/Cas9 and CRISPR/Cas12 has gained significant prominence compared to other gene-editing technologies. CRISPR/Cas has led to breakthroughs in various applications, and these systems are increasingly recognized as potential methods for species improvement. In this review, two types of CRISPR/Cas systems and CRISPR/Cas9 applications in livestock germ cells were analyzed. A concise overview of the developmental processes of germ cells in livestock was provided, elucidating the principles, composition, and derivative techniques associated with CRISPR/Cas9 and CRISPR/Cas12, and the implementation of CRISPR/Cas9 systems in livestock germ cells was discussed. Furthermore, the current review anticipate the extensive application of gene-editing techniques in this domain, aiming to offer a valuable reference for future research in the field of gene editing in livestock.

The hosts of Sarcoptes scabiei (S. scabiei) are extensive and distributed globally. Scabies infections can threaten the health of the host and result in significant economic losses to the aquaculture industry. Currently, the pathogenic mechanism of the S.scabiei remains unclear, and there are no ideal diagnostic or treatment strategies for S.scabiei infections. Investigating the gene function of the S.scabiei is crucial for addressing this issue. Advances in molecular biology over recent years have facilitated an in-depth exploration of the gene function and pathogenic mechanism of S.scabiei, providing a robust data foundation for further study. This review encapsulates the latest research on the applications of genomics, transcriptomics, and proteomics, as well as significant functional genes in S.scabiei, with the aim of offering novel insights into understanding the pathogenesis of S.scabiei and identifying effective molecular-level treatment approaches.

This article summarizes the research progress in precise nutritional supply for growing-finishing pigs in China, where most intensive and large-scale pig farms encounter issues such as excessive breeding density, low feed utilization efficiency, high breeding costs, and environmental pollution. The key to addressing these challenges lies in the precise nutritional supply for pigs. By developing individualized feeding plans based on the characteristics of pigs, including gender, dietary needs, physiological status, and production requirements, can significantly improve pig growth performance, enhance feed utilization efficiency, reduce breeding costs, and mitigate environmental pollution. This approach is conducive to promoting the sustainable development of the pig farming industry. The article summarizes the research progress in precise nutrition supply of growing-finishing pigs, mainly elaborates on the dynamic nutritional requirement system, precise nutritional supply methods, and intelligent equipment. Additionally, it forecasts future research trends, aiming to provide a reference for the research on precise nutritional supply for growing-finishing pigs.

Lipid droplets (LDs) are unique and dynamic organelles formed from the endoplasmic reticulum membrane, primarily responsible for storing neutral lipids within cells. Additionally, LDs interact with other organelles and participate in biological processes such as lipid metabolism, membrane biogenesis, cell signaling, and immune responses, etc. In recent years, a growing body of evidence has shown that LDs play a critical role in pathogen infection, particularly in the process of viral infection. This review summarizes the interaction between LDs and viruses, including the impact of LDs on viral replication, and on their regulation of innate immune responses. Understanding the role of LDs in viral infection is of great importance for uncovering the pathogenic mechanisms of viruses, developing novel antiviral drugs, and preventing viral transmission.

The resistance of Haemonchus contortus to anthelmintics such as ivermectin and albendazole has become increasingly serious, causing major losses to the aquaculture industry in most countries and regions of the world. Currently, research on drug resistance in Haemonchus contortus focuses on epidemiological studies, drug resistance mechanisms and drug resistance interventions, and has made some progress. In this paper, we review the distribution of drug resistance and its influencing factors, the mechanism of drug resistance and reversal of drug resistance based on cell membrane pumps, autophagy level, cellular respiratory chain, parasite substitution, combined deworming with plant extracts and anthelmintics, and breeding of resistant hosts, to provide a new way of thinking for the study of drug resistance in Haemonchus contortus, and to provide reference bases for the scientific control of Haemonchus contortus, the rational use of drugs, and the development of new drugs. It also provides a reference base for scientific control, rational use of drugs and development of new drugs.

Aluminum (Al) is one of the most abundant metallic elements on Earth and is widely used in production and life due to its excellent properties. The accumulation of Al in the environment is gradually increasing, and Al pollution has become one of the major problems facing human society. The accumulation of Al in the body, resulting from long-term chronic Al intake, can damage many organs such as the heart, liver, spleen, kidney, and brain. Immunotoxicity is one of the most sensitive health effects of Al exposure and has attracted widespread attention. This paper reviews the toxic effects of Al exposure on the spleen, bursa of Fabricius, bone marrow, intestinal mucosa, lymph, and thymus, and summarizes the mechanisms of Al-induced immunotoxicity. The accumulation of Al in animals can damage immune organs, lead to apoptosis of immune cells, induce oxidative stress, trigger the expression of inflammatory factors, disrupt the metabolism of micronutrients, and suppress immune function. Pathological damage and oxidative stress induced by Al exposure are important pathological mechanisms of Al-induced immunosuppression, yet the molecular mechanisms of immunotoxicity are not yet systematically understood and require further exploration.

In order to investigate a live, non-invasive, simple, and efficient method for the assessment of abdominal fat deposition traits and selection of hens, the present study took Qingyuan partridge chickens as research object and combined the multiple body size trait selection method with eight machine learning models to construct regression prediction models and classification prediction models for the abdominal fat deposition of hens at different days of age. Using multiple early body size traits at various ages between 58 and 136 days of age combined with machine learning methods, the accuracy of body size traits at different ages in predicting abdominal fat content of adult Qingyuan partridge hens did not show significant differences. The RF model had the best prediction effect for regression prediction, with a fitting effect of R² of 0.821-0.861 and a prediction error MAE of 6.32-7.27. In terms of classification prediction, the Bagging model exhibited superior performance in both binary and tertiary classification. The binary classification accuracy ACC reached 94.54% to 100%, while the tertiary classification accuracy ACC reached 99.58% to 100%. In this study, live prediction models for abdominal fat deposition in high-quality chickens were established and optimized based on machine learning. These models can serve as a technical foundation for early live selection on abdominal fat deposition and breeding of high-quality chickens as well as for the exploration of the related technology for the construction of prediction models for abdominal fat content.

This study aimed to identify key candidate genes and pathways in cardiac and pulmonary tissues that mediate the development of ascites syndrome in broiler chickens.Taking the sires of fast-growing white-feathered broilers at 33 days of age as the research materials, 5 roosters with ascites syndrome and 3 normal roosters were selected. The occurrence of ascites syndrome in the roosters was determined through necropsy and the measurement of blood biochemical indicators. The subjects were categorized into an ascites group (5 roosters) and a control group (3 roosters).The results of blood biochemical analysis indicated that hematocrit (HCT), hemoglobin (HGB), and granulocyte percentage (GRA) in the ascites group were significantly higher than those in the normal group (P < 0.01).The lymphocyte percentage (LYM) in the ascites group was significantly lower than that in the normal group (P < 0.01).Additionally, the partial pressure of carbon dioxide (PCO₂) and total calcium (Ca) levels were significantly elevated in the ascites group compared to the normal group (P < 0.01), while the base excess (Beb) content was significantly lower in the ascites group (P < 0.05).The results of HE staining showed that in the normal group, the myofibrils in the myocardial tissue were arranged neatly, with no evidence of blood cell deposition between cells. In the ascites group, myocardial fibers exhibited structural disarray, and a large number of blood cells were deposited between the myofibrils. Transcriptome analysis identified 969 differentially expressed genes (DEGs) in lung tissue, including 417 upregulated genes and 552 downregulated genes. In cardiac tissue, 809 DEGs were identified, comprising 397 upregulated genes and 412 downregulated genes. Pathway enrichment analysis revealed significant enrichment in processes such as the mitotic cell cycle, centromere complex assembly, oxygen binding, neuroactive ligand-receptor interaction, cell adhesion molecules, cellular senescence, extracellular matrix (ECM)-receptor interaction, and motor proteins. Genes associated with ascites syndrome in broiler chickens were selected from enriched pathways and validated using real-time quantitative PCR. The results showed that the expression trends of KIF20A, NUSAP1, HBAD, TFRC, and HBBA genes differed between the ascites and normal groups, consistent with transcriptome sequencing findings, and these genes could be regarded as important candidate genes for ascites syndrome in broilers.The study, utilizing transcriptome technology, has identified significant pathways and candidate genes related to the development of ascites syndrome in broiler chickens, providing important insights into the disease mechanism and prevention strategies.

The aim of this study was to analyze that whether the accuracy of breeding value prediction for cashmere traits in Inner Mongolia cashmere goats was improved if dominance effects were added in animal genetic evaluation models. Based on the Illumina GGP_Goat_70K BeadChip sequencing data from 2 256 individuals of Inner Mongolia cashmere goats (Aerbas type), the phenotype and pedigree records of cashmere traits(cashmere diameter, cashmere production) were collected from individuals at age 1 to 8 years old.Two animal models were established according to whether dominance effects were taken into account or not. The genetic parameters and breeding values were estimated using the BLUP and GBLUP methods.And the accuracy of the estimated breeding values of cashmere traits was further evaluated by the cross-validation method, which was used to determine the optimal model. Finally, a multivariate animal model was established to estimate the genetic correlation and accuracy of breeding value prediction for both of the traits.And the differences in genetic parameters and breeding value accuracy between single trait and multi trait models were compared. The results showed that when the dominance effects were not considered in the animal model, the additive heritabilities for cashmere production using the BLUP and GBLUP methods were 0.156 and 0.215, and the accuracy of breeding value estimation was 27.58% and 60.33%, respectively; and the additive heritabilities for cashmere diameter were 0.252 and 0.292, and the accuracy of breeding value estimation was 28.08% and 62.33%, respectively. When both additive and dominance effects were considered in the animal model, the additive heritabilities for cashmere production using the BLUP and GBLUP methods were 0.150 and 0.200, and the dominant heritabilities were 0.147 and 0.070, with breeding value estimation accuracies of 38.50%and 72.62%, respectively; And the additive heritabilities for cashmere diameter were 0.263 and 0.290, and the dominant heritabilities were 0.288 and 0.026, and the accuracy of breeding value estimation was 39.66% and 65.97%, respectively.Under the best model, the genetic correlation between cashmere production and cashmere diameter was 0.340 0, and the phenotypic correlation was 0.038 5.Compared to that in single trait animal model, the accuracy of breeding value estimation for cashmere production and cashmere diameter was increased by 9.81%-19.43% and 14.01%-21.97%, respectively. In summary, the cashmere production have moderate to low heritability, and the cashmere diameter have moderate to high heritability. The genetic correlation between the two traits is relatively small, indicating that genetic improving for cashmere production will not cause thickening of cashmere diameter. The accuracy of breeding values estimation for the two traits by GBLUP method is significantly higher than that with BLUP method, with a 10.92% to 12.29% increase in cashmere production and a 3.64% to 11.58% increase in cashmere diameter. The accuracy of breeding value estimation under multi trait animal models is higher than that with single trait animal models. When both additive and dominance effects are considered in the animal model, the accuracy of genomic prediction can be significantly improved, indicating that the influence of dominance effects should be considered in the breeding process of Inner Mongolia cashmere goats in terms of cashmere production and cashmere diameter.

This study aimed to use molecular biological methods to explore the effect of miR-665 targeting BCL2L11 on the myoblasts proliferation of goat. Three healthy male Wu'an goats aged 1 month and 9 months each, with similar body weights and feeding environments for each age group was selected.The expression of BCL2L11 and miRNA-665 in the longest dorsal muscle tissue of Wu'an goats at different ages was detected using RT-qPCR; The expression of BCL2L11 in different tissue of Wu'an goats was also detected by RT-qPCR; Wild-type and mutant vectors of BCL2L11 3'UTR, as well as miR-665 mimics and control groups, were constructed and cotransfected into HEK293T cells. The targeting relationship between miR-665 and BCL2L11 was identified using a dual luciferase reporter gene detection system. The goat myoblasts were transfected with miR-665 mimic and negative control, and the expression levels of apoptosis markers factors XIAP and Fas were detected by RT-qPCR, and the effect of miR-665 overexpression on the proliferation of goat myoblasts was analyzed by EdU test simultaneously. The results showed that BCL2L11 was highly expressed in the longissimus dorsi muscle tissue; There were differences in the expression of miR-665 and BCL2L11 in the longissimus dorsi muscle tissue of goats aged 9-months and 1-months, and the expression of miR-665 at 1-month was higher than that at 9-month, while the expression of BCL2L11 was opposite, and the two were negative regulated; The dual luciferase report analysis showed that, after transfecting overexpressed miR-665 mimic, the luciferase activity of BCL2L11 was significantly inhibited, and the mRNA expression level of the target gene BCL2L11 was also significantly reduced. In order to further verify the function of miR-665, the expression of apoptosis marker genes XIAP and Fas was also significantly reduced after transfection of miR-665 mimic; EdU test analysis showed that the proliferation of myoblasts increased significantly after overexpression of miR-665. To sum up, miR-665 as a regulatory element of BCL2L11 in goat longissimus dorsi muscle tissue, could significantly inhibit the expression level of BCL2L11 in goat myoblasts, when miR-665 was overexpressed, it could promote the proliferation of myoblasts.

This study systematically compared the effects of different marker densities on the predictive accuracy and explored methods for combining these two chips of genetic evaluations in Huaxi cattle based on the practical application of the Illumina BovineHD 770K and Cattle 110K chips. In this study, a genomic selection reference population of 3 948 Huaxi cattle was used. The 770K chip data were imputed to 790K by using resequencing data. Then 4 marker densities were selected: 90K (intersection of the two chips), 110K, 770K, and 790K (union of the two chips). The heritability of 5 traits (weaning weight, daily weight gain during the fattening period, calving ease, carcass weight, and dressing percentage) were estimated, and the predictive accuracy of genomic evaluations was compared using the GBLUP model, identifying the optimal marker density for genetic evaluations in Huaxi cattle. The results showed that: 1) There were no significant differences in the estimated heritability of the 5 traits across the 4 marker densities. The heritability for weaning weight and daily weight gain during fattening period ranged from 0.47 to 0.50 (high heritability), carcass weight ranged from 0.37 to 0.39 (moderate heritability), and calving ease and dressing percentage ranged from 0.14 to 0.21 (low to moderate heritability). 2) The Cattle 110K chip demonstrated significantly better predictive accuracy for all traits compared to the Illumina BovineHD 770K chip (P < 0.05) in the GBLUP model. The improvements were particularly notable for carcass weight, calving ease and dressing percentage, with increases of 14.9%, 13.8%, and 8.4%, respectively. For weaning weight and daily weight gain during fattening period, the improvements were 2.8% and 4.5%, respectively. 3) The predictive accuracy of traits increased with higher heritability under the 4 marker densities. The regression coefficients for the different marker densities were 0.439 2 (90K), 0.374 1 (110K), 0.413 6 (770K), and 0.459 3 (790K). Therefore, the Cattle 110K chip can be used directly, achieving good predictive accuracy while reducing costs in practical genetic evaluations of Huaxi cattle.

The aim of this study was to investigate the effect of silent information regulator 2-related enzyme1 (Sirt1) on proliferation and myogenic differentiation of BSMSCs. In this experiment, small fragments of interfering RNA (siRNA) and overexpressed plasmid targeting Sirt1 were transfected into primary satellite cells stripped from the thigh muscle of 3-4-month-old fetal cattle. RNA and protein samples were collected at specific time points during the proliferation and differentiation stages, with 3 biological replicates per treatment. Real-time quantitative PCR (qRT-PCR) and Western blotting were used for analysis. In addition, EdU staining experiments were performed to evaluate cell proliferation.The results showed that the number, length, and diameter of muscle ducts were significantly increased by Sirt1 knockdown under a 40-fold microscope during cell differentiation. qRT-PCR and Western blotting confirmed that compared with the control group, mRNA levels of differentiation markers myosin heavy chains (MyHC) and myogenin (MyoG) were significantly up-regulated after interfering Sitr1 (P < 0.01). The protein expression level of MyoG was also significantly increased (P < 0.05) after interfering Sitr1. Conversely, Sirt1 overexpression resulted in a significant reduction in the number, length, and diameter of muscle ducts. Western blotting showed that the expression of MyoG in Sirt1 overexpression group was significantly lower than that in control group (P < 0.01). At the proliferation stage of BSMSCs, qRT-PCR, Western blotting and EdU tests showed that Sirt1 knockdown and overexpression had no significant effects on cell proliferation (P>0.05). In summary, Sirt1 knockdown can effectively promote cell differentiation, while overexpression can inhibit the process, but has no significant effect on proliferation. These findings confirm that Sirt1 is an important factor regulating the myogenic differentiation of BSMSCs, and plays a key role in coordinating myoblast differentiation and promoting myotube formation.

This study aimed to explore the effects of the screted frizzled related protein 4 (SFRP4) gene on the differentiation of bovine preadipocytes, the Qinchuan cattle perirenal preadipocytes was used as the experimental subject. Both treatment and control groups had 3 biological replicates. The full-length protein-coding sequence of the SFRP4 gene was cloned and analyzed via bioinformatics. Quantitative real-time PCR assessed SFRP4 expression across various tissues and differentiation stages in perirenal preadipocytes. Effective bovine SFRP4 siRNA sequences were screened, and an adenoviral overexpression vector was constructed for transfection and differentiation induction in bovine preadipocytes. Oil Red O staining, qRT-PCR, and Western blotting were used to evaluate the function of SFRP4. Results demonstrated the CDS of SFRP4 gene was 1 041 bp; There were high homology between Qinchuan cattle SFRP4 amino acid sequences and those of sheep and goats, with more distant relationships to mice. This protein was classified as a hydrophilic protein and had a total of 24 phosphorylation modification sites. SFRP4 was broadly expressed across tissues, with higher expression in kidney and subcutaneous fat. Temporal expression patterns of SFRP4 during perirenal preadipocyte differentiation indicated an initial increase followed by a decrease. SFRP4 knockdown resulted in reduced lipid droplet accumulation in perirenal preadipocytes, with significant downregulation of adipogenic differentiation marker genes PPARγ, FABP4, PLIN2, and SCD1 (P < 0.05), and a highly significant reduction in CEBPβ expression (P < 0.01). PPARγ (P < 0.05) and CEBPβ (P < 0.01) protein levels were also downregulated. Conversely, SFRP4 overexpression led to significant upregulation of PPARγ (P < 0.01), CEBPβ (P < 0.01), FABP4 (P < 0.05), PLIN2 (P < 0.01), and SCD1 (P < 0.01) genes expression and increased PPARγ, CEBPβ, FABP4, and SCD1 protein levels (P < 0.05). These findings provide critical insights into the regulatory role of SFRP4 in bovine perirenal preadipocyte adipogenic differentiation and lipid deposition.

This study aimed to identify differential metabolites and pathways associated with blood oxygen saturation (BOS) by analyzing blood metabolites between the high and low BOS groups of dairy cows. Blood samples were collected from 60 Chinese Holstein cows in two Tibet's commercial farms, then 43 blood metabolite profiles and other phenotypes were obtained from 30 Holstein cows in each of the high and low BOS groups based on absolute quantification by ¹H NMR. Fold Change analysis, t-test and variable importance in projection were used to investigate the differential metabolites and related pathways related to BOS. A total of 17 metabolites were found to be associated with high and low BOS, with 3 up-regulated metabolites (glycine, mannitol, urea; P < 0.05) and 14 down-regulated metabolites (citrate, formate, pyruvate and others; P < 0.05) in high BOS group. The 16 metabolic pathways were found that related to the glyoxylate and dicarboxylate metabolism, glycine, serine and threonine metabolism and others. Differential metabolites were related to a variety of metabolisms such as amino acids and organic acids in the cow body and had a critical role in the protection and treatment of tissue damage, the regulation of cellular homeostasis, and the alleviation of stress caused by hypoxia. In general, this study identified the differential metabolites as well as pathways related to BOS levels, providing a foundation for further research on the mechanism of high-altitude adaptation in cows and alleviating high-altitude hypoxia stress.

This study aimed to investigate the genetic structure, diversity, and kinship of geese at the Daozhou gray goose conservation farm to guide future conservation efforts.The Daozhou gray goose conservation farm used a single-sire family rotation model, involving 49 families. A healthy 200-day-old male goose from each family was selected for DNA extraction via venous blood sampling. The DNA samples were subjected to whole-genome resequencing, and the resulting data were analyzed using bioinformatics software to assess the genetic structure and diversity of the population.A total of 15 245 050 SNPs were detected in the Daozhou gray goose conservation population, reducing to 14 628 485 after quality control. Genetic diversity analysis revealed an average observed heterozygosity (Ho) of 0.280, expected heterozygosity (He) of 0.288, inbreeding coefficient (Fis) of 0.070, average polymorphism information content (PIC) of 0.262, and gene diversity index (Nei) of 0.331. A total of 802 runs of homozygosity (ROH) were identified, with an inbreeding coefficient of 0.010, indicating low inbreeding levels. IBS distance matrix and G-matrix results showed that only a few individuals had close kinship. Population admixture and principal component analysis indicated that most individuals clustered into one subpopulation, while a few formed two small subpopulations. Fst calculation result showed moderate differentiation between the two smaller subpopulations.In summary, the conserved population of Daozhou gray goose exhibits relatively rich genetic diversity and a low level of inbreeding, reflecting good conservation outcomes. It is recommended to decrease breeding between family lines with excessively close kinship relationships in order to maintain genetic diversity and ensure the long-term health of the population.

To investigate the differences in muscle growth among different populations of Mongolian horses, the muscle phenotype and molecular-level differences between the Mongolian horse populations with the most significant environmental differences and the greatest geographical distance(Baerhu horse and Wushen horse) was compared. The experimental animals in this study consisted of 3 Baerhu horses raised in the Chenbaerhu Banner and 3 Wushen horses raised in the Wushen Banner. All horses were healthy stallions, free-range grazing in the wild. The average age of the horses in each population was 5 years, with similar body conditions within each population. The pre-slaughter live weight of the Baerhu horses was (303.10±14.10) kg, and the carcass weight was (148.29±15.43) kg. The pre-slaughter live weight of the Wushen horses was (287.90±37.5) kg, and the carcass weight was (140.83±5.04) kg. The experimental animals were divided into two populations based on their breed, with 3 repetitions per group. All animals were slaughtered, and their gluteus medius muscles were collected. After the muscle samples were embedded in paraffin, HE and immunohistochemistry staining were performed. The muscle fiber area and the proportion of slow-twitch muscle fibers were statistically analyzed. Additionally, transcriptome sequencing was conducted on the collected muscle samples. The sequencing data were analyzed for differentially expressed genes using the DESeq2 software, and GO and KEGG enrichment analyses were performed using the David online tool. Finally, qRT-PCR was used to validate the sequencing results. In this experiment, HE staining and immunohistochemical staining revealed that the average muscle fiber area of the gluteus medius in Baerhu horses was (2 592±180.92) μm², while in Wushen horses it was (1 997±73.39) μm², with a significant difference between the two populations (P < 0.05). The proportion of slow-twitch muscle fibers in the gluteus medius of Baerhu horses was (10.34±0.59)%, while in Wushen horses it was (8.14±0.81)%, with no significant difference between the two populations (P>0.05).This study identified a total of 1 103 differentially expressed genes in the gluteus medius of Baerhu and Wushen horses, with 460 upregulated genes and 643 downregulated genes expressed in the gluteus medius of Wushen horses. The study found that MYH15, MYOZ2, several glutamate receptor genes, and several GABAA receptor genes were highly expressed in the gluteus medius of Baerhu horses, while MYH6 and FOXO1 genes were highly expressed in the gluteus medius of Wushen horses. Enrichment analysis of the differentially expressed genes revealed 208 enriched items in the GO analysis and 65 pathways in the KEGG analysis. These enriched items and pathways were mainly associated with glutamate signaling, GABA signaling, and muscle growth and development. This study investigates the gluteus medius muscle of the Baerhu horse and the Wushen horse, finding that the fiber area of the gluteus medius in Baerhu horses is significantly larger than that in Wushen horses, while the proportion of slow muscle fibers shows no significant difference. At the molecular level, there are notable differences between the two populations, particularly in muscle fiber types, fiber area, and neuromuscular signaling in the muscle spindles.

The aim of this study was to study the developmental patterns of behavioral characteristics of Kunming dogs and to explore their associations with age and gender. A total of 100 Kunming puppy dogs (50 males and 50 females) were selected and longitudinally studied from 3 to 12 months of age. Their behavioral characteristics were tested and analyzed for 4 core indicators: running speed, possession, social adaptation and olfactory ability, with tests conducted once a month. The results showed that the running speed of Kunming dogs gradually increased from the puppy stage to the adult stage, with gender having a minimal impact on running speed; in terms of possession, female dogs exhibited a stronger desire to possess at specific ages, while male dogs maintained a stable trend; in terms of social adaptation, Kunming dogs showed a steady upward trend from 3 to 12 months of age, but there was a significant gender difference, with female dogs being relatively less socially adaptable than male dogs, except at specific ages; in terms of olfactory ability, Kunming dogs maintained a relatively stable high level and did not show a clear upward trend. Based on the physiology, behavior, and training content of working dogs, the 4 core indicators of running speed, possession, social adaptation and olfactory ability are used to characterize the behavioral characteristics of Kunming dogs. These 4 core indicators each have their own characteristics in terms of age and gender, showing the strengths and weaknesses of Kunming dogs, and providing a more comprehensive and scientific basis for the breeding selection, population development, and training use of Kunming dogs.

This study aimed to reveal the bidirectional transcriptional regulation character of prolactin receptor gene (PRLR) and sperm flagellar protein 2 (SPEF2) in chicken embryonic gonads. The left and right gonads of 180 Dawu Jinfeng chicken embryos at 4 embryonic ages were collected, and each 3 samples mixing pools formed a repeat. Except for the complete degeneration of the right ovary of E21.5 hens, the other ipsilateral gonads of the same embryonic age and sex were divided into 14 groups. The expression changes of PRLR and SPEF2 in gonads of Dawujinfeng chickens at 4 embryonic ages (E12.5, E16.5, E18.5 and E21.5) were detected using real-time quantitative PCR (RT-qPCR). The transcription start sites (TSS) of the two genes were identified using rapid amplification of cDNA ends (5'RACE), their core promoter region was identified using dual luciferase reporter assay (DLRA). The methylation level in promoter region was detected using bisulfite genomic sequencing PCR (BSP). Results were as follows: The expression of PRLR in testis from E12.5 to E21.5 was higher than in ovary (P<0.05), while the expression of SPEF2 in ovary from E18.5 to E21.5 was higher than in testis (P<0.05). Among the 10 TSSs of PRLR, 5 had promoter activity, and all the 3 TSSs of SPEF2 had promoter activity. PA1 promoter region of PRLR and SC of SPEF2 exhibited the highest promoter activity (P<0.05), respectively. Further detection found that 565 bp of PA1 and 478 bp of SC in reverse complement had the highest promoter activity, respectively. The bidirectional promoter activity of 478 bp region was significantly higher than 565 bp (P<0.05), and both had significantly higher promoter activity for PRLR than for SPEF2 (P<0.05), which was in conformity with that the expression of PRLR was significantly higher than SPEF2 in chicken embryonic gonads at E12.5-E21.5 (P<0.05). That the methylation level of 443 bp CpG island in the bidirectional promoter region was significantly higher in E21.5 ovary than in testis (P<0.05), was in accordance with the higher expression of PRLR in testis. The methylation level of 159 bp CpG island in the first intron of SPEF2 was significantly higher in testis than in ovary (P<0.05), correlated with the higher expression of SPEF2 in ovary. In summary, the transcriptional expression of PRLR and SPEF2 in chicken embryonic gonads was regulated by bidirectional promoter with 478 bp core region, which had higher promoter activity for PRLR than SPEF2, and the expression of PRLR in gonad was higher than SPEF2. The methylation of the CpG island in BDP participated in regulating the transcriptional expression of PRLR, with ovary at E21.5 had higher methylation level, and PRLR was expressed lower in ovary. These results provided a theoretical basis for revealing transcriptional regulation mechanism of PRLR and SPEF2 in chicken gonadal development.

This study aimed to explore the effect of bone morphogenetic protein 4 (BMP4) on the expression of gap junction protein alpha 1 (GJA1) in sheep ovarian granulosa cells and its molecular regulatory mechanism. Granulosa cells were isolated from the ovaries of healthy 2-4-year-old sheep obtained from a slaughterhouse in Langfang city. Immunofluorescence staining was used to localize the distribution of GJA1 in granulosa cells. The cells were randomly divided into 4 groups, each treated with recombinant BMP4 protein at concentrations of 0, 10, 50, and 100 ng·mL^-1, with 3 replicates per group, and cultured for 24 hours. Cell viability was assessed using the CCK-8 assay, while RT-qPCR and Western blot were employed to examine the effects of BMP4 on GJA1 mRNA and protein expression levels. To investigate the potential mechanism by which BMP4 regulates GJA1 expression, the cells were divided into 3 groups with 3 replicates per group. In addition to the control group, cells were treated with 10 μmol·L^-1 BMP type I receptor inhibitor (Dorsomorphin) and small interfering RNA to knock down SMAD family member 4 (SMAD4), all treated with 100 ng·mL^-1 BMP4 for 24 hours. RT-qPCR was used to measure the expression of GJA1 and SMAD4, and Western blot was used to assess the expression levels of GJA1 and SMAD4 as well as the phosphorylation levels of SMAD1/5/8. Finally, a scratch dye tracking assay was performed to assess the gap junction activity between sheep ovarian granulosa cells. The results showed that BMP4 significantly inhibited the expression of GJA1 and gap junction activity in sheep ovarian granulosa cells (P < 0.05). This inhibitory effect was significantly weakened after treatment with Dorsomorphin and SMAD4 knockdown (P < 0.05). Moreover, BMP4 treatment significantly increased the phosphorylation levels of SMAD1/5/8 (P < 0.05). In conclusion, BMP4 regulates GJA1 expression and subsequently affects the gap junction activity of granulosa cells through the SMAD1/5/8-SMAD4 signaling pathway. These findings enhance the understanding of how the BMP/SMAD pathway regulates gap junction activity in sheep granulosa cells, providing a foundation for improving in vitro follicle maturation methods and molecular breeding of high-fertility ewes.

Based on 16S rRNA sequencing technology, this study aimed to analyze the differential changes of vaginal microbial communities in sheep during pre- and endometrial receptivity, and screen the marker flora of endometrial receptivity in sheep. In this experiment, 12 healthy multiparous female Hu sheep aged two years old with similar body weight were selected as the research objects. The experiment was divided into 2 groups, 6 sheep in each group. The vaginal secretions and endometrial tissues were collected on the 5th day (pre-receptive endometrium group, PE group) and the 15th day (peceptive endometrium group, RE group) after mating. RT-qPCR was used to detect the expression of endometrial tissue-related receptivity genes, and the 16S rRNA V3V4 variable region was selected for sequencing analysis of vaginal secretions to compare the differences in microbial composition between the 2 groups. RT-qPCR results showed that the expression of HOXA10, LIF and VEGFA genes in the endometrium on the 15 th day after mating was significantly higher than that on the 5 th day after mating (P < 0.01 or P < 0.05), indicating that the endometrium on the 15 th day after mating entered the receptive state. Alpha diversity analysis showed that there was no significant difference in vaginal microorganisms between the pre-receptive group and the receptive group (P>0.05). Comparative analysis of Beta diversity showed that there were differences between the pre-tolerance group and the tolerance group. The results of relative abundance of species showed that Proteobacteria and Firmicutes were the dominant phyla at the level of phylum classification, accounting for more than 70%. Compared with the pre-tolerance group, the abundance of Proteobacteria and Fusobacteriota in the tolerance group decreased, the abundance of Firmicutes increased; at the genus level, unidentified _ Enterobacteriaceae and PorpHyromonas were the dominant genera. Compared with the pre-tolerance group, the abundance of unidentified _ Enterobacteriaceae, Streptobacillus and other bacteria in the tolerance group decreased; the abundance of PorpHyromonas and Ureaplasma increased. The results of differential flora comparison showed that there were significant differences in the abundance of Fusobacteriota and Acidobacteriota at the phylum level (P < 0.05). At the genus level, there were significant differences in the abundance of Occallatibacter, EdapHobacter and Roseiarcus (P < 0.05). Functional prediction analysis showed that compared with the pre-receptive group, progesterone-mediated oocyte maturation, estrogen signaling pathway, antigen processing and presentation were significantly increased in the receptive group (P < 0.05). There was no significant difference in microbial Alpha diversity between the pre-receptive and receptive groups of sheep endometrium, and Beta diversity indicated that there were differences between the two groups. At the same time, there were significant differences in flora at multiple classification levels. Facklamia, Turicibacter and Firmicutes could be used as the marker flora of endometrial receptivity in sheep.

The study aimed to investigate the effect of genistein (GEN) added to the semen extender on the cryopreservation of semen from cattle, and to provide a theoretical basis for optimizing the semen extender of cattle and improving the sperm quality of cattle after freezing and thawing. In this study, the fresh semen of 6 health Qinchuan bulls aged 3-5 years old, weighing (586±20) kg were collected, and the semen that passed the test was mixed, diluted with different concentrations of genistein for cryopreservation. According to the different concentrations of GEN in the semen extender, this study was divided into 5 groups, 0 μg·mL^-1 GEN group (control group), 30 μg·mL^-1 GEN group, 60 μg·mL^-1 GEN group, 90 μg·mL^-1 GEN group, and 120 μg·mL^-1 GEN group, with 3 replicates in each group. After thawing, bovine automatic sperm quality analyzer was used to measure the motility performance of sperm, hypotonic swelling detection method and peanut lectin fluorescent labeling method were used to detect sperm acrosome integrity and plasma membrane integrity, and the kits were used to detect mitochondrial membrane potential (MMP), antioxidant performance, malondialdehyde (MDA) content and reactive oxygen species (ROS) level. Compared with the control group, the forward motility sperm rate of the 30, 60 and 90 μg·mL^-1 GEN groups was significantly increased (P < 0.05), and the acrosome integrity of sperm in the 30 and 60 μg·mL^-1 GEN groups was significantly increased (P < 0.05). The ROS level in the 60 μg·mL^-1 GEN group was significantly lower than that in the other groups (P < 0.05). Compared with the control group, the contents of T-AOC and SOD were significantly increased in the GEN group (P < 0.05), the MDA content was significantly decreased in the 30, 60 and 90 μg·mL^-1 GEN groups (P < 0.05), and the GSH-px content was significantly increased in the 60, 90 μg·mL^-1 GEN groups (P < 0.05), the CAT content was significantly increased in the 60, 120 μg·mL^-1 GEN groups (P < 0.05). The MMP of the 60 μg·mL^-1 GEN group was significantly higher than that of the other groups (P < 0.05). The results showed that the supplementation of the semen extender with genistein could improve the antioxidant performance of sperm in bovine semen, inhibit the oxidative stress of sperm during cryopreservation, alleviate the freeze-thaw damage of sperm acrosomes and mitochondria, and improve the motility performance of sperm after thawing, so as to improve the quality of bull semen after freezing-thawing, and the concentration of genistein was 60 μg·mL^-1.

The purpose of this study was to investigate the effect of 450 mg·kg^-1 ursolic acid(UA) on breast muscle quality of broilers and to explore its possible mechanism of action on wooden breast (WB). A total of 120 Arbor Acres (AA) broilers with similar initial body weight were selected as experimental animals and randomly divided into 2 groups with 6 replicates in each group and 10 chickens in each replicate. One group was fed a basal diet (CON group), and the other group was fed a basal diet added with 450 mg·kg^-1 UA (UA group). The results of this experiment showed that: 1) UA significantly reduced the incidence of wooden breast and had the most significant effect in slightly or moderately WB (P<0.05). 2) UA could improve the breast muscle quality of broilers, which showed a significant decrease in shear force, yellowness and drip loss, but a significant increase in pH_45min (P<0.05). 3) The results of tissue section observation showed that UA significantly reduced muscle fiber diameter, muscle fiber cross-sectional area and collagen volume ratio, and significantly increased muscle fiber density (P<0.01).4) UA significantly decreased the contents of CK and HYP in serum (P<0.01), significantly decreased the activities of PFK and LDH in breast muscle, and significantly increased the content of glycogen in breast muscle (P<0.01), but had no significant effect on the content of GLU in serum (P>0.05). 5) UA not only significantly increased T-AOC level, GSH-Px activity and decreased MDA content in breast muscle (P<0.05), but also significantly decreased the contents of TNF-α, IL-6 and TGF-β1 in breast muscle (P<0.05). 6) At the gene expression level, UA significantly reduced the expression of TGF-β1, IL-6, TNF-α, LDHA, PFK1, PI3K, HIF-1α, Akt1, P65 genes in breast muscle tissue (P<0.05), and significantly increased the expression of Smad7 gene (P<0.01). In summary, UA can improve the quality of breast muscle in broilers, inhibit glycolysis, increase muscle glycogen content, reduce the adverse effects of oxidative stress and inflammation of breast muscle on broilers, and effectively reduce the incidence of WB in broilers. This result may be achieved by inhibiting the PI3K/Akt/HIF-1α signaling pathway and enhancing the negative feedback regulation of the TGF-β1/Smads signaling pathway. Therefore, adding 450 mg·kg^-1 UA to the basal diet of broilers is expected to be an effective strategy to alleviate the occurrence of WB.

This study aimed to analyze the nutrients content of corn from ten different sources, determine the apparent ileal amino acid digestibility (AID AA) and standardized ileal amino acid digestibility (SID AA) of corn in 28-day-old white-feathered broilers and construct the prediction equations of SID AA. A total of 330 1-day-old healthy male broilers (Arbor Acres) were randomly divided into 11 groups with 6 replicates per group and 5 broilers per replicate. At the age of 25 d, broilers in 11 treatments were fed 10 experimental diets which the protein sources only came from corn and a nitrogen-free diet, respectively. All broilers were euthanized on 28 d, and the ileal digesta were collected to determine the standardized ileal amino acid digestibility. The results showed that, the average content of dry matter(DM), crude protein(CP), ether extract(EE), Ash, crude fiber(CF), neutral detergent fiber(NDF), acid detergent fiber(ADF), nitrogen-free extract(NFE), phytic acid(PA), total phosphorous(TP), total starch(TS) of corn from 10 different sources were 87.14%, 9.04%, 3.94%, 1.36%, 1.97%, 10.07%, 2.61%, 70.82%, 0.84%, 0.26% and 74.56%, respectively. The content of 18 amino acids ranged from 0.20% to 1.99%. Pro had the highest coefficient of variation (22.84%) and the coefficient of variation of Cys (10.34%) was the lowest. The AID and SID were the lowest for threonine (44.80% and 68.15%), and the highest for leucine (83.93% and 89.56%). The AID AA and SID AA were significantly affected by the sources(P < 0.05). A total of 16 SID AA prediction equations of corn in 28-day-old white-feathered broilers were constructed, among which SID Phe had the highest fitting degree (R²=0.962) and SID Gly had the lowest fitting degree (R²=0.478). In conclusion, the prediction equations established in this study had a better reference value for realizing precise nutrition and efficient utilization of corn in white-feathered broilers.

The aim of this experiment was to investigate the key genes and signaling pathways of regulation of energy-restricted feeding during rearing and conversion to ad libitum on the reproductive organ development and hormone level of laying hens at the initiation of laying period using transcriptome sequencing technology. A total of 720 6-week-old Hyline-Brown chicks were allocated equally to three groups with six replicates of 40 chicks each, and were fed one of three diets that were nutritionally equal with the exception of ME levels. From 6 to 17 weeks of age, the chicks in control group were given diet with 12.34 MJ·kg^-1 ME, and fed ad libitum. The levels of ME in diet of chicks in the experimental groups were 90% [11.11 (12.34×90%) MJ·kg^-1] and 80% [9.87 (12.34×80%) MJ·kg^-1] of that in control group, and the daily amount of feed was restricted to the absolute quantity of the diet consumed by chicks in control group. From 18 to 20 weeks of age, all laying pullets were fed a basal diet ad libitum. At 20 weeks of age, four chicks in each of the ad libitum feeding group and 80% energy-restricted feeding group with significant differences (P < 0.05) in reproductive organ development and plasma progesterone concentrations were selected to screen the novel mRNA implemented by the RNA-seq. The results showed as follows: 1) The body weight and body weight CV of chicks at 20 weeks of age decreased linearly with increasing energy restriction (P < 0.001), the ADFI and F/G (P < 0.001) from 6 to 20 weeks of age increased linearly, while the ADMEI and ADG (P < 0.001) decreased linearly with increasing the degree of energy restriction. 2) A gradual increase in the degree of energy restriction resulted in a gradual increase in the serum UN (P=0.007), but a gradual decrease in serum TG and LDL (P=0.045, 0.029), and in liver TC and NEFA (P=0.024, 0.003). 3) With the increase of energy restriction during rearing period, the length, ratio of length to body weight, weight and index of oviduct (P= 0.012, 0.016, 0.042, 0.045), the number and index of small yellow follicle (P=0.017, 0.039), and the weight and index of ovarian stroma (P=0.046, 0.047) decreased linearly, while the plasma progesterone level (P < 0.001) increased linearly. 4) The ovary stroma of chicks at 20 weeks of age in the ad libitum feeding group (ALF20W) and 80% energy-restricted feeding group (ERF20W) were used to screen the novel mRNA implemented by the RNA-seq. The proportion of pure reads matching to chicken reference genome was more than 93.77%, and the content of Q20 and Q30 was more than 97.03% and 92.14%, respectively. A total of 1 488 differential genes were screened in ALF20W and ERF20W, of which 600 were down-regulated and 888 were up-regulated in ERF20W. The GO functional enrichment analysis found that differentially expressed mRNAs were involved in biological processes such as biological regulation of cell proliferation, development, and reproduction. The KEGG pathway were significantly enriched in 28 pathways, among these pathways, the steroid hormone biosynthesis pathway, estrogen signaling pathway, ovarian steroidogenesis pathway, and cAMP signaling pathway were related to energy metabolism or reproduction. The differentially expressed genes including cAMP response element-binding protein (CREB), steroid-producing acute regulatory protein (StAR), cytochrome P450 1B1 (CYP1B1), insulin-like growth factor-1 (IGF-I), adrenocorticotropic hormone (MC2R), cytoskeletal keratin (KRT18), and progesterone receptor (PGR) were found to be enriched in the above signaling pathways, which maybe the potential target gene and pathway of energy restriction regulating reproductive organ development and estrogen production in laying chicks. The qRT-PCR results showed that the expression trends of 10 randomly selected differentially expressed genes were consistent with RNA-Seq results. The results showed that the body weight, body weight CV, serum TG and LDL, liver TC and NEFA, the length, ratio of length to body weight, weight and index of oviduct, the number and index of small yellow follicle, and the weight and index of ovarian stroma of chicks at 20 weeks of age decreased linearly with increasing the degree of energy restriction, while the plasma progesterone level increased linearly. It is suggested that energy restriction during the rearing period may regulate the expression of StAR, CREB1, CYP1B1, IGF-I, MC2R, KRT18 and PGR genes in ovarian tissues, and act on steroid hormone biosynthesis pathway, estrogen signaling pathway, ovarian steroidogenesis pathway, and cAMP signaling pathway, to regulate the energy metabolism, reproductive organ development and estrogen production of laying chicks at the initiation of laying period.

The aim of this study was to investigate the effects of chlorogenic acid (CGA) on the reproductive performance of female rabbits and the growth of suckling rabbits under heat stress conditions, and to screen the optimum dosage of CGA supplemented in basal diet. Four hundred and sixty Hyla female rabbits with similar body weight and parity were randomly assigned into five groups: Control group (basal diet), CGA-200 group (basal diet+200 mg·kg^-1 CGA), CGA-400 group (basal diet+400 mg·kg^-1 CGA), CGA-600 group (basal diet+600 mg·kg^-1 CGA) and CGA-800 group (basal diet+800 mg·kg^-1 CGA). The pre-feeding period lasted for 7 days, and the experimental period lasted for 71 days. During the experiment, the environmental temperature and relative humidity of the rabbit house were monitored and recorded to calculate the temperature-humidity index (THI), and the reproductive performance and serum antioxidant capacity were determined. The results of THI showed that the proportions of heat stress conditions were 67%, in which extreme heat stress, severe heat stress and moderate heat stress were 27%, 20% and 20%, respectively. Results showed that the conception rate and farrowing rate in CGA-400 group were significantly elevated by 29.08 percentage points (71.25% vs. 42.17%) and 18.77 percentage points (52.50% vs. 33.73%), respectively, when compared with the control group (P < 0.05). Compared with the control group, the litter size and number of kits born alive in 400 mg·kg^-1 CGA group were significantly enhanced by 0.80 (8.36 vs. 7.56) and 1.01 (7.64 vs. 6.63) (P < 0.05). Moreover, 400 mg·kg^-1 CGA supplementation significantly enhanced overall weight of the litter, the weight of litters born alive, and the average body weight of kits born alive when compared with control group (P < 0.05). Furthermore, dietary supplemented with 400 mg·kg^-1 CGA significantly increased the litter weight and average body weight of suckling rabbits at 7, 14, 21 and 35 days after birth (P < 0.05). Additionally, dietary supplemented with 400 mg·kg^-1 CGA significantly enhanced the concentration of progesterone (P), total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities(P < 0.05), and significantly reduced MDA concentration in the serum of females rabbits when compared with that of the control group at the 15th day of gestation (P < 0.05). Collectively, dietary supplemented with 400 mg·kg^-1 CGA significantly improved reproductive performance of female rabbits and the growth of suckling rabbits under heat stress. In conclusion, 400 mg·kg^-1 CGA can alleviate heat stress-induced oxidative stress, improve reproductive performance of female rabbits and the growth of suckling rabbits.

To establish a rapid TaqMan fluorescent quantitative PCR detection method for duck plague virus (DPV), specific primers and probes were designed based on the conserved sequence of the DPV US6 gene, and a recombinant plasmid DPV-US6 standard was constructed. The sensitivity, specificity, and reproducibility of the method were evaluated, and it was applied to study the proliferation of DPV in chicken embryo fibroblast (CEF) cells and the distribution pattern of virus load in the organs and tissues of artificially infected ducks. The results showed that the DPV TaqMan fluorescent quantitative PCR detection method was successfully established, with a minimum detection limit of 10 copies·μL^-1. There was no cross-reaction with Muscovy duck parvovirus, duck circovirus, duck Tembusu virus, duck reovirus, H9N2 subtype avian influenza virus, duck hepatitis A virus types Ⅰ and Ⅲ, or duck chlamydia. The inter-batch and intra-batch coefficient of variation were both less than 2.0%. After DPV-AX strain infected CEF cells, the viral nucleic acid copy number detection results indicated that the virus proliferated slowly from 4 to 8 hours, rapidly increased from 12 to 60 hours, peaked at 60 hours, and gradually declined from 72 to 144 hours. Compared with the virus titer determined by the TCID₅₀ method, the two detection methods showed good correlation, allowing the copy number to replace TCID₅₀. The virus load distribution detection in organs and tissues of ducks artificially infected with DPV showed the highest virus load in the liver. The detection method established in this experiment provides a tool for studying the proliferation pattern of the DPV-AX strain in CEF cells.

Avian influenza, caused by avian influenza viruses, is a highly contagious disease characterized by high mortality rates among infected poultry, posing significant health risks to poultry and economic burdens. However, the majority of duck farming comprises small-scale and free-range practices, where environmental conditions vary and ducks are prone to diseases. In such scenarios, traditional vaccination practices present challenges such as inconvenient injection and immunological stress. To tackle this issue, this study devised a method to produce a convenient and safe oral biological agent of hemagglutinin (HA) tailored specifically for ducklings. We constructed the anchoring sequence (pgsA, BmpA, cA, and M6)-GFP reporter expression system, enabling the visualization of different anchoring sequences' effects on surface-displayed proteins in Lactococcus lactis (L. lactis). With this system, we successfully identified BmpA and cA as efficient anchoring sequences in L. lactis. After comparing the expression efficiency of HA in L. lactis and optimizing the codons, we selected the optimized HA gene sequence and connected it with BmpA and cA. Then, we co-integrated them into the expression plasmids of L. lactis. Subsequently, we introduced the constructed expression plasmids into L. lactis containing the integrated HA1 gene in the genome, thereby generating recombinant L. lactis capable of both inducible secretory expression and surface display of HA(NZB-HA1-pNZ8148-BmpA-HA1 and NZB-HA1-pNZ8148-HA1-cA). The ELISA results indicated that orally administered inducible secretory-surface display recombinant L. lactis eliciteds superior levels of HA-specific IgG in duckling serum compared to single-expression recombinant L. lactis treatment. This study successfully established the Anchoring sequence-GFP screening system for Lactobacillus, identifying efficient anchoring sequences suitable for L. lactis, thereby achieving efficient expression of HA on the surface of L. lactis. Subsequently, in conjunction with the genomic secretion expression, we successfully obtained an oral biological agent with satisfactory HA immunogenicity for ducklings. This achievement offers a practical and feasible strategy for the subsequent development of convenient and safe avian influenza oral vaccines.

Avian metapneumovirus (aMPV) subtype B is associated with the development of swollen head syndrome in chickens, which can lead to immunosuppression, severe secondary infections, and a subsequent decline in the productive performance of the poultry. The establishment of a reverse genetics system for domestic aMPV isolates is of considerable significance for advancing virological research and facilitating the development of targeted vaccines. The amplicons of the viral genome were sequenced individually, and the complete genome sequence of the aMPV B1 strain was deduced through sequence assembly. The genome was divided into four segments based on enzyme cleavage sites and sequentially inserted into the pTH vector to construct the viral genome plasmid pTH-B1. A synonymous mutation introduced at position 7 474 of the viral genome to silence the Sal I cleavage site as a genetic marker. A Pme I cleavage site was introduced between the M and F genes of the pTH-B1 plasmid, and the EGFP expression cassette was inserted using this site to obtain the plasmid pTH-B1EGFP. The expression cassettes of the viral N, P, and M2.1 genes were co-cloned into the pCI-Neo expression vector, yielding the auxiliary plasmid pCI-NPM2.1. The L gene was individually cloned into the pCI-Neo expression vector, yielding the auxiliary plasmid pCI-L. The pTH-B1 and pTH-B1EGFP plasmids were co-transfected with the two auxiliary plasmids into BSR/T7 cells, respectively. Subsequently, the transfected cells were co-cultured with Vero cells and continuously passaged for further analysis. The successful rescue of the rB1 and rB1-EGFP strains was confirmed through the observation of cytopathic effects (CPE) and green fluorescence, along with the detection of vrial N gene and genetic marker, indirect immunofluorescence assay (IFA) and Western blot. The proliferation levels and trends of the rB1 and rB1-EGFP strains were found to be similar to those of the parental virus strain. This study successfully obtained the infectious clone of the subtype B avian metapneumovirus B1 strain using a three-plasmid rescue system. The intergenic non-coding region between the M and F genes is capable of accommodating gene expression sequences, which lays a necessary foundation for further investigation into the pathogenesis of subtype B aMPV and the development of vaccines.

This study aimed to design an mRNA vaccine vector for feline infectious peritonitis virus (FIPV) and evaluate the immunogenicity of the transcribed FIPV mRNA vaccines. The nucleocapsid (N) protein of FIPV was selected, and its sequence was optimized, Using CureVac's mRNA technology platform, appropriate non-coding sequence, signal peptide sequences, and poly A tails were chosen. The synthesized target sequence was cloned into the pBluescript Ⅱ KS(+) vector, linearized by a single enzyme digestion, and transcribed in vitro to synthesize mRNA encoding the FIPV N gene. The transcribed mRNA sequence was capped, purified, and analyzed by agarose gel electrophoresis. In vitro transfection assays were used to verify the expression of antigen proteins in cells. Subsequently, mice were immunized with the FIPV N-mRNA vaccine and their humoral and cellular immune responses were assessed. Agarose gel electrophoresis confirmed the successful preparation of a stable single mRNA sequence using the designed mRNA transcription vector. After transfection into cells, the target antigen protein could be expressed stably within 12-24 hours. ELISA results showed a robust humoral immune response to the FIPV N-mRNA vaccine in mice, with significantly higher levels of specific antibodies, IL-4, and TNF-α compared to the control groups. Elispot assay results demonstrated significantly higher levels of IFN-γ secreted by spleen cells in the FIPV N-mRNA group compared to the control groups. The mRNA vaccine transcribed by the vector targeting the FIPV N protein efficiently expressrs the desired protein in cells and exhibits good immunogenicity, eliciting both humoral and cellular immune response in mice. The FIPV N-mRNA vaccine may be a preparation of mRNA transcription vector provides important reference value for the design and development of mRNA vaccines against infectious diseases.

In this study, a strain of F-type enterovirus was isolated from bovine enterovirus(BEV) positive feces through plaque purification by inoculating BHK cells. After being correctly identified by RT-PCR, it was sent to a biological company for whole-genome sequencing and TCID₅₀ determination. The whole genome of BEV was analyzed and a genetic evolution tree was constructed using bioinformatics software such as MEGA11.0. The recombinant protein containing the VP3 gene of the BEV isolate was obtained through prokaryotic expression and used as a coating antigen to establish an indirect ELISA method for detecting antibodies. The specificity, sensitivity, and repeatability of the established ELISA method were measured and used for the detection of clinical samples. The results showed that the TCID₅₀ of the BEV isolate was 10^-6.26·mL^-1, and the full length of the virus isolate was 7 439 nt, with an ORF size of 6 504 bp, encoding 2 168 amino acids. It had the closest genetic relationship and the highest nucleotide homology with F-type enterovirus BEV4. Therefore, the isolated virus was an F-type enterovirus, temporarily named BEV-QD. Compared with other F-type strains, the BEV-QD strain had amino acid mutations and deletions, mostly concentrated in the P2 and P3 regions. The indirect ELISA method for antibody detection established in this study had good repeatability; the results remained positive after the positive serum was diluted 1 600 times, indicating good sensitivity; it did not specifically bind to positive sera such as BVDV, indicating good specificity; among the 40 clinical samples tested, 13 were positive, with a positive rate of 32.5%. This study isolated an F-type bovine enterovirus mutant strain and established an indirect ELISA method for antibody detection, providing a basic means for the detection and prevention of bovine enterovirus.

This study was aimed at screening Tibetan medicines exerting inhibitory effects on the in vitro replication of Bovine coronavirus (BCoV). To achieve this, a prokaryotic expression vector encoding the BCoV nucleocapsid (N) protein was constructed. Following induced expression, the purified N protein was used to immunize mice and rabbits, resulting in the production of mouse anti-N protein polyclonal antibodies and rabbit anti-N protein antibodies. Western blot and indirect immunofluorescence assay (IFA) techniques were subsequently developed to assess BCoV replication. The water extract of 21 candidate Tibetan medicines was obtained by water decoction method. The maximum safe concentration of water extract on human colorectal adenocarcinoma cells (HCT-8) was determined by CCK-8. HCT-8 cells infected with BCoV were used as the research object, and BCoV infection group, Tibetan medicine water extract treatment group and ribavirin treatment group were set up respectively. After 24 h of BCoV infection, real-time fluorescence quantitative PCR, Western blot and IFA were used to determine the replication of BCoV in cells. The inhibitory effect of Tibetan medicine on virus replication was statistically analyzed, and Tibetan medicine with significant inhibitory effect on BCoV was screened. The polyclonal antibodies of mouse anti-BCoV N protein and rabbit anti-BCoV N protein were prepared respectively, and the Western blot and IFA methods for detecting BCoV were established. Among the 21 Tibetan medicines, 15 had significant inhibitory effects on BCoV replication (P < 0.01). Among them, the inhibitory effects of three Tibetan medicines, Euphorbia altotibetica Paulsen, Terminalia chebula Retz., and Stellera chamaejasme L. on BCoV replication were better than those of ribavirin. In this study, 15 Tibetan medicines with inhibitory effects on BCoV replication in vitro were screened. Among them, three Tibetan medicines, Euphorbia altotibetica Paulsen, Terminalia chebula Retz., and Stellera chamaejasme L. had significant inhibitory effects, which laid a foundation for the development of BCoV prevention and treatment drugs.

This study aimed to express the C-terminal binding domain (S1-CTD) of the porcine epidemic diarrhea virus (PEDV) S1 protein in a soluble prokaryotic system and to screen DNA aptamers targeting S1-CTD using the systematic evolution of ligands by exponential enrichment (SELEX) technique, laying the foundation for the development of therapeutic drugs and detection methods for the PEDV. The PEDV-S1-CTD plasmid was cloned into prokaryotic expression vectors containing maltose-binding protein (MBP), small ubiquitin-like modifier (SUMO), transcription termination factor (NusA), and glutathione S-transferase (GST) tags. Recombinant plasmids pET21b-MBP-S1, pET21b-SUMO-S1, pET21b-NusA-S1, and pET21b-GST-S1 were constructed and transformed into E. coli BL21 for expression. The rMBP-S1 recombinant protein with good solubility and high expression was selected for further experiments. Western blot and indirect immunofluorescence assay (IFA) confirmed the correct expression and receptor binding of the protein. Aptamer Apt-S1-3 and Apt-S1-11 were enriched with the highest affinity (18% and 16.2%, respectively) and specificity, respectively. ELASA determined a dissociation constant of 2.09 nmol for Apt-S1-3, which showed no cross-reactivity with rMBP, rMBP-gD, rMBP-GP5, or BSA proteins. Mfold analysis and molecular docking simulations revealed the aptamer's ability to form stable complexes with the target protein. IFA and flow cytometry confirmed the aptamer's recognition and inhibition of PEDV infection. This study successfully screened a DNA aptamer that binds to the PEDV S1-CTD protein with high affinity and specificity, inhibiting virus infection and providing a foundation for targeted therapy and detection methods for PEDV.

In order to better understand the differences in material metabolism and virulence changes between virulent and attenuated strains of Mycoplasma hyopneumoniae (Mhp), this study was based on the virulent strain ES-2 and the attenuated strain ES-2L induced by passaging of strain ES-2.The growth characteristics, cell adhesion ability, metabolic production of hydrogen peroxide level, biofilm formation ability and other biological characteristics and comparative genomics of the two strains were studied. In terms of growth characteristics, the growth rate and titer of ES-2L strain were higher than that of ES-2 strain, and its color change unit (CCU) was 1.0×10¹⁰ CCU·mL^-1, which had better adaptability on medium. In terms of virulence changes, both ES-2 and ES-2L strains could form biofilm, but the biofilm formation capacity of ES-2L strain was weaker than that of ES-2 strain. The level of metabolizing glycerol to produce hydrogen peroxide of ES-2L strain was lower than that of ES-2 strain, and its adhesion ability to bronchial epithelial cells was weaker than that of ES-2 strain. The results of genome collinearity comparison between ES-2 and ES-2L showed that the overall collinearity of the two strains was good, but there were several gene deletion and inversion in ES-2L strains. The localization analysis of the deletion genes in ES-2L strains showed that a total of 9 large fragments (>500 bp) of gene deletion were detected, and these 9 fragments contained a total of 18 deletion genes. Analysis of SNP (single nucleotide polymorphism) and Indel (Insertion and deletion variation) showed that 348 SNVs (single nucleotide variations) were detected, which were mainly distributed in 99 genes, and 22 dels (deletion variations) were mainly distributed in 19 genes. The results of gene island analysis showed that ES-2L had a gene island coding sequence ranging from 395 to 425 kb, and ES-2 had two gene island coding sequences ranging from 354 to 364 kb, and 920 to 945 kb.These deletion or SNP genes and gene island changes may be related to the decrease of virulence and the change of growth characteristics of ES-2L. In summary, this study clarified the differences in material metabolism and virulence changes between virulent and attenuated strains of Mhp, and analyzed the changes of the two strains at the gene level by using comparative genomics method, providing a perspective for further understanding the pathogenic mechanism of Mhp and mining virulence factors.

The study aims to construct Glaesserella parasuis (G. parasuis) mutants overexpressing oligopeptide permease gene through improvement of its expression capacity, and to evaluate its immunogenicity and efficacity in mice. G. parasuis CF7066 strain being used as the parent strain, the overexpressing mutans were constructed via natural transformation and Flp-FRT markless mutation system. The OppA expression capacity of the overexpression strains was detected by WB, and then strains were induced by arabinose to excise Kan^R cassette and were cultured at high temperature to eliminated Flp expression plasmid. The mutant of ΔnanH :: oppA^R was selected for conductind immunization and challenge experiments in mice. Comprehensive evaluations were conducted on serum antibody levels, spleen lymphocyte proliferation and the murine survival rate. Three overexpressing mutans of ΔnanH :: oppA^R, ΔnanH :: oppA andΔnanH : : P_qseB-oppA were constructed. Because ΔnanH :: oppA^R had the highest capacity for expression of OppA, it wase used for murine immunization experiment. The rOppA-ELISA showed that the mice immunized with ΔnanH :: oppA^R strain produced the higher level of OppA antibody than CF7066 and ΔnanH, but lower than the commercial inactivated vaccine group. After being stimulated with rOppA, the number of lymphocyte proliferation were significantly larger than before immunization among the mice immunized with ΔnanH :: oppA^R and with CF7066(P < 0.05), but not in the mice immunized with ΔnanH, the inactivated vaccine and PBS control. The challenge experiment showed that the survival rate were 100% in the mice immunized with ΔnanH :: oppA^R and CF7066, and it was 75% in ΔnanH group. Three overexpressing mutants of G. parasuis were constructed, in which ΔnanH :: oppA^R has the highest expression capacity of OppA. Immunization and challenge experiments showed that ΔnanH :: oppA^R elicited murine antibody production and splenic lymphocyte proliferation specific for OppA protein, and simultaneously provided substantial protection against the wild strain CF7066. Thus, ΔnanH :: oppA^R possessed potential as an attenuated vaccine strain for G. parasuis.

The purpose of this study is to investigate the effects of sheep milk and goat milk on the liver and kidney of type 2 diabetic mice, and provide theoretical reference for clinical prevention and treatment of diabetes and its complications. Dairy analyzer was used to detect the nutritional components of sheep milk and goat milk. The type 2 diabetes mellitus(T2DM) mouse models were induced by high-sugar and high-fat diet combined with streptozotocin (STZ, STZ injection was conducted in week 5). The diabetic mice were randomly divided into diabetes model control (DMC) group, sheep milk group (Sheep) and goat milk group (Goat) with 10 mice in each group. A control group (Con) was also set up. Mice in Sheep group and Goat group were fed sheep milk and goat milk respectively from the first week until the end of the ninth week, and the body weight of mice and their food intake were measured every week. Glucose tolerance of mice was measured at the end of the 4th and 9th week. At the end of the 9th week, blood and urine were collected from mice, and the levels of blood lipids, blood urea nitrogen, serum creatinine, urinary albumin, and urinary creatinine were determined. Using Real time-PCR to test the expression abundance of mRNA of IL-6, TNF-α, IL-1β and TGF-β1 in the liver and kidneys. It was found that the fat, protein, lactose and total solid content of sheep milk were significantly higher than those of goat milk (P < 0.01) based on dairy analyzer analysis. Before the injection of STZ, the body weight of DMC mice at week 2 to week 4 increased significantly compared with the Con group (P < 0.01). At the 3rd and 4th week, the body weight of mice in Sheep group decreased significantly compared with DMC group (P < 0.01). After STZ injection, the body weight of DMC mice decreased significantly (P < 0.05) compored with Con group. However, at the end of the 9th week, compared with the DMC group, the weight loss rate of mice in Sheep group slowed down significantly (P < 0.01). In the glucose tolerance test, it was found that the area under the glucose tolerance test curve of mice in Sheep and Goat groups decreased significantly compared with the DMC group (P < 0.05) at the end of the 9th week. Analysis of serum and urine samples showed that the blood lipid levels and urine albumin levels of mice in Sheep and Goat groups tended to decrease compared with the DMC group, but there was no significant difference (P>0.05). However, the serum urea nitrogen and blood creatinine levels, as well as the urine creatinine level, decreased significantly (P < 0.05). In addition, the HE staining results showed that sheep and goat milk could alleviate liver steatosis, glomerular lesions and renal tubular epithelial cell vacuolar degeneration in diabetic mice. Compared with the DMC group, the mRNA content of IL-6, TNF-α, IL-1β, and TGF-β1 in the liver and kidneys of the Sheep group mice showed a significant decrease, while in the Goat group mice, the content of other inflammatory factors also significantly decreased, except the TNF-α of kidney. In conclusion, sheep milk and goat milk can significantly inhibit the intake of T2DM mice, delay the weight loss of diabetic mice, improve glucose tolerance as well as effectively reduce the histopathological damage of liver and kidney, significantly inhibit the inflammation of liver and kidney, and play an effective role in the treatment of diabetes mellitus and its complications, and the mitigating effect of sheep milk is better than that of goat milk.

The aim of this study was to establish an indirect ELISA method for the detection of porcine teschovirus (PTV) type 5 and to provide a material reserve for its prevention and control. PTV is an acute virulent pathogen of pigs, which is characterized by high morbidity and mortality. The clinical manifestations include encephalitis, pneumonia, reproductive disorders, myocarditis, and diarrhea. PTV can be divided into various subtypes. The clinical symptoms vary from each other. Therefore, lacking of efficient and specific serological detection methods blocks the control and prevention of PTV-5. This study inserted the VP1 gene of PTV-5 into pET-30a plasmid and obtained the VP1 protein. After optimizing the conditions, an indirect ELISA method against the VP1 of PTV-5 was successfully established. The method has no cross-reactivity with the standard positive sera of common porcine viruses. The lowest detection dilution was 1∶3 200, and the coefficients of variation were less than 10% both intra-and inter-batch, showing good specificity, sensitivity, and reproducibility of the method. A total of 279 clinical samples collected from the northeast were detected by this method. The results showed that the positive rate was 67.74%, which was in consistent with previous report. The indirect ELISA assay established in this study has a good overall evaluation and provides technical support for the clinical detection of PTV-5.

IL-1β mediates inflammatory pathology, and blocking IL-1 activity has important anti-inflammatory effects. In this study, recombinant protein was prepared by constructing porcine IL-1β prokaryotic expression plasmid, expression by Escherichia coli and protein purification. Four hybridoma cell lines (1E2, 2H3, 5H3 and 7E7) with stable IL-1β monoclonal antibody secretion were prepared by cell hybridoma technique. The ELISA titer of the 4 mAbs in ascites were up to 1: 1 024 000. The epitope of 5H3 mAb was ¹⁴⁹DLKREVVFCM¹⁵⁸. The epitope recognized by 1E2, 2H3 and 7E7 mAb was ²⁰²KRYPKRD²⁰⁸. The anti-inflammatory test results of lipopolysaccharide (LPS) mice inflammation model showed that compared with LPS control group, serum NO, TNF-α, IL-6 and IL-1β contents and clinical symptom score of mice treated with IL-1β monoclonal antibody were significantly reduced, indicating that all the four strains of monoclonal antibody had better anti-inflammatory effect, and 1E2 monoclonal antibody had the best anti-inflammatory effect. It laid an important foundation for the study of porcine IL-1β blocking drugs.

The purpose of this study was to find out the specific effects of porcine epidemic diarrhea virus (PEDV) on intestinal goblet cells in piglets, and to reveal its potential mechanism. The model of PEDV infection in piglets was established, and the specific effects of virus infection on the number of goblet cells and mucus secretion in piglets' intestines were revealed by using Alcian blue-periodic acid-Schiff (AB-PAS) staining and immunofluorescence staining. In order to verify the results of in vivo experiments, an in vitro experimental model of goblet cells was further constructed by using intestinal stem cells. In this model, firstly, the replication characteristics of PEDV are explored. Then, the effects of PEDV infection on transcription of functional genes (Muc2, TFF3, SPDEF) in goblet cells were studied. Finally, the influence of virus infection on the core regulatory pathway (MAPK signaling pathway) in the biological process of goblet cells was detected. Through the observation of tissue sections, it was found that PEDV mainly infected the jejunum and ileum of piglets, and jejunum was more susceptible to virus. Infection leads to severe atrophy of intestinal villi, significant decrease of goblet cells and serious loss of mucin. In addition, the in vitro culture model of goblet cells was successfully established, and the replication characteristics of PEDV in this model were revealed. Further research based on this in vitro infection model showed that PEDV infection significantly reduced the transcription level of Muc2, TFF3 and SPDEF genes in goblet cells, and at the same time, the activity of MAPK signaling pathway was also inhibited. PEDV infection can lead to the decrease of goblet cells and mucin secretion level in piglets. The inhibition of MAPK signaling pathway by virus may be the key reason for this phenomenon.

The purpose of this study was to investigate the effect of ten eleven translocation 1 (TET 1) on the methylation of the DNA of mouse uterine natural killer (uNK) cells, and to gain insight into its molecular regulatory mechanism. Mouse uterine metaphase was aseptically collected on the 10th day of gestation, and the uNK cells were isolated and purified for culture. The expression of TET 1 gene was knocked down by RNA interference technology. Then, total DNA from cells of the interference group and the normal control group were extracted, respectively. Sequencing was performed using reduced representation bisulfite sequencing (RRBS) method, and the results were analyzed by bioinformatics software for the statistics and annotation of differentially methylated region (DMR) in the two group samples, and further analyzed by the GO database for DMR-related genes to understand their functions. Also, the sequencing results were analyzed for the differentially methylated region (DMR) annotation, and the enriched signaling pathways in which regulated by the KEGG database. The results showed that there were 14 120 DMRs in the TET 1 interference group when compared with the control group, of which 4 897 were hypermethylated DMRs and 9 223 were hypomethylated DMRs. The largest number of DMRs were distributed in the genebody, with a total of 9 762 DMRs, accounting for 69.14% of the total number of DMRs. DMRs were widely distributed in the different elements of the genome, and some genes had DMRs with both hypermethylated and hypomethylated in different elements. GO annotation results showed that the DMRs were mainly concentrated in ATP binding, nucleic acid binding, cell formation, cell differentiation, embryonic development, RNA polymerase II transcriptional regulation and negative regulation of cell proliferation, etc. KEGG database analysis results revealed that the DMRs were significantly enriched in the metabolic pathways, with a total of 12 key molecules involved in metabolism in the pyruvate metabolic pathway, and 54 DMRs appeared, which was the most significant enrichment of DMRs in the metabolic pathway. Among the 12 key molecules, acetyl-coA synthetase (Acss), lactate dehydrogenase B (Ldhb), and pyruvate kinase L/R (Pklr) exhibited a single hypermethylated status. In addition, significant enrichment of DMRs were also found in the PI3K/AKT signaling pathway and HIF-1 signaling pathway, indicating that DMRs play important roles in mediating pyruvate metabolism. Thus, TET 1 has a methylation regulatory effect mouse uNK cells, and pyruvate metabolism is the main pathway for its regulatory effect. What's more, Acss, Ldhb and Pklr are potential regulatory target molecules, while PI3K/AKT and HIF-1 are important signaling pathways which involved in regulation.

In order to reveal the male reproductive toxicity of polystyrene nanoplastics (PS-NPs), this study investigated the effects of PS-NPs on duck testicular tissue using male Shaoxing ducks. In this study, 32 Shaoxing ducks were randomly divided into 4 groups(8 ducks in each group), namely, blank Control group, L-NPs group, M-NPs group, and H-NPs group, and were fed with PS-NPs at dietary concentrations of 0, 1, 10 and 100 mg·kg^-1. After 45 days, the body weight of the ducks were weighed, the testes of the ducks were collected, testis coefficients were calculated, the pathological changes of testis tissues of each group were observed by HE staining. The testicular tissues were tested for malondialdehyde (MDA) content and the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) by kit. The expression levels of oxidative damage-related proteins Nrf2, NQO1 and HO-1 were detected by Western blot. The mRNA levels of inflammation-related genes TNF-α and IL-6 were detected by real-time fluorescent quantitative PCR. The results showed that the testicular coefficient was significantly reduced in the H-NPs group with the increase of PS-NPs treatment concentration compared with the control group (P<0.05). The pathological results showed that the interstices between the convoluted seminiferous tubules were continuously enlarged, the number of interstitial cells was gradually reduced, and the structure of convoluted tubule wall was blurred. The testicular tissues of the M-NPs group and H-NPs group had a significant (P<0.05) and highly significant (P<0.01) increase in the content of MDA. The activity of GSH, SOD and CAT decreased significantly (P<0.05) and very significantly (P<0.01). Western blot results showed that the protein expression levels of Nrf2, NQO1 and HO-1 present a trend of significant (P<0.05) or highly significant decrease (P<0.01) in the M-NPs group and H-NPs group. The real-time fluorescence quantitative PCR results showed that the mRNA transcript levels of TNF-α and IL-6 genes were significantly (P<0.05) and highly significant increased (P<0.01). The results showed that PS-NPs could cause morphological and structural changes, inflammation and oxidative stress in the testicular tissues of Shaoxing ducks. The degree of damage was positively correlated with the dose of PS-NPs exposure. The inflammatory response caused by PS-NPs may be closely related to the Nrf2/HO-1 signaling pathway.

Periodontitis is one of the common oral diseases in dogs, which can cause irreversible periodontal tissue loss and reduced periodontal adhesion, and seriously affect animal welfare. This study was designed to understand the drug resistance status oral flora in canine periodontitis; and to explore the drug sensitivity and drug resistance genes of characteristic pathogenic bacteria of canine periodontitis. This trial will be randomly selected periodontitis-infected dogs and healthy dogs to collect dental plaque and conduct drug sensitivity tests. In addition, 16S rDNA sequencing was performed on oral samples. The differences in oral flora between periodontitis-infected dogs and healthy dogs were explored, and the possible characteristic pathogenic bacteria causing canine periodontitis were identified. The drug susceptibility of the oral flora was determined. Then, the isolation, identification and drug sensitivity test were performed. The results showed that the relative abundance of Bacteroidetes and Spirochaeta increased at the level of oral flora, and the relative abundance of Porphyromonas at the genus level was significantly increased. It was speculated that the potential pathogenic bacteria of canine periodontitis was Porphyromonas. The subgingival aerobic bacteria had the highest sensitivity to amoxicillin and enrofloxacin, and had the lowest sensitivity to metronidazole. The sensitivity of subgingival anaerobes to enrofloxacin was medium, and the sensitivity to metronidazole was lowest. P. clinical is sensitive to enrofloxacin and contains two resistance genes ermF and tetQ. Enrofloxacin is preferred in the clinical treatment of canine periodontitis.

The purpose of the study was to investigate the effects of magnolol solid dispersion on growth performance, serum antioxidant capacity and gut microorganism of calves. Using the antisolvent co-precipitation method, a solid dispersion of magnolol was prepared using hydroxypropyl acetate methylcellulose succinate as the carrier, with a ratio of 2∶8 between magnolol and the carrier. Fifteen healthy 30 day old Holstein calves with similar physical conditions were randomly divided into 3 groups with 5 replicates in each group. The control group (CON group) was fed pasteurized milk, while the experimental group (MAG group, ASD group) was fed pasteurized milk with 150 mg·d^-1 magnolol and 750 mg·d^-1 solid dispersion of magnolol. The weight of the calves was measured on day 0 and 30 of the experiment, and the average daily weight gain was calculated. On the 30th day of the experiment, rectal feces were collected to detect gut microorganism, jugular vein blood was collected and serum was separated, and antioxidant capacity was measured using ELISA. The characterization results showed that the solid dispersion of magnolol was successfully prepared, and magnolol was dispersed in an amorphous form in the carrier. The pharmacodynamic results showed that magnolol solid dispersion significantly increased final weight and average daily gain of calves (P < 0.05), significantly decreased MDA content (P < 0.05), and significantly increased SOD activity, GSH-Px activity, CAT activity and T-AOC (P < 0.05), significantly increased the relative abundances of Firmicutes and Actinomycetes (P < 0.05), significantly decreased the relative abundances of Proteobacteria (P < 0.05), and significantly increased the Chaol index, Observed_otus (observed feature number), Shannon index and Simpson index (P < 0.05). The effect of magnold ASD was better than magnolol raw material. The results showed that the supplementation of magnolol solid dispersion at 750 mg·d^-1 could improve the growth performance of calves, enhance serum antioxidant capacity, increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, increase the diversity of gut microorganism and improve the composition of gut microorganism.

The study aimed to investigate the effects of different levels of enzymatic corn gluten meal (MCGM) on the growth performance, intestinal barrier, digestive enzyme activities, and intestinal microbial composition of weaned piglets. A total of 240 healthy weaned piglets (21 days-age, Duroc × Landrace × Yorkshire) with an average body weight of 5.71 ± 0.79 kg were selected. Based on the principle of body weight, the piglets were randomly assigned to five experimental groups: 5% conventional corn gluten meal (CGM) group, 5% fish meal (FM) group, and 5%, 10%, and 15% enzymatic corn gluten meal groups (MCGM1, MCGM2, and MCGM3), respectively. The feeding trial lasted for 14 days. The results showed that: 1) The feed-to-gain ratio (F/G) of weaned piglets in the MCGM1 and FM groups were significantly lower than that in the CGM, MCGM2 and MCGM3 groups (P<0.05). The fecal score in the MCGM2 group was significantly higher than that in the other experimental groups (P<0.05). 2) Compared to the CGM, MCGM2 and MCGM3 groups, the activities of trypsin and chymotrypsin in the duodenum and jejunum of the MCGM1 and FM groups were significantly increased (P<0.05). 3) The villus-to-crypt ratio in the duodenum of the MCGM1 and MCGM3 groups were significantly higher than that in the CGM and FM groups (P<0.05). Additionally, compared to the MCGM2 and MCGM3 groups, the mRNA expression levels of intestinal barrier genes (Occludin and ZO-1) in the MCGM1 group were significantly elevated (P<0.05). 4) The characteristic bacteria in the ileum of piglets in the MCGM1 group were f__Peptostreptococcaceae, o__Peptostreptococcales-Tissierellales, and g__Terrisporobacter, and in the cecum were o__Veillonellales-Selenomonadales; the characteristic bacteria in the ileum of piglets in the FM group were g__Lactobacillus and g__Prevotella. In conclusion, the addition of 5% MCGM can significantly enhance the digestive enzyme activities in the duodenum and jejunum of weaned piglets, strengthen the intestinal barrier function, and thereby improve growth performance.