Gene editing is an important tool for precise modification of specific gene sequences in the genome to achieve molecular breeding. The rapid development of gene editing technology has provided a new approach for modern molecular breeding in pigs， significantly improving breeding efficiency. This technology has undergone three generations of evolution： ①Zinc finger nuclease （ZFNs） and transcription activator-like effector nucleases （TALENs） laid the foundation for targeted editing but had the drawbacks of complex operation and high cost. ②The clustered regularly interspaced short palindromic repeats （CRISPR） system made a breakthrough， among which iGeoCas9 improved thermal stability and editing activity by a hundredfold through structural modification， and the miniaturization of Cas protein optimized delivery efficiency. ③Base editors achieved precise single-base conversion， and the guide editing system broke through type limitations， supporting free base conversion and small fragment modification. In pig breeding， this technology improves meat quality， enhances growth performance， optimizes reproductive efficiency， and strengthens disease resistance， it also enables innovative applications such as cold resistance and environmental protection. Compared with traditional breeding， gene editing technology can shorten the cycle and maintain genetic diversity， but it faces challenges such as insufficient editing efficiency， off-target risks， and industrialization barriers. This article systematically reviews the gene editing technological development and breeding applications， aiming to provide references for optimizing the performance of editing tools and establishing a safe industrialization system， promoting technological innovation， and supporting the progress of pig breeding technology and the sustainable development of the industry.

Cells are the fundamental units that make up living organisms， possessing unique developmental trajectories and molecular features. Single cell sequencing technology， as an efficient method for analyzing genetic information at the cellular level， can sequence the genome， transcriptome， proteome， metabolome， and epigenetics， and support integrated analysis strategies such as multi-omics sequencing to accurately capture the molecular characteristics of individual cells. This technology breaks through the limitations of traditional population cell research， and has become a powerful tool for analyzing the molecular regulatory mechanisms and cell fate transition trajectories of animal cells， providing a new perspective for animal genetics breeding. The artical focuses on five single-cell sequencing technologies—Single-cell RNA sequencing， single-cell DNA sequencing， single-cell epigenome sequencing， single-cell proteomics sequencing， and single-cell metabolomics sequencing， systematically elaborates on the developmental history and underlying principles， and discusses in depth the application cases of single-cell sequencing technology in livestock and poultry genetic breeding， aiming to provide a theoretical reference for subsequent research in this field.

In the context of the comprehensive ban on the addition of growth-promoting antibiotics in feed， finding safe and effective antibiotic alternatives is the research focus of the livestock industry. Probiotics have gained prominence due to their green nature， lack of harmful substances， and excellent efficacy. Bacillus coagulans （BC）， integrating functional traits of lactic acid bacteria and Bacillus， stabilizes intestinal micro-ecological homeostasis， enhances immune responses， and improves nutrient utilization efficiency in animals. Clostridium butyricum （CB）， as a Gram-positive anaerobic Bacillus， plays a key role in regulating the intestinal flora，short-chain fatty acid metabolism，and anti-inflammation and antioxidant due to its biological characteristics such as resistance，alkali resistance and heat resistance. The author briefly described the functions of the two types of probiotics， and analyzed the combined mechanism of their effects on improving animal growth performance， optimizing the structure of the intestinal flora， and enhancing immune function. The progress of the combined application of the two types of bacteria in poultry and pig production was reviewed. In order to provide a theoretical basis for the precise matching and in-depth development of future composite probiotics.

Heat stress is a common environmental issue in poultry production. Chickens’ skin lacks sweat glands， and their body heat regulation ability is limited under high-temperature conditions. In severe cases， this can have a negative impact on their production performance. This article expounds the key physiological and behavioral responses of chickens under high-temperature stress， including a sharp increase in respiratory rate， elevated body temperature， a significant reduction in feed intake， and a sharp increase in water intake. The underlying mechanism was analyzed， covering the activation and disorder of the neuroendocrine system （hypothalamic-pituitary-adrenal axis， hypothalamic-pituitary-gonadal axis and hypothalamic-pituitary-thyroid axis）. Since heat stress induces a series of negative effects in chickens，impairing intestinal health， antioxidant capacity and immune function， it ultimately compromises production performance， reproductive performance and product quality. Therefore， a thorough understanding of the impact of heat stress on the body heat regulation and production performance of chickens is of vital importance in chicken production. The author reviewed the mechanism by which heat stress affects the body heat regulation and production performance of chickens， with the aim of providing theoretical basis and practical guidance for the health management and production efficiency improvement of chickens.

As a woody plant with significant economic， medicinal， and ecological value， the main parts utilized from the mulberry tree include its fruits， leaves， branches and root bark. These parts are rich in active substances such as flavonoids， polysaccharides， polyphenols and alkaloids， which exhibit various biological functions including growth promotion， reduction of blood sugar and lipid， anti-inflammatory effects， enhanced antioxidant capacity and immune function， as well as improved meat quality. With the rapid development of technological methods， extraction techniques for these active components have been continuously optimized， common methods include solvent extraction， ultrasound-assisted extraction， and enzyme-assisted extraction. Owing to their high content of bioactive components， natural origin， residue-free nature and renewability， mulberry trees show broad application potential in the post-antibiotic era and have been increasingly used in aquaculture in recent years. However， the efficacy of specific monomers within mulberry bioactive substances and appropriate dosage standards across different species require further clarification. Issues such as the standardization and scaling of processing technologies for mulberry resources also need to be urgently addressed. Based on this， this review summarizes the classification and value of mulberry resources， extraction techniques for bioactive components， and their effects on the growth performance， antioxidant capacity， immunity function， glucose and lipid metabolism， and meat quality of aquatic animals. It also offers perspectives on future research directions， with the aim of providing reference and insights for the developing new feed resources and advancing the utilization of mulberry in feed applications.

As a by-product of Astragalus， the yield of Astragalus straw is more than ten times that of its root. As a feed resource with both nutritional value and biological activity， its application in ruminant breeding has attracted significant attention. The author systematically reviews the nutrient content， composition and levels of bioactive compounds in Astragalus straw， as well as its application in ruminant feeding. Astragalus straws are rich in crude protein and minerals， and are high quality roughage resources， but shows significant differences in nutrient content depending on origin and cutting period. Astragalus straw contains multiple bioactive substances such as polysaccharides， flavonoids and saponins. Except for polysaccharides， the content of other active substances is similar to that of the root. Studies have shown that the bioactive compounds present in Astragalus straw can enhance growth and fattening performance， improve rumen fermentation function， modulate the rumen microbial community structure， and boost the antioxidant and immune functions in ruminants. Consequently， it holds promising application prospects and significant practical implications in ruminant production. Future research should continue to explore the metabolism mechanism of Astragalus straw in ruminants， and optimize its processing and utilization technology， to provide a more solid theoretical and practical foundation for the wide application of Astragalus straw in animal husbandry.

Camel milk is highly regarded for its distinctive flavor and rich nutritional composition. Volatile flavor compounds， which are key carriers of its unique sensory characteristics， play a central role in determining the quality of camel milk and influencing the development of the related industry. This review systematically elaborates on the composition and characteristics of major flavor compounds in camel milk， such as aldehydes， ketones， acids， and esters. It also examines the formation mechanisms of these flavor substances through pathways including lipid metabolism， protein hydrolysis， and carbohydrate metabolism. Furthermore， the impact of various factors （such as storage conditions， packaging methods， heat treatment intensity， feed composition， seasonal variations， as well as candidate genes and regulatory factors） on the flavor profile of camel milk is summarized. The review aims to provide a theoretical foundation for the high-value utilization of camel milk resources， the development of distinctive flavored dairy products， and the regulation of flavor and quality in camel milk.

Recombinant live vector vaccines utilize attenuated pathogens as carriers to deliver target antigens， offering distinct advantages including potent immunogenicity， cost-effective production， and flexible immunization strategies. These vaccines demonstrate broad prospects in infectious disease prevention and tumor immunotherapy. This review systematically summarizes recent advances in bacterial and viral vector-based vaccines， with particular focus on their antigen expression characteristics， suitable animal models， and immunization efficacy. Regarding bacterial vectors， genetically modified Bacillus Calmette-Guérin expressing Mycobacterium tuberculosis antigens or immunomodulators significantly enhances immunization efficacy. Salmonella vectors exhibit outstanding performance in mucosal immunization and cancer therapy due to their suitability for oral administration. Lactic acid bacteria， as safe delivery platforms， effectively induce both mucosal and systemic immune responses. Additionally， Escherichia coli and Bacillus subtilis have shown promising progress in developing anti-infection and anti-tumor vaccines. Among viral vectors， Adenoviruses have been successfully employed in Coronavirus disease 2019 （COVID-19） and swine influenza vaccines owing to their high transduction efficiency. Poxviruses are ideal for multivalent vaccine design due to their large capacity for foreign gene insertion. Herpesviruses demonstrate excellent immunogenicity when expressing Foot-and-mouth disease virus or Influenza virus antigens， while maintaining high safety profiles due to their restricted host range. RNA virus vectors such as Newcastle disease virus and Porcine reproductive and respiratory syndrome virus serve as promising platforms for polyvalent vaccines， leveraging their unique replication features. Furthermore， enterovirus vectors show potential for treating neurological disorders. Despite these achievements， further improvements in immunogenicity， safety optimization， and scalable manufacturing remain key research priorities. This review provides valuable insights to guide future development and clinical translation of live vector vaccines.

Porcine epidemic diarrhea virus （PEDV） is one of the high-risk pathogens in the pig farming industry and poses a significant threat to global pig farming. More seriously， due to the high variability of the virus and its immune escape characteristics， it poses a more severe challenge to traditional vaccines and chemical control drugs. Based on the multi-target synergistic intervention effect of traditional Chinese medicine （TCM） formulas， TCM has become one of the hot directions in the current antiviral research field. This paper comprehensively reviews the domestic and international research progress of TCM in the prevention and control of porcine epidemic diarrhea （PED）， and systematically summarizes the prevention and control effects and mechanisms of action of the latest TCM formulas. At the same time， combined with modern TCM technology， involving technologies such as artificial intelligence， network pharmacology， TCM nanotechnology， and TCM probiotic fermentation， it sorts out and summarizes the multi-path and multi-strategy enhancement of TCM， and looks forward to the development direction and application prospects of TCM in the prevention and treatment of PED， providing safer and more effective TCM compound preparations for the prevention and control of PED， and also providing reference for the prevention and control of other animal viral diseases.

Canine myxomatous mitral valve disease （MMVD） is the most common heart disease and the most common cause of cardiac death in domestic dogs. Myxomatous degeneration of the mitral valve cannot be separated from the histological lesions of mitral valve cells. The most predominant lesion is deposition and degeneration of the extracellular matrix （ECM） of the valve. Activation of valve interstitial cells （VICs） into a myofibroblast phenotype is the source of the ECM lesion. There are multiple mechanisms for the activation of VICs. In the pathogenesis of MMVD， 5-hydroxytryptamine （5-HT） pathway is associated with changes in the physical and chemical environments of valves. The transforming growth factor-β （TGF-β） pathway is the main signalling pathway controlling the pathogenesis of MMVD and is associated with inflammation， mechanical forces and angiotensin in the animal organism. Bone morphogenetic proteins （BMPs） are associated with the developmental process of the animal organism. Reactive oxygen species （ROS） are involved in myocardial remodelling and heart failure and amplify the activation of MMVD by TGF-β， and a disintegrin and metalloproteinase thrombospondin motifs （ADAMTS） are associated with valve structure and function. At this stage， the treatment of MMVD antagonistic pathway drugs has emerged. 5-HT receptor antagonists attenuate VICs activation and MMVD-associated valve tissue lesions， and cytokines such as the cell membrane repair protein MG53 and fibroblast growth factor inhibit the TGF-β signalling pathway thereby treating MMVD. This article discusses three major aspects of MMVD histopathology， the mechanism of VICs activation， and the progress of research on therapeutic drugs for MMVD antagonistic pathways， in order to provide reference for in-depth research and clinical treatment of this disease.

The somatic cell nuclear transfer （SCNT） technology， as a significant breakthrough in the field of developmental biology in the 20th century， its landmark achievement was the birth of the first cloned mammal Dolly， which marked a new era in mammalian cloning technology.This technology accomplishes complete nuclear reprogramming through the transplantation of a differentiated somatic cell nucleus into an enucleated oocyte， ultimately producing genetically identical organisms. The author systematically reviewed the scientific history， key technological optimizations， and significant application values of sheep cloning research. It focus on pivotal scientific issues such as the selection of donot cells， strategies for improving nuclear transfer efficiency， the molecular mechanisms of epigenetic reprogramming， and the regulation of the oocyte microenvironment. The study also evaluates the technology’s potential across biomedical domains such as accelerated livestock breeding， endangered species preservation， human disease model construction， and therapeutic cloning. Concluding with informed deliberations on future technological trends， this review aims to furnish a framework for both theoretical investigation and the industrial utilization of SCNT.

Ticks， as significant ectoparasites of dogs， can spread various zoonotic diseases， such as Lyme disease and babesiosis， posing a serious threat to public health. At present， insecticides are the main means to control ticks， but the long-term use has led to an increase in the resistance of ticks and has also caused environmental pollution problems. Therefore， it is urgent to develop safer and more effective prevention and control measures. The canine anti-tick vaccine has a broad application potential as it interferes with the feeding， reproduction and attachment functions of ticks through specific antibodies. The research has found that multiple candidate proteins （such as Bm86， ATAQ， SUB， TIM， HK， LIP， FER1， FER2， P0， 64TRP， AQP， L7LTU1 and GST） can serve as vaccine antigens， reducing the reproduction and infection ability of ticks by decreasing tick numbers， weight， and oviposition. However， the cross-protection effect of the existing antigens against various ticks is still not satisfactory， and the application effect in dogs still needs further verification. Future research should focus on canine vaccine trials， develop more candidate protective antigens， utilize new technologies such as vaccine genomics and quantum vaccine genomics to enhance vaccine efficacy， and combine with breeding management， rational use of pesticides and anti-tick vaccines to form a comprehensive prevention and control system.The author summarized the various types of ticks and their hazards to the health of dogs and humans， reviewed the application of insecticides in tick control and its limitations， and focused on analyzing the current research status of canine tick vaccines.

Objective This study aimed to investigate the effects of dietary supplementation with Eucalyptus globulus essential oil （EGO） on the growth performance， serum biochemical indicators， antioxidant capacity and intestinal health of broilers under heat stress. Method A total of 100 healthy 21-day-old Cobb male broilers with similar body weights were randomly divided into 5 groups with 3 replicates per group and 15 broilers per replicate. Broilers in control group （CON） was housed at （24±1）℃ throughout the day and fed a basal diet， and broilers in the other groups were subjected to heat stress （（32±1）℃ from 09：00 to 17：00 daily and （24±1）℃ otherwise） and fed basal diets supplemented with 0 （HS group）， 100 （HS+EGO-L group）， 200 （HS+EGO-M group）， and 300 （HS+EGO-H group） mg/kg EGO. The experiment lasted 21 d. During the trial period， the growth performance was statistically analyzed. At the end of the trial， subwing vein blood， intestinal tissues and cecal contents of broilers were collected to determine serum biochemical indicators， intestinal tissue morphology， and cecal microflora， respectively. Result ①Compared with CON group， the final body weight （FBW）， average daily feed intake （ADFI）， and average daily gain （ADG） of broilers in HS group were significantly decreased （P<0.05）， and the F/G was significantly increased （P<0.05）. The total bilirubin （TBIL） content and alkaline phosphatase （ALP） activity in serum of broilers in HS group were significantly increased （P<0.05）， while the calcium and phosphorus levels in serum were significantly decreased （P<0.05）. Serum superoxide dismutase （SOD） activity of broilers in HS group was significantly decreased （P<0.05）， and malondialdehyde （MDA） content was significantly increased （P<0.05）. Heat stress significantly reduced the villus height （VH） in duodenum and jejunum of broilers in HS group （P<0.05）. Cecal microflora analysis revealed a significant decrease in Simpson index （P<0.05） and a significant increase in Shannon index （P<0.05）， along with altered Beta diversity. The relative abundance of Firmicutes of broilers in HS group increased， the relative abundance of Bacteroidetes decreased， the ratio of Firmicutes to Bacteroidetes （F/B） increased， and the relative abundance of Eisenbergiella and Butyricicoccus of broilers in HS group were specifically proliferated. ②Compared with HS group， FBW and ADG of broilers in HS+EGO-M and HS+EGO-H groups were significantly increased （P<0.05）， and F/G in HS+EGO-H group was significantly decreased （P<0.05）. Serum ALP activity of broilers in HS+EGO-H group was significantly decreased （P<0.05）， while serum phosphorus level in HS+EGO-M and HS+EGO-H groups was significantly increased （P<0.05）. Serum calcium level in each EGO group was significantly increased and MDA content was significantly decreased （P<0.05）. VH in duodenum of HS+EGO-L and HS+EGO-M groups and jejunum of HS+EGO-H group was significantly increased （P<0.05）. The relative abundance of Firmicutes in cecum of broilers in each EGO group was decreased， and the relative abundance of Bacteroidetes in HS+EGO-M and HS+EGO-H groups was increased， with a decrease in F/B ratio. The relative abundance of Christensenellaceae_R-7_group， Faecalibacterium， Blautia， and norank_o_RF39 in cecum of broilers in HS+EGO-H group were specifically proliferated. ③The Tuzzerella， norank_f_Oscillospiraceae， and Escherichia-Shigella showed significant or extremely significant positive correlation with serum MDA content （P<0.05 or P<0.01）， and Christensenellaceae_R-7_group， Lachnoclostridium， norank_o_Clostridia_UCG-014 and Akkermansia showed significant or extremely significant negative correlation with serum MDA content （P<0.05 or P<0.01）. Conclusion Under the experiment conditions， addition of EGO to feed could alleviate liver damage and oxidative stress in broilers under heat stress， improve the antioxidant capacity of the body， improve intestinal morphology， and modulate gut microbiota balance， and then improve the growth performance of broilers. A dosage of 300 mg/kg EGO was recommended for optimal efficacy.

Objective The aim of this study was to investigate the effects of Glycyrrhiza crude polysaccharides （GP） supplementation on alfalfa silage quality， antioxidant activity， microbial community structure， and in vitro rumen fermentation parameters， so as to provide theoretical and practical references for utilizing local characteristic Chinese herbal medicine active ingredients to modulate high-quality alfalfa silage and develop functional forage products. Method Alfalfa （Medicago sativa L.） at the early flowering stage was used as the experimental material. The experiment was divided into 4 treatments with 4 replicates. Each treatment was supplemented with 0 （control group， CK）， 0.5% （GP1）， 1.0% （GP2）， and 1.5% （GP3）GP， respectively. After 60 days of ensiling， the nutritional composition， fermentation quality， antioxidant activity， microbial community structure， and in vitro rumen fermentation parameters of alfalfa silage were analyzed. Result Compared with CK group， ①GP supplementation significantly increased the contents of dry matter （DM）， ether extract （EE）， and total polysaccharides （TP）， the number of lactic acid bacteria （LAB）， total antioxidant capacity （T-AOC）， glutathione peroxidase （GSH-Px） activity， and DDPH free radical scavenging rate of alfalfa silage （P<0.05）. Conversely， GP supplementation significantly reduced silage pH and yeast counts （P<0.05）. The contents of water-soluble carbohydrates （WSC） and lactic acid （LA）， and superoxide dismutase （SOD） activity in GP2 and GP3 groups were significantly increased （P<0.05）. The contents of neutral detergent fiber （NDF） and propionic acid （PA） in GP3 group were significantly decreased （P<0.05）. ②GP supplementation decreased the relative abundances of Cyanobacteria and unclassified_p_Cyanobacteria in alfalfa silage while increased the relative abundances of Firmicutes， Enterococcus mundtii， Pediococcus acidilactici， and Lactiplantibacillus plantarum. Specifically， the relative abundance of Fructilactobacillus sanfranciscensis in GP2 group showed a marked increase， whereas the relative abundances of Staphylococcusequorum and Staphylococcus epidermidis in GP1 and GP3 groups exhibited significant increase. Correlation analysis results revealed that the relative abundance of unclassified_p_Cyanobacteria was significantly positively correlated with pH and yeast counts （P<0.05）， while was significantly negatively correlated with LA content and LAB counts （P<0.05）. The relative abundance of Lactiplantibacillus plantarum was significantly negatively correlated with yeast counts （P<0.05）. ③GP supplementation significantly increased the in vitro dry matter digestibility （IVDMD） and in vitro neutral detergent fiber digestibility （IVNDFD） of alfalfa silage （P<0.05）. Conclusion Under the experimental conditions， GP supplementation could improve alfalfa silage quality， antioxidant activity， and in vitro rumen fermentation parameters， and enhance the abundance of beneficial bacteria. Overall， adding 1.0% GP provided the optimal improvement in alfalfa silage quality.

Objective This study aimed to investigate the effects of diets with different protein levels on growth performance and rumen metabolism regulation of yaks， so as to provide theoretical basis for feeding yaks with different diet protein levels. Method 20 healthy yaks with average body weight of （210.60±11.75） kg were selected for this trial. The comparable net energy level （7.73 and 7.65 MJ/kg）， were divided into 2 groups according to different protein levels （18.88% and 12.77%）： Low-protein diet group and high-protein diet group， each group consists of 10 heads. The trial period was 20 days and the formal period was 90 days. Then the growth performance and nutrient digestibility were measured， and the rumen fluid was collected for non-targeted metabolome analysis. Result After 90 days of fattening， the average daily gain of yaks in high protein diet group was extremely significantly higher than that in low protein diet group （P<0.01）. The crude protein digestibility and crude ash digestibility of yaks in high protein diet group was significantly or extremely significantly higher than that in low protein diet group （P<0.05 or P<0.01）. A total of 29 up-regulated and 28 down-regulated differential metabolites were screened by gas chromatography and mass spectrometry， and 287 up-regulated and 244 down-regulated differential metabolites were screened by liquid chromatography and mass spectrometry， respectively. Analysis of the correlation of differential metabolites showed that 21 pairs of positively correlated and 18 pairs of negatively correlated metabolites. Pathway enrichment analysis showed that phenylalanine metabolism， tyrosine metabolism， vitamin B₆ metabolism， biosynthesis of ubiquinone and other terpenoid quinones， and riboflavin metabolism were more significant. Conclusion The average daily gain of yaks fed 18.88% crude protein and 7.65 MJ/kg net energy was significantly higher than that of 12.77% crude protein diet group， which could effectively shorten the fattening cycle and improve the level of rumen microbial metabolism of yaks.

Objective This study aimed to investigate the effects of glycerol monolaurate （GML） on the growth performance， rumen fermentation parameters， serum biochemical indices， and gas emissions in dairy cows. Method Forty-eight seven-month-old healthy Holstein cows with similar body weights were selected and randomly assigned to four groups with twelve cows per group. Based on the daily dry matter intake of the test animals， 0 （control group）， 0.6%， 1.2% and 1.8% GML were added to the basic feed， respectively. The pretest period was 14 days， the main test period was 56 days. The body weight of dairy cows were weighed on the 0 and 50th days， and the average daily weight gain was calculated. Fecal samples of dairy cows were collected using rectal fecal sampling on the 50th to 54th days for the determination of nutrient apparent digestibility. Rumen fluid of dairy cows was collected on the 0 and 51st days for the determination of rumen fermentation parameters. Blood samples were collected on the 52nd days for the determination of serum biochemical indices， and the respiratory mask was used to determine the rate of gas emission from dairy cows on the 49th to 56th days. Result ①The final body weight and average daily gain of dairy cows in 0.6% GML group were significantly lower than those in other groups （P<0.05）. The apparent digestibility of neutral detergent fiber of dairy cows in 0.6% and 1.8% GML groups were significantly lower than that in control and 1.2% GML groups （P<0.05）， and the apparent digestibility of acid detergent fiber and crude protein were significantly higher than those in other groups （P<0.05）. ②There were no significant differences of microbial protein content and pH in rumen fluid of dairy cows in each group （P>0.05）. The contents of acetic acid （A） and propionic acid （P） and A/P value in rumen fluid of dairy cows on the 51st days showed significant linearly changes with the amount of GML additive （P<0.05）. ③There were no significant differences of total protein， albumin， urea， glucose， triglyceride and lipase in serum of dairy cows in each group （P>0.05）. The white globule ratio and total cholesterol content in 1.2% GML group were significantly lower than those in other groups （P<0.05）. ④Methane （CH₄） and carbon dioxide （CO₂） emission rates measured by respiratory mask showed a slight decreasing trend in 0.6% GML group compared with control group， whereas 1.2% and 1.8% GML groups showed marginally higher emission. However， no significant differences were detected in each group （P>0.05）. Conclusion Supplementing the diet with 0.6% GML exerted a certain emissions-reducing effect by lowering CH₄ and CO₂ emission rates， while also affecting growth performance. However， higher doses of GML （1.2% and 1.8%） demonstrated potential advantages in improving the utilization rate of crude protein and acidic fiber in rumen， as well as promoting daily weight gain.

Objective This study aimed to investigate the effects of dietary vitamin B₆ （VB₆） contents on the growth performance， biochemical parameters， and digestive and antioxidant capacity in juvenile largemouth bass （Micropterus salmoides）， and determine the optimal dietary VB₆ requirement， providing nutrient requirements parameters and theoretical basis for the development of formulated feed in juvenile largemouth bass. Method A total of 2 400 juvenile largemouth bass with an initial body weight of （92.50±0.28）mg were randomly divided into six groups： P0 （control group）， P1， P2， P3， P4， and P5， with four replicates per group and 100 fish per replicate， which were fed six types of formulated feed containing 3.66， 9.89， 17.80， 31.03， 46.36， and 58.91 mg/kg VB₆， respectively， for a 30 days culture period. After the test， the effects of VB₆ on the growth performance， biochemical parameters， and digestive and antioxidant capacity in juvenile largemouth bass were measured， and the water stress test was carried out. Result The weight gain and specific growth rates of juvenile largemouth bass in P1 group were significantly higher than those in P4 and P5 groups （P<0.05）， and there was no significant difference in feed coefficient （P>0.05）. The crude protein content of juvenile largemouth bass in P4 group was significantly higher than that in P2 group （P<0.05）， the crude fat content was significantly lower than that in P0， P1， and P3 groups （P<0.05）. There was no significant difference in the contents of glucose and triglycerides， and alanine aminotransferase activity in visceral mass of juvenile largemouth bass in each group （P>0.05）. The aspartate aminotransferase activity in P4 and P5 groups was significantly higher than that in P0 group （P<0.05）. The intestinal muscular thickness of juvenile largemouth bass in P2， P3， and P5 groups was significantly lower than that in P0 group （P<0.05）. The activities of trypsin and α-amylase in P4 and P5 groups were significantly lower than those in P0 group （P<0.05）， while the lipase activity in P1 group was significantly higher than that in other groups （P<0.05）. The survival rate of juvenile largemouth bass in P2-P5 groups was significantly lower than that in P1 group post-air-exposure stress （P<0.05）， the total superoxide dismutase （T-SOD） activity in visceral mass was significantly lower than that in P0 and P1 groups （P<0.05）， and the catalase （CAT） activity was significantly lower than that in P0 group （P<0.05）. The malondialdehyde （MDA） content in P3 group was significantly lower than that in P0 group （P<0.05）. Compared with pre-air-exposure stress， the T-SOD activity and MDA content in visceral mass of juvenile largemouth bass in each group were significantly increased post-air-exposure stress （P<0.05）， while CAT activity only significantly increased in P0-P2 groups （P<0.05）. Conclusion Under the conditions of this experiment， line regression analysis showed that the VB₆ requirement of juvenile largemouth bass in feed was 5.34 mg/kg using weight gain rate as the evaluation index， and the optimal amount of VB₆ addition to the feed was 9.89 mg/kg with survival rate after post-air exposure as the evaluation index.

Objective This study aimed to investigate the differences in egg quality among six local chicken breeds （Chahua chickens， Tibetan chickens， Silkies chickens， Jining Bairi chickens， Wenshang Barred chickens， and Rugao Yellow chickens）， and evaluate their egg production performance during the peak laying period， so as to provide references for breeding and utilization of local chicken breeds. Method A total of 60 hens per breed at 25 weeks of age were selected， with 6 replicates per breed and 10 hens per replicate. At 30 weeks of age， 45 fresh eggs from each breed were collected. Among these， 30 eggs were used to determine conventional indices such as egg weight （EW）， eggshell strength （ESS）， and Haugh unit （HU）， while the remaining 15 eggs were used to measure albumen water content （AWC）， yolk water content （YWC）， and yolk crude fat content （YCF）. Comparative analysis， correlation analysis， and principal component analysis （PCA） were performed. Result Chahua chickens exhibited significantly lower EW and yolk weight （YW） compared with the other five breeds （P<0.05）. All breeds showed excellent HU， with Tibetan chickens having the highest albumen height （AH） and HU. Silkies chickens had the highest ESS at 41.90 N. Compared with the other five breeds， Wenshang Barred chickens had the thickest eggshell thickness （EST） and the lowest AWC （P<0.05）. Rugao Yellow chickens had the highest yolk ratio （YR， 29.96%）， but the lowest yolk color （YC）， YWC， and YCF. The average coefficients of variation for egg quality traits across breeds ranged from 6.40% to 7.81%， indicating relatively stable characteristics. Correlation analysis revealed significant or extremely significant correlations among 11 pairs of indicators （P<0.05 or P<0.01）， including AH and HU， EW and YW， and ESS and EST. The strongest correlation was observed between AH and HU （r=0.974）. PCA results indicated that Tibetan chickens was the optimal choice due to its significant advantage in the albumen factor （score： 1.333）. Although Rugao Yellow chickens had the highest shape index （SI） score （0.626）， it performed poorly in terms of albumen， eggshell， and yolk quality， resulting in the lowest comprehensive score. Conclusion Six local chicken breeds exhibited distinct genetic potential， with significant differences in traits such as EW， ESS， AH， and YCF. Dynamic correlations were observed among various egg quality indicators. Among all breeds， Tibetan chickens demonstrated the best comprehensive egg quality.

Objective This experiment aimed to investigate the effects of different mixing ratios of fresh alfalfa and oat hay on fermentation quality and in vitro rumenfermentation characteristics when used for mixed silage. Method Fresh alfalfa and oat hay were uniformly mixed at fresh weight ratios of 8∶2 （AO1）， 7∶3 （AO2）， and 6∶4 （AO3）， and ensiled for 60 days. The silage effects were analyzed through sensory evaluation， and measurement of composition， fermentation quality， aerobic stability， and in vitro rumen fermentation parameters. Result ①The sensory evaluation score of the AO2 mixed silage reached 18.87， was significantly higher than that of AO1 and AO3 groups （P<0.05）.②AO1 silage had the highest contents of crude protein （CP ） and ether extract （EE）， and the lowest contents of acid detergent fiber （ADF）， Ash， and water soluble carbohydrates （WSC）. There were no significant differences in neutral detergent fiber （NDF） among the groups （P>0.05）. The forage quality scores of AO1 and AO2 groups were significantly higher than that of AO3 group （P<0.05）， with AO1 silage having the highest score of 60.39. Aerobic stability results showed that the AO2 silage maintained stability for the longest time under aerobic exposure （204.5 h）， followed by AO3 （166.45 h）， and AO1 had the shortest stability time （133.17 h）.③The lactic acid content in the mixed silage of both AO1 and AO2 groups were significantly higher than that of AO3 group （P<0.05）.④The gas production of AO3 group was higher than the other treatment groups at all time points. The dry matter degradation rate （DMD） and crude protein degradation rate （CPD） of AO1 group were significantly higher than those of AO2 and AO3 groups （P<0.05）.As the proportion of fresh alfalfa decreases，the neutral detergent fiber degradation rate （NDFD） showed a decreasing trend among treatments （P＝0.07）， while no significant difference was found in acid detergent fiber degradation rate （ADFD） among groups （P>0.05）. The total volatile fatty acid （TVFA）， butyrate， and ammonia nitrogen （NH₃-N） concentrations in AO1 group were significantly higher than those in AO2 and AO3 groups. Conclusion When the mixing ratio of fresh alfalfa to oat hay was controlled at 8∶2 and 7∶3， the silage fermentation quality was optimal， and the in vitro digestibility was high. It could provide a reference for producing mixed silage of fresh alfalfa and oat hay in Northeast China.

Objective The study aimed to investigate the effects of astaxanthin （AST） on reproductive performance， antioxidant capacity of female rabbits and the growth of offspring under heat stress conditions， as well as to screen optimal dosage of AST in diet. Method A total of 240 New Zealand female rabbits with similar body weight and parity （2-3） were randomly divided into four groups： Control group （basal diet）， AST5 group （basal diet+5 mg/kg AST）， AST15 group （basal diet+15 mg/kg AST） and AST25 group （basal diet+25 mg/kg AST）. The test started from 6 d before female rabbits mating artificially to the 35 d postpartum （weaning of suckling rabbits）， which lasted for 72 d. During the experiment， the environmental temperature and relative humidity in the rabbit house were monitored and recorded to calculate the temperature-humidity index （THI）. Feed intake of female rabbits at different pregnant stages， the reproductive performance， serum antioxidant capacity and the concentrations of estradiol （E₂）， progesterone （P） and prolactin （PRL）， and growth performance of the offspring were determined. Result 63%（≥50%） of the experimental period was under heat stress conditions （THI≥27.8）， which achieved conditions of heat stress. Compared with control group， ①Female rabbits in AST25 group had higher average feed intake at all stages （P<0.05）. Conception rate and litter rate in AST25 group were significantly improved （P<0.05）. The feed intake and other reproductive performance of does showed no significant difference between groups. ②Serum concentration of malondialdehyde （MDA） in AST15 and AST25 groups were significantly decreased （P<0.05）， and the activity of superoxide dismutase （SOD） was significantly enhanced （P<0.05）. Furthermore， higher activity of serum glutathione peroxidase （GSH-Px） was observed in AST25 group （P<0.05）. The activity of catalase （CAT） in serum showed no significant difference among the groups （P>0.05）. ③Moreover， reduced serum estradiol level at 15 d of gestation （P<0.01） and enhanced serum prolactin level at 21 d postpartum （P<0.05） were observed in AST25 group. Serum progesterone levels at 15 d of gestation in the all experimental groups were significantly reduced （P<0.05）. ④Additionally， the average number of kits per litter at 7 and 14 d in AST5 group， at 14 d in AST15 group， at 14， 21 and 35 d in AST25 group were all increased （P<0.05）， and the litter weight in AST25 group was increased （P<0.05）. The other growth performance indicators of kits showed no significant difference among the groups （P>0.05）. Conclusion Under the heat stress conditions in this experiment， adding AST to the diet could improve the reproductive performance of female rabbits and the growth of their offspring by enhancing their antioxidant capacity. The appropriate amount of AST added was 25 mg/kg.

Objective This experiment aimed to investigate the effects of different levels of fat powder on the egg-laying performance， egg quality， serum biochemical indicators， and nutrient apparent metabolic rate of Hy-Line Brown laying hens， in order to provide a scientific basis for the application of fat powder in the feed ration of laying hens. Method A total of 270 Hy-Line Brown laying hens， aged 44 weeks， were selected and randomly divided into three groups， with six replicates per group and 15 hens in each replicate. The hens in control group were received a basic feed， while in the experimental groups were fed with 2% and 4% fat powder in the basic feed， respectively. The pre-feeding period was 1 week， and the experimental period was 8 weeks. The number of eggs， abnormal eggs and egg weight were recorded every day， and the feed consumption was recorded every week to calculate the average daily feed intake， egg production rate， average egg weight and feed-egg ratio. Eggs and serum were collected at 48 and 52 weeks of age to determine egg quality and serum biochemical indexes. Feces were collected at 52 weeks of age to determine the apparent metabolic rate of nutrients. Result Compared with control group， ①The mean daily feed intake of laying hens at 49-52 weeks of age was significantly higher in 2% fat powder group （P<0.05）. ② Eggshell thickness was significantly lower at 48 weeks old in 2% and 4% fat powder groups （P<0.05）， while egg specific gravity was significantly higher at 52 weeks old （P<0.05）. ③Alkaline phosphatase activity was significantly higher （P<0.05）， while the content of immunoglobulin A was significantly lower （P<0.05） in serum of 48 weeks old laying hens in 2% fat powder group. ④Glutamine aminotransferase activity and immunoglobulin G content were significantly increased （P<0.05）， while immunoglobulin A content was significantly decreased （P<0.05） in serum of 52 weeks old laying hens in 2% fat powder group. Conclusion Addition of 2% and 4% fat powder significantly improved feed intake and egg quality in Hy-Line Brown laying hens， but there might be a risk of inhibiting lipid metabolism.Based on the results of this experiment， the recommended amount of fat powder to be added was 2%.

Objective This study aimed to investigate the effects of dietary supplementation with Lycium ruthenicum Murr. extract of different concentrations on lipid profiles， adipose tissue morphology， and the expression of lipid metabolism-related genes and proteins in Bamei ternary hybrid pigs. Method Forty-eight Bamei ternary hybrid pigs aged （50±2） days with an average weight of 7.83 kg±0.52 kg were randomly divided into four groups， with three replicates per group and four pigs per replicate. The pigs were fed either a basal diet （CON group） or diets supplemented with 5， 10， and 15 g/kg of Lycium ruthenicum Murr. extract （LE， ME， and HE groups）， respectively. The experimental period was 42 days. At the end of the experiment， two pigs from each replicate were slaughtered， and blood， abdominal subcutaneous fat， and epididymal fat tissue were collected for the measurement of serum lipid levels， observation of fat tissue cell morphology， and mRNA and protein expression of lipid metabolism-related factors in fat tissues. Result Compared with CON group， ①Serum triglyceride （TG） levels of pigs in LE， ME， and HE groups were significantly reduced （P<0.05）， serum total cholesterol （TCH） levels were significantly reduced （P<0.05）， and serum high-density lipoprotein cholesterol （HDL-C） levels in LE group were significantly increased （P<0.05）. ②The area of lipid droplets in the subcutaneous adipose tissue of the abdomen of pigs in ME group was significantly reduced （P<0.05）， while there was no significant difference in the area of lipid droplets in the epididymal adipose tissue （P>0.05）. ③ In LE， ME， and HE groups， the mRNA and protein expression of lipid synthesis-related factors （PPARγ， ACC， and SREBP1） in abdominal subcutaneous fat and epididymal fat were significantly or extremly significantly reduced （P<0.05 or P<0.01）， while the mRNA and protein expression of lipid degradation-related factors （CPT1 and HSL） were significantly or extremly significantly up-regulated （P<0.05 or P<0.01）. Conclusion Adding Lycium ruthenicum Murr. extract to the diet could significantly reduce lipid deposition in the abdominal subcutaneous fat of Bamei ternary hybrid pigs by inhibiting the expression of lipid synthesis-related factors （PPARγ，ACC，and SREBP1） and promoting the expression of lipid degradation-related factors （CPT1 and HSL）， with the 10 g/kg addition showing the best effect.

Objective This study aimed to screen out Bacillus from sika deer that could efficiently degrade ZEN， and to provide a strain resource for ZEN detoxification in deer feed. Method Bacillus strains were isolated from faeces of sika deer by enrichment culture and streak plate separation techniques. The above strains were co-cultured with ZEN， and the degradation rate of ZEN was detected by high-performance liquid chromatography （HPLC）. The strains were identified by morphological， physiological， biochemical and molecular biological methods. The effects of time， temperature， initial pH of medium and toxin concentration on the degradation efficiency of ZEN were investigated. The safety of the strain was evaluated by intragastric test in mice. Result One ZEN highly efficient degrading bacterium RDY21 was isolated from the feces of sika deer. This bacterium could degrade 93.83% of ZEN （10 μg/mL） after 72 hours of incubation. Strain RDY21 could grow in an 8% sodium chloride environment and could utilize various sugars. The starch hydrolysis test， citric acid utilization test， propionate utilization test and V-P test were all positive. Whole genome sequencing analysis revealed that the average nucleotide identity between strain RDY21 and the type strain Bacillus licheniformis ATCC 14580 was 99.02%， and it was identified as Bacillus licheniformis. The optimal degradation temperature， time and initial pH of the culture medium for strain RDY21 to degrade ZEN were 37 ℃， 72 h and 7.0， respectively. It had a certain toxin tolerance under the environment of ZEN concentration ranging from 1 to 40 μg/mL．Compared with control group， there were no significant effects of Bacillus licheniformis RDY21 on average daily weight gain， average daily feed intake， organ coefficients， blood physiological and biochemical indices of mice after 28 days of administration （P>0.05）. Conclusion The Bacillus licheniformis RDY21 isolated in this study had the ability to degrade ZEN efficiently. It had no toxic side effects on mice and could be used as a candidate strain for microbial degradation of ZEN.

Objective This study aimed to establish an accurate method for determining the sterculic acid content in cottonseed meal and feed to facilitate the rational utilization of cottonseed meal protein resources. Method By optimizing the reaction conditions of 2-pyridinecarboxylic hydrazide with sterculic acid， the extraction process of sterculic acid and mass spectrometry parameters， a liquid chromatography tandem mass spectrometry （LC-MS/MS） method for quantifying sterculic acid using external standards was successfully established. The performance of the method was evaluated by examining its linear range， limit of detection， limit of quantification， recovery， precision （intra-assay and inter-assay） and accuracy， along with application to analyze actual samples. Result After optimization， the optimal reaction mixture composition for the reaction of sterculic acid with 2-pyridinecarboxylic hydrazide was determined as 500 mmol/L EDC， 15 mmol/L HOAt and 40 mmol/L 2-pyridinecarboxylic hydrazide. The optimal reaction temperature and time were established as 20 ℃ and 60 min， respectively. Methanol was found to be the best extracting solution for free sterculic acid， while the best hydrolysis conditions for bound sterculic acid were 50 ℃ for 12 h. The method demonstrated a good linear relationship for sterculic acid over the range of 1 to 1 000 ng/mL， with correlation coefficients （r） all greater than 0.99. The limit of detection and limit of quantification were were determined to be 5 and 12.5 μg/kg， respectively. For free sterculic acid， the recovery ranged from 80.13% to 95.30%， with intra coefficient of variation of 1.01% to 2.21% and inter coefficient of variation of 1.61% to 2.38%. For esterified sterculic acid， the recovery ranged from 85.46% to 105.20%， with intra coefficient of variation of 0.79% to 2.01% and inter coefficient of variation of 3.40% to 6.22%. The optimized method had been successfully applied to quantified sterculic acid in cottonseed oil， cure cottonseed meal， degossypoled cottonseed meal and cottonseed protein samples， with concentrations ranging from 0.09 to 1 812.76 mg/kg. Conclusion The established LC-MS/MS method had the advantages of high sensitivity， high accuracy and high precision. It was suitable for quantifying free and esterified sterculic acid in feed and provides technical support for the appropriate utilization of cotton meal.

Objective The aim of this study was to elucidate the dynamic changes and correlations between the plasma and fecal metabolomes before and after exercise in Turkmen horses， identify potential metabolic biomarkers and key pathways， and provide a scientific basis for evaluating exercise adaptation in Turkmen horses. Method Ten healthy Turkmen horses were selected for 1 h standardized exercise training， plasma and fecal samples were collected pre-exercise and post-exercise within 30 min. Untargeted liquid chromatography-mass spectrometry （LC-MS） metabolomics was employed for sample analysis. A partial least squares discriminant analysis （PLS-DA） model was established to screen differential metabolites. KEGG pathway enrichment analysis was performed， and Spearman correlation analysis was used to assess plasma-fecal metabolite associations. Result After exercise， plasma exhibited significant alterations in 106 metabolites （47 upregulated，59 downregulated），with lipid metabolites predominating. Upregulated plasma metabolites included fatty acid esters of hydroxy fatty acids （FAHFAs） （e.g.， FAHFA （18∶1/18∶2））， palmitic acid， and decanoylcarnitine. Downregulated metabolites were represented by 12-oxophytodienoic acid. KEGG pathway enrichment analysis revealed that the differential metabolites were significantly enriched in the fatty acid biosynthesis， arginine and proline metabolic pathways. 52 differential metabolites were identified in fecal analysis （8 upregulated， 44 downregulated）. Adrenic acid FAHFA （17∶0/18∶0） and kynurenic acid were significantly upregulated in feces， while carbohydrates， short-chain fatty acid precursors， and amino acid metabolites were decreased. Trans-petroselinic acid and 2-ketohexanoic acid were also significantly downregulated. Fecal differential metabolites were primarily enriched in the valine/leucine/isoleucine biosynthesis pathway. A significant positive correlation between plasma and fecal adrenic acid levels was identified by cross-tissue correlation analysis（r=0.92， P<0.05）. Furthermore， six FAHFAs， such as FAHFA （18∶1/18∶2）， accumulated significantly in plasma， while fecal FAHFA （17∶0/18∶0） level concurrently increased. Plasma oxidized lipids and flavin mononucleotide （FMN） increased synchronously， and the anti-inflammatory metabolite kynurenic acid was upregulated in feces. Conclusion Energy metabolism in Turkmen horses after exercise was characterized by fatty acid dominance and amino acid supplementation. Accumulation of oxidized lipids in plasma could induce oxidative stress. Concurrent elevation of adrenic acid in both plasma and feces， along with cross-tissue regulation of anti-inflammatory metabolites like FAHFAs and kynurenic acid， suggested a coordinated multi-tissue defense mechanism against exercise stress. Decreased fecal levels of energy metabolites such as trans-petroselinic acid and 2-ketohexanoic acid imply enhanced intestinal absorption supporting energy demands. This study had for the first time discovered the cross-tissue response characteristics of FAHFA metabolites， providing novel targets for the development of metabolic markers and anti-fatigue strategies.

Objective The aim of this study was to investigate the effects and regulatory mechanisms of ring finger protein 20 （RNF20） on the metabolism of branched-chain amino acids （BCAAs） in gonadal white adipose tissue （gWAT） in mice and 3T3-L1 adipocytes. Method Male adipocyte-specific Rnf20 gene knockout mice （Rnf20^flox/flox； adiponectin Cre⁺， ASKO） and littermate wild-type mice （Rnf20^flox/flox； adiponectin Cre^-， WT） were used in this study. Targeted metabolomics was used to detect the levels of BCAAs in gWAT and serum， and Real-time quantitative PCR was used to analyze the expression of BCAAs catabolic genes （Bcat2， Bckdha， etc.） in gWAT， liver， and gastrocnemius muscle from both groups of mice. To examine the effect of Rnf20 gene on BCAAs metabolism in vitro， siRNAs were designed to down-regulate the expression of Rnf20 gene in 3T3-L1 preadipocytes and differentiated into mature adipocytes. The adipogenic differentiation efficiency of interference group （siRNF20） and control group （siNC） was evaluated through oil red O staining， and the expression of adipogenic differentiation marker genes， lipolysis genes， and BCAAs catabolism genes were detected. The contents of BCAAs in adipocyte culture medium of siRNF20 and siNC groups were detected. Result Compared with WT mice， the contents of BCAAs （leucine， isoleucine and valine） in gWAT of ASKO mice were extremely significantly increased （P<0.01）， and the expression of BCAAs catabolism related genes Bcat2， Acad5， Ehhand and Hibch were significantly or extremely significantly decreased （P<0.05 or P<0.01）. However， there was no significant difference in the content of BCAAs in serum and the expression of BCAAs catabolism related genes in liver and gastrocnemius muscle （P>0.05）. Compared with siNC group， knockdown of Rnf20 gene in vitro could inhibit adipogenic differentiation of 3T3-L1 cells，with a significant decrease in the expression of the adipogenic marker gene Pparγ （P<0.05）， and an extremely significant increase in the expression of the lipolytic gene Adrb3 （P<0.01）. The expression of key genes Bcat2 and Bckdha involved in BCAAs catabolism in mature adipocytes decreased with the knockdown of Rnf20 gene， and the BCAAs content in siRNF20 group culture medium was extremely significantly increased （P<0.01）. Conclusion Rnf20 gene in adipocytes regulated the key genes Bcat2 and Bckdha involved in BCAAs catabolism through transcriptional regulation， maintaining the homeostasis of BCAAs within the cell. Knockdown of Rnf20 gene could inhibite BCAAs catabolism， leading to the accumulation of BCAAs in adipose tissue. The results provided a new direction for in-depth exploration of the function of Rnf20 gene and new genetic materials for genetic breeding research on fat deposition traits in large animals.

Objective The trial aimed to investigate the changes in blood gas indices and plasma differential metabolites in tumbler pigeons before and after exercise， analyze their blood acid-base balance and metabolic differences， and provide data support for exploring the exercise and fatigue recovery mechanisms of this breed. Method Twelve healthy 180-day-old tumbler pigeons （6 males and 6 females） were selected. Blood samples were collected in a resting state before exercise and after exhaustive exercise for blood gas analysis and non-targeted metabolomics analysis. Differential metabolites before and after exercise in the pigeons were screened， and metabolic pathway analysis was conducted. Result Blood gas analysis revealed that， compared to pre-exercise levels， the partial pressure of carbon dioxide in blood （PvCO₂） and total carbon dioxide （TCO₂） levels were significantly decreased after exercise （P < 0.05）， while the hematocrit （Hct） and hemoglobin （Hb） concentration were extremely significantly decreased （P<0.01）. No significant differences were observed in bicarbonate （HCO₃^―） and sodium ion （Na⁺） levels （P>0.05）.Plasma metabolomics analysis identified differential metabolites in both positive and negative ion modes following exercise compared to pre-exercise levels. The levels of the metabolites allopurinol riboside， sphingomyelin， trans-aconitic acid， lysophosphatidic acid， loganetin and succinyladenosine were significantly higher than before exercise （P<0.05）. Conversely， the levels of the metabolites glycyl-leucine， aminohexanoic acid， decursinol， heptadecanoic acid， and palmitoleic acid were significantly lower than before exercise （P<0.05）. Differential metabolites were significantly enriched in pathways including phenylalanine metabolism， biosynthesis of unsaturated fatty acid， linoleic acid metabolism， and fatty acid biosynthesis. Conclusion Under the conditions of this experiment， compared to pre-exercise levels，the concentrations of PvCO₂ and TCO₂ in the blood of tumbler pigeons were significantly decreased after exercise， the concentrations of Hct and Hb were extremely significantly decreased. Moreover， exercise mainly affected the glucose metabolism， lipid metabolism， and amino acid metabolism of tumbler pigeons. The experimental results provided a theoretical basis for the post-exercise recovery and breeding selection of tumbler pigeons.

Objective This study aimed to investigate the effects of adding different concentrations of rutin to the diluent on the cryopreservation of bull semen. Method Semen was collected from three Simmental bulls and three Wagyu bulls using an artificial vagina. Samples with motility >75% were pooled and divided into five groups： A control group without rutin and four treatment groups supplemented with 0.4， 0.8， 1.2 and 1.6 g/L rutin in the diluent. Post-thaw sperm motility and kinematic parameters （including average path velocity， curvilinear velocity， straight-line velocity， wobble， linearity and straightness） were evaluated using a computer-assisted sperm analysis （CASA） system. Sperm malformation rate was assessed by eosin staining. Plasma membrane integrity was determined using the hypo-osmotic swelling test （HOST）. Acrosome integrity was examined by peanut agglutinin staining. Reactive oxygen species （ROS） level was measured by fluorescence assay. Antioxidant enzyme activities were also determine using the reagent kit. Result 0.8 g/L rutin group showed significantly higher post-thaw sperm motility， curvilinear velocity， straight-line velocity， wobble， linearity， plasma membrane integrity and acrosome integrity compared with control and other treatment groups （P<0.05）， and higher average path velocity and straightness compared with control group （P<0.05）. 0.8 g/L rutin group exhibited significantly lower sperm abnormality， ROS levels and malondialdehyde （MDA） content compared with control group （P<0.05）. The activities of catalase （CAT） and glutathione peroxidase （GSH-Px） in 0.8 and 1.2 g/L rutin groups were significantly higher than in control group （P<0.05）. Total antioxidant capacity （T-AOC） was significantly increased in 0.8 g/L rutin group compared with all other groups （P<0.05）， and 1.2 and 1.6 g/L rutin groups also showed significantly higher T-AOC than in control group （P<0.05）. Conclusion The addition of 0.8 g/L rutin to the diluent provided the best cryoprotective effects on bull semen， significantly improving post-thaw sperm motility， kinematic performance， plasma membrane and acrosome integrity and antioxidant enzyme activities （T-AOC， GSH-Px and CAT）， while significantly reducing sperm abnormality， ROS level and MDA content.

Objective This study aimed to establish a fibroblast growth factor receptor 1 （FGFR1） gene knockout bovine mammary epithelial cell line （bMECs） using CRISPR/Cas9 technology， and investigate the effects of FGFR1 gene deletion on cell viability， proliferation， and lactation function， thereby elucidating its molecular regulatory mechanisms in mammary gland development and lactation. Method sgRNAs were designed targeting exons 3 and 4 of FGFR1 gene. Following in vitro assessment of cleavage efficiency， a knockout plasmid for FGFR1 gene was generated. Genomic DNA was collected 7 d post-screening， and PCR amplification along with Sanger sequencing were employed to confirm FGFR1 gene editing. Monoclonal cells were isolated from the edited cell population using the limiting dilution method. FGFR1 gene knockout cell lines with a singular genotype were subsequently identified through TA cloning. The knockout efficiency of monoclonal cell lines was assessed via Western blotting， while cell viability was determined using CCK-8 assay. Additionally， Real-time quantitative PCR was utilized to evaluate the expression of genes associated with proliferation and lactation in the monoclonal cell lines. Result The in vitro cleavage assay demonstrated cleavage efficiencies of 82.87% and 27.80% for the two sgRNAs， confirming successful construction of the knockout plasmid. Subsequently， over 95% of bMECs displayed red fluorescence 72 h post-electrotransfection， with multiple double peaks observed in the target region following drug screening， indicating a pronounced editing effect. From a pool of 384 monoclonal cell lines， 92 were successfully propagated， with 72 （78.26%） exhibiting FGFR1 gene editing， 31 （33.70%） showing sgRNA1 site editing， and 55 （59.78%） displaying sgRNA2 site editing. Ultimately， 8 robust monoclonal cell lines were isolated. Sequencing analysis revealed diverse editing patterns among the monoclonal cell lines， including fragment deletions and base substitutions. Notably， the KO#2 cell line exhibited editing at two sites with a single editing type. Western blotting and CCK-8 results showed that compared with control group， the knockout effect of KO#2 strain cells was extremely significant （P<0.01）， while FGFR1 gene knockout did not significantly impact cell viability （P>0.05）. Real-time quantitative PCR analysis results revealed that compared with control group， there was no significant differences in the expression of genes associated with cell proliferation （P>0.05）. However， the significant reduction in the expression of genes related to lactation function was observed（P<0.05 or P<0.01）. Conclusion This study successfully constructed one strain of bMECs with FGFR1 gene knockout， indicate that the absence of FGFR1 gene did not notably impact cell survival and proliferation， but significantly inhibited lactation synthesis function. The results provided a basis for future investigations into the role and mechanisms of FGFR1 gene in bMECs.

Objective In vitro embryo production and embryo transfer （IVP-ET） is one of the most important reproduction technology in modern dairy industry. This study aimed to analyze the influencing factors of pregnancy rate of in vitro embryo transfer in Holstein cattle， examine the phenotype characteristic of the pregnancy rate of in vitro embryo transfer using embryos transfer records from large-scale dairy operation， providing actionable information to optimize the technical system for somatic cell nuclear transfer. Method A total of 5 155 in vitro embryo transfer records were collected along with the corresponding pregnancy check results from 4 534 Holstein cattle in 11 large-scale dairy farms. The logistic regression model was used to analyze the effects of non-genetic factors （such as embryo-related factors） on embryo transfer successful rate. Result The overall pregnancy rate of in vitro produced embryo transfer in Holstein heifers was 51.29%. Several factors had an extremely significant effect on the pregnancy rate， including embryo-related factors （preservation status， developmental stage， in vitro culture duration） and the recipient’s transfer number. Specifically， the pregnancy rate of fresh embryos was extremely significantly higher than that of frozen embryos （P<0.01）. At different developmental stages， expanded blastocysts resulted in an extremely significantly higher pregnancy rate compare with blastocysts （P<0.01）. Among embryos cultured in vitro for different days， the transfer pregnancy rate of embryos cultured until the afternoon of the 6th day， morning and afternoon of the 7th day was extremely significantly higher than that of embryos cultured until the afternoon of the 8th day （P<0.01）. Furthermore， first-time recipients achieved an extremely significantly higher pregnancy rate compare with undergoing subsequent transfers （P<0.01）. Recipient age and the sire of the embryo had a significant effecton the pregnancy rate， heifers aged >14.5 to ≤15 months achieved the highest pregnancy rate （52.59%）， which was extremely significantly higher than that of heifers aged <14 months （P<0.01）. The sire of the embryo also significantly influenced the pregnancy rate （P<0.05）， indicating that the intrinsic quality of the embryo， as determined by the sire， was a critical upstream factor for transfer success， which underscored the strong interrelationship between the processes of embryo production and the final outcomes of embryo transfer. Conclusion Utilizing extensive embryo transfer data， this study demonstrated that embryo preservation status， developmental stage， in vitro culture duration， and recipient transfer number were critical determinants of pregnancy rate. The results underscored that enhancing embryo quality and rigorous recipient selection were key to increasing the efficiency of IVP-ET in Holstein cattle.

Objective The aim of this study was to amplify the sequence of scavenger receptor class B member 2 （SCARB2） gene in Lijiang pigs and conduct bioinformatics analysis，and detect its expression in various tissues of Lijiang pigs， providing a theoretical basis for exploring the function of this gene in the future. Method Adipose tissues from 6-month-old Lijiang pigs were collected， the mRNA sequence of SCARB2 gene from wild boar （Sus scrofa） in GenBank （accession No.： NM_001244155.1） was be used as a reference sequence to design primers， the CDS region sequence of SCARB2 gene was amplified and sequenced， and bioinformatics analysis software was used to analyze the sequence characteristics and codon preferences of SCARB2 gene， the similarity of SCARB2 gene sequence between multiple species and Lijiang pigs， as well as between domestic and foreign pig breeds and Lijiang pigs， was analyzed，and the phylogenetic tree was constructed. The physicochemical properties and protein structure of SCARB2 protein were predicted. Real-time quantitative PCR was used to detect the expression of SCARB2 gene in the back fat， heart， liver， lung， kidney， and other tissues of 2， 4， and 6-month-old Lijiang pigs and 6-month-old Duroc pigs. Result The total length of the CDS region sequence of SCARB2 gene in Lijiang pigs was 1 437 bp， encoding 478 amino acids. The homology between Lijiang pigs and Capra hircus， Ovis aries， Bos taurus， Equus caballus， Rattus norvegicus， Mus musculus， Homo sapiens and Gallus gallus was 91.3%， 91.2%， 91.0%， 89.8%， 82.6%， 82.5%， 76.0%， and 66.0%， respectively. Among 6 local pig breeds， except for Bama Miniature pigs， the similarity between Lijiang pigs and local pigs in China was 99.4%， and the similarity with foreign pigs ranged from 99.1% to 99.3%. Phylogenetic tree analysis showed that Lijiang pigs were grouped with Sus scrofa， Bos taurus， Capra hircus and Ovis aries， while Gallus gallus form a separate branche. SCARB2 protein was a hydrophilic protein with 2 transmembrane helices， 1 signal peptide cleavage site， 10 N-glycosylation sites， and 39 phosphorylation sites. The secondary and tertiary structures were mainly characterized by random coil. The expression of SCARB2 gene in back fat and liver of 6-month-old Lijiang pigs was extremely significantly higher than that in other tissues （P<0.01）. With the age increasing， the expression of SCARB2 gene in abdominal fat， back fat and shoulder fat of Lijiang pigs showed a significant increase， reaching significant or extremely significant levels between 2 and 6 months of age （P<0.05 or P<0.01）. The expression in back fat of 6-month-old Lijiang pigs was extremely significantly higher than that of Duroc pigs （P<0.01）. Conclusion This study successfully amplified the SCARB2 gene sequence of Lijiang pigs and analyzed its molecular characteristics. SCARB2 gene was expressed in multiple tissues such as subcutaneous fat， liver， and lung， and its expression significantly increased with age in subcutaneous fat tissue. Therefore， SCARB2 could be used as a candidate gene for studying fat deposition traits in Lijiang pigs， and the results could further provide reference for studying the molecular mechanism of pig fat deposition.

Objective This experiment aimed to construct an overexpression vector of heme oxygenase 1 （HMOX1） gene in Wuliangshan Black-boned chickens and transfect it into chicken primary preadipocytes， and investigate the effect of HMOX1 gene overexpression on the proliferation of chicken primary preadipocyte. Method Collect the subcutaneous fat tissue from 1-day-old chicks of Wuliangshan Black-boned chicken， extract total RNA and reverse transcribe it into cDNA， and amplify the CDS region of HMOX1 gene. The overexpression vector pcDNA3.1-HMOX1-mRFP was constructed using the plasmid pcDNA3.1-mRFP， followed by restriction enzyme digestion and sequencing for identification. The pcDNA3.1-HMOX1 overexpression vector was transfected into chicken primary preadipocytes by using transfection reagents， and the overexpression efficiency was detected. Cell viability was measured using CCK-8 method， and the cell cycle was analyzed by flow cytometry. Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of proliferation-related genes （P21， CDK1 and PCNA）. Result Restriction enzyme digestion and sequencing indicated that the pcDNA3.1-HMOX1-mRFP overexpression vector was successfully constructed. Real-time quantitative PCR and Western blotting test results showed that， compared with control group， the mRNA and protein expression of HMOX1 gene in cells transfected with pcDNA3.1-HMOX1-mRFP were extremely significantly upregulated （P<0.01）， demonstrated that HMOX1 was successfully overexpressed in chicken primary preadipocytes. CCK-8 assay results showed that compared with control group， the cell viability of HMOX1 overexpression group significantly increased at 12 hours after transfection （P<0.05）， and decreased extremely significantly at 36 and 48 hours after transfection （P<0.01）. The results of flow cytometry test showed that，compared with control group， the number of cells in the S phase in HMOX1 overexpression group increased extremely significantly at 12 hours （P<0.01）， while the number of cells in the G1 phase decreased extremely significantly （P<0.01）. The number of cells in the S phase at 48 hours was significantly reduced （P<0.05）. Detection results of proliferation-related genes showed that there was no significant difference in the mRNA and protein expression of P21 and CDK1 genes in the cells between the two groups （P>0.05）. The mRNA and protein expression of PCNA gene in overexpression group were significantly lower than those in control group （P<0.05）. Conclusion The HMOX1 gene overexpression vector was successfully constructed. After transfection of the original preadipocytes of Wuliangshan Black-boned chicken， cell proliferation was inhibited. The experimental results could provide theoretical guidance for further study of the effect of HMXO1 on the differentiation of primary preadipocytes of Wuliangshan Black-boned chicken.

Objective The ORF2 protein of Goose astrovirus （GAstV） was expressed using a prokaryotic expression system， and a rabbit polyclonal antibody against this protein was prepared，to provide a material basis for the study of the pathogenic mechanism and diagnostic methods of GAstV. Method The recombinant prokaryotic expression plasmid pGEX-6P-1-ORF2 was constructed. After the recombinant plasmid was identified by PCR and double enzyme digestion， it was transformed into Escherichia coli BL21（DE3） competent cells. The protein expression was induced by IPTG and the reaction conditions were optimized to obtain the GAstV ORF2 protein. Polyclonal antibody against GAstV ORF2 were generated in New Zealand White rabbits with the purified recombinant protein GAstV ORF2. The antibody titer was determined by indirect ELISA. The immunogenicity and specificity of the polyclonal antibody were assessed using Western blotting and indirect immunofluorescence assay （IFA）. In order to clarify the tropism and localization of GAstV in the tissues of goslings， using the prepared polyclonal antibody， the distribution of the virus in the liver and kidney tissues of infected goslings was detected by immunohistochemistry （IHC） method. Result PCR and double enzyme digestion assays indicated that the recombinant prokaryotic expression plasmid pGEX-6P-1-ORF2 was successfully constructed. The GAstV ORF2 protein was successfully obtained through prokaryotic expression， and the protein concentration after purification was 1.38 mg/mL. The immunization of New Zealand White rabbits successfully generated polyclonal antibodies against GAstV ORF2 protein， with a high antibody titer reaching 1∶100 000. Western blotting analysis demonstrated specific reactivity between the polyclonal antibodies and purified GAstV ORF2 protein， as evidenced by distinct immunoreactive bands. IFA results confirmed the antibody’s specific recognition of both native GAstV and eukaryotic-expressed GAstV ORF2 protein. IHC test results showed that GAst was widely distributed in the liver and kidney of infected goslings. GAstV was specifically expressed in the cytoplasm of parenchymal cells and inflammatory cells. Conclusion This study successfully constructed the recombinant prokaryotic expression plasmid pGEX-6P-1-ORF2， and carried out in vitro induction expression to prepare rabbit polyclonal antibody against GAstV ORF2 protein. This polyclonal antibody had a high titer and strong specificity， and could be used for the detection of viral antigens in GAstV-infected goslings.

Objective The purpose of this experiment was to isolate and identify the Porcine epidemic diarrhea virus （PEDV） strains in Heilongjiang province. Phylogenetic analysis based on the S and ORF3 gene sequences of PEDV isolates， genetic evolution analysis was conducted to enrich the existing PEDV materials， providing a reference basis for the prevention and control of porcine epidemic diarrhea （PED） and the development of vaccines. Method Collect samples from suspected swine epidemic diarrhea cases in the pig farm， and use Vero-E6 cells for adaptive cultivation to obtain the virus， and isolate and identify the virus strains through RT-PCR and indirect immunofluorescence assay （IFA）. S1， S2 and ORF3 genes of PEDV isolates were respectively amplified by PCR， and recombinant plasmids were constructed and sequenced for identification. The sequences of S and ORF3 genes of PEDV isolates were obtained. The nucleotide sequence similarities of S and ORF3 genes of the isolates were compared with those of 26 reference strains， and genetic evolutionary analysis was conducted to determine the subgroup of the isolated strains. Result PEDV isolates were cultured on Vero-E6 cells， and exhibited typical cytopathic effects， deformation and rounding， decreased refractivity， cell fusion， and followed by cell detachment and plaque formation. RT-PCR and IFA tests showed that specific bands of approximately 600 bp in size were amplified. After the isolated strains infected cells， they all reacted specifically with the PEDV N protein monoclonal antibody， indicating that the isolated strains were all PEDV， and were respectively named PEDV HLJ， HLJ2020 and HLJ2021 strains. Recombinant plasmids of S1， S2， and ORF3 genes of PEDV isolates were successfully constructed and sequenced to obtain the corresponding gene sequences. The results of nucleotide sequence similarity alignment and genetic evolutionary analysis indicated that the PEDV HLJ and HLJ2020 strains belonged to GⅠb subgroup， while PEDV HLJ2021 strain was classified into GⅡc subgroup. Conclusion In this study， 3 prevalent PEDV strains were successfully isolated from the small intestinal tissues of diarrhea pigs in Heilongjiang region. Among them， the isolated strains PEDV HLJ and PEDV HLJ2020 belonged to GⅠb subgroup， while the PEDV HLJ2021 strain belonged to GⅡc subgroup.

Objective Since August 2021， breeding duck flocks in Anhui， Shandong and other regions had experienced unexplained decreases in egg production， accompanied by symptoms such as depression and ovarian hemorrhage. This study aimed to clarify the pathogenic characteristics of the causative agent and provide a basis for the prevention and control of decreased egg production in breeding ducks. Method Virus isolation was performed using duck embryo fibroblasts （DEF） on ovarian tissue samples from diseased ducks collected in Yishui， Shandong， in September 2022. Next-generation sequencing technology was used to sequence the viral genome， which was compared with the reference virus genome database to determine the pathogen species. The viral genome structure was analyzed， and the encoded proteins were clarified. Specific primers were designed based on the sequenced viral sequences for pathogen identification. The viral genes were compared with reference sequences for similarity and phylogenetic analysis to explore the genetic characteristics and evolutionary relationships of the virus. Regression experiments were carried out using breeding ducks at the peak of egg production to verify the pathogenicity of the virus. Result After inoculating the diseased tissue samples into DEF cell， the cells showed typical cytopathic effects. Whole-virome comparison showed that there were virus sequences closely related to Orbiviridae， and after sequence assembly， it was designated as Duck orbivirus （DORV）. The genome consisted of 10 double-stranded RNA segments， encoding 12 proteins. PCR results confirmed that the pathogen in this study was DORV， and the isolate strain was named DORV-SDYS. The DORV-SDYS strain was closely related to Parry Lagoon virus （PLV）， Corriparta virus （CORV） and Acado virus （ACAV）. The nucleotide sequence similarity of the VP5 fragment was 80.1%-82.4%， and the amino acid sequence similarity was 88.1%-91.1%. Phylogenetic analysis showed that DORV-SDYS had distinct evolutionary characteristics. Animal regression experiments confirmed that DORV-SDYS could lead to a decrease in the egg production rate of breeding ducks， and caused pathological damage to ovarian follicles. Conclusion In this study， a virus was successfully isolated from the ovarian tissue of laying ducks with decreased egg production in Yishui， Shandong province， and it was designated as DORV. The characteristics of the full coding region of its genome were clarified. It was confirmed that DORV infection was the etiological factor causing decreased egg production in breeding ducks， filled the gap in the research on the pathogenicity of Orbivirus in waterfowl hosts， and was of great significance to the healthy development of the waterfowl industry.

Objective Bovine respiratory disease complex （BRDC） is a multi-pathogen disease that can lead to high morbidity and mortality rates in cattle herds， causing severe economic losses to the cattle industry. To evaluate the viral pathogen infection in BRDC-affected cattle in Xinjiang， this study conducted viral pathogen detection on BRDC cases from six regions of Xinjiang. Method From January 2023 to December 2023， 561 nasal swabs were collected from cattle with respiratory symptoms in six regions of Xinjiang， including Yanqi， Kashgar， Changji， Bole， Ili and Aksu. PCR technology was used to detect seven common viruses causing BRDC， which were Infectious bovine rhinotracheitis virus （IBRV）， Bovine adenovirus type 3 （BAV-3）， Bovine coronavirus （BCoV）， Bovine rhinitis A virus （BRAV）， Bovine rhinitis B virus （BRBV）， Bovine viral diarrhea virus （BVDV）， and Bovine parainfluenza virus type 3 （BPIV-3）. The infection status of these seven pathogens in cattle of different seasons， in different regions and in different breeds was analyzed. Result The results demonstrated that all seven pathogens were detected. The detection rate was highest for IBRV at 14.4%， followed by BVDV at 13.0%， with the lowest detection rate observed for BAV-3 at 7.0%. Mixed infections were predominantly double infections （13.4%）， with the main pathogens including BAV-3+BCoV （1.8%）， IBRV+BPIV-3 （1.6%）， and BRBV+BVDV （1.6%）. Among triple infections （3.7%）， the highest detection rate was observed for IBRV+BPIV-3+BVDV （0.5%）. Quadruple infections （0.6%） and quintuple infections （0.6%） were also detected， such as IBRV+BRAV+BRBV+BPIV-3 （0.4%） and IBRV+BAV-3+BCoV+BRAV+BPIV-3 （0.2%）. There were significant differences in virus positivity rates across seasons， with the highest detection rate of BAV-3 in spring （11.0%）， BVDV in summer （34.0%）， IBRV in autumn （61.3%）， and BPIV-3 in winter （38.7%）. Geographically， the highest detection rate of BRBV was in Yanqi （50.0%）， BAV-3 in Kashgar （15.3%）， IBRV in Changji and Bole （28.8% and 22.6%， respectively）， BVDV in Ili （55.7%）， and an equal proportion of BRBV and BPIV-3 in Aksu （35.5% each）. Additionally， when categorizing the samples by cattle use into beef and dairy groups， dairy cattle were primarily infected with IBRV （29.5%）， while beef cattle were mainly infected with BVDV （12.9%）. Conclusion The investigation results showed that there were obvious seasonal， regional and breed differences in viral pathogen infection of BRDC in some areas of Xinjiang in 2023. This study would be helpful for formulating prevention and control strategies for BRDC in Xinjiang.

Objective This study was conducted to investigate the effects and potential mechanisms of Fusobacterium necrophorum subsp. necrophorum （Fnn） infection on mitochondrial dynamics and mitophagy of bovine dermal fibroblasts（BDFs）. Method This study isolated and cultured BDFs from the skin between cow toes and co-cultured them with 1×10⁸ CFU/mL Fnnfor 12 h to establish an infection model group （Fnn）. Uninfected cells were used as control group （CON）， the morphological changes of BDFs afterFnninfection were observed. The cell activity of BDFs was detected by CCK-8 kit. Real-time quantitative PCR was used to detect the changes in the expression of genes related to mitochondrial dynamics， biosynthesis and mitophagy， and immunofluorescence assay was used to detect the changes in the expression of related proteins. Result Compared with CON group， cells in Fnn group showed swelling， bulging， and deformation， and Fnn infection significantly inhibited the activity of BDFs. Real-time quantitative PCR results showed that compared with CON group， after Fnn infection， the relative expression levels of peroxisome proliferator receptor gamma coactivator alpha （PGC-1α）， nuclear respiratory factor-1 （Nrf1）， mitochondrial transcription factor A （Tfam）， mitochondrial fusion factor gene-2 （Mfn-2）， phosphatase and tensin homolog induced protein kinase 1 （PINK1）， E3 ubiquitin ligase （Parkin） genes in BDFs were significantly or extremely significantly decreased （P<0.05 or P<0.01）， while the relative expression levels of optic nerve atrophy protein 1 （Opa1）， Mfn-1， and Sequetosome 1 （P62） genes showed a decreasing trend （P>0.05）. The relative expression of genes Fis1， light chain-3α （LC3α）and LC3β were extremely significantly increased （P<0.01）. The results of immunofluorescence assay detection showed that compared with CON group， after Fnn infection， the fluorescence signals of dynein related protein 1 （Drp1）， PINK1， Parkin and P62 in BDFs were enhanced， the fluorescence signals of Opa1， Mfn-1 and LC3 were weakened. Conclusion Fnn infection could disrupt the mitochondrial biosynthesis of BDFs， disrupt mitochondrial fusion and fission processes， and induce mitophagy. This results provided a new theoretical basis for a deeper understanding of the pathogenesis of hoof rot disease.

Objective This study aimed to investigate whether the intestinal mucosa of laying hens developed enhanced resistance to secondary injury after initial injury and to explore the underlying mechanisms， thereby promoting new perspectives for developing approaches to repair intestinal mucosal damage in laying hens. Method Seventy-eight 7-day-old Hyline White chicks were divided into 13 treatment groups （6 chicks per group）. Six chicks were radomly selected and received an intraperitoneal injection of PBS as the first negative control group （1^st NC）. The remaining 72 chicks were subjected to intestinal mucosal injury via intraperitoneal injection of lipopolysaccharide （LPS， 10 mg/kg BW）. Six chicks were radomly selected， euthanized and sampled at 1， 2， 4， 6， 8 and 24 hours post-injection （hpi）， designated as 1^st Xhpi groups （X represents the time of sampling after injection）. At 48 hours post-injection， six chicks from the LPS-injected group were radomly selected and injected with PBS as the second negative control group （2^nd NC， equivalent to the 1^st 48hpi group）， the remaining chicks received a second injection of the same dose of LPS. Six chicks were radomly selected， euthanized and sampled at 1， 2， 4， 6 and 8 hours post the second LPS challenge， designated as 2^nd Xhpi groups. Blood， duodenal tissues were collected， and intestinal crypts were isolated for analysis of barrier function， intestinal stem cells （ISCs） activity， and crypt niche changes. Result Following the first LPS challenge， inflammatory cell infiltration， decreased villus height/crypt depth， increased permeability， and decreased mucus layer thickness were observed in the intestinal mucosa of the 1^st 4hpi group. At 48 hours post injection， the degree of injury in the 2^nd NC group was reduced， and the barrier function was enhanced， which was manifested as the expression level of the tight junction Ocln gene， the density of goblet cells， the thickness of the mucus layer， and the expression level of the mucin Muc2 gene were all higher than those in the 1^st NC group. After the second LPS challenge， compared with 2^nd NC group，in the 2^nd 4hpi group， inflammatory cell infiltration and permeability did not significantly increase， but Claudin-1 and complement protein C5 gene levels were markedly upregulated （P<0.05）. The ISCs analysis results showed that the activity of active intestinal stem cells （aISCs） in the 1^st 4hpi group decreased and then gradually recovered， returning to normal at 48 hours post injury.The reserve ISCs （rISCs） in the 2^nd 4hpi group could be rapidly activated and maintain high activity. The analysis results of intestinal crypts showed that the Notch signal pathway of intestinal crypts was inhibited in both injury processes. Conclusion Following initial injury and repair， the intestinal mucosa exhibited enhanced barrier function and suppressed Notch signaling pathway in crypts. Secondary injury triggered rapid activation of rISCs and promoted differentiation of ISCs into secretory epithelial cells.

Objective This study aimed to explore the effect of icaB gene on biofilm-formation ability and antimicrobial resistance of methicillin-resistant Staphylococcus aureus （MRSA）. Method MRSA N315 standard strain was selected to construct icaB gene knockout strain N315ΔicaB by homologous recombination technology， and the differences between strain N315ΔicaB and wild strain N315 WT in terms of growth tendency， colony morphology， cell wall thickness， biofilm-formation ability and antimicrobial resistance were comparatively analysed. Result There was no difference in the growth trend， colony shape and color in N315ΔicaB compared to strain N315 WT. The results of cell wall thickness measurement showed that there was no significant difference in cell wall thickness in strain N315ΔicaB compared to strain N315 WT （P>0.05）. The results of biofilm-formation ability showed that there was no significant difference in biofilm-formation ability between strains N315ΔicaB and N315 WT （P>0.05）. The antimicrobial resistance results showed that the minimum inhibitory concentrations of penicillin， oxacillin， gentamicin， ciprofloxacin and nitrofurantoin against strain N315ΔicaB were lower than those in strain N315 WT. Conclusion The knockout of icaB gene did not affect the growth status and biofilm-formation ability of MRSA. However， it increased its susceptibility to several antimicrobials. This study successfully constructed icaB gene knockout strain N315ΔicaB， which provided an ideal cell model to explore the new functions of icaB gene， and laid a foundation for in-depth study of biofilm regulatory networks at the genetic level. Meanwhile， it also provided a scientific basis for enhancing the efficacy of antibacterial drugs against MRSA by targeting the icaB gene.

Objective To confirm an acute death case of Pavo cristatus at Anyang zoo， this study carried out clinical diagnosis and laboratory tests. Method The clinical symptoms and pathological changes of dead peacocks were observed. The pathogenic bacteria were isolated and identified by methods such as identification medium， Gram staining， biochemical identification， 16S rRNA gene PCR amplification， phylogenetic tree construction and animal experiments. The biofilm-forming ability of the isolated bacteria was detected by crystal violet staining method， and the drug sensitivity test was conducted by K-B disk method. Drug resistance genes were detected by PCR. Result There was no obvious symptoms on the body surface of dead peacocks. The results of the peafowl necropsy showed that there was an increase and turbidity in pericardial effusion， and the liver was enlarged. There was urate deposition in the kidneys， and bleeding in the intestines. The liver tissues of dead peacocks were aseptically collected and streaked and inoculated on MacConkey medium， and red colonies were isolated. Microscopic examination revealed that the pathogenic bacteria were pink short rods， scattered singly or in pairs， and suspected to be bacteria of the Enterobacteriaceae family. The results of biochemical identification indicated positive reactions for indole， MR， pentose， sorbitol， and so on， and negative reactions for Simonsons citrate， V-P， urea， hydrogen sulfide， and so on. 16S rRNA gene PCR amplified fragment was sequenced to obtain a sequence of about 1 500 bp in length （GenBase accession No.： C_AA110994.1）. The phylogenetic tree indicated that the isolated strain had the highest similarity （100%） to Escherichia coli （E. coli）， confirming that the isolated bacterium was E. coli. The isolated bacteria had stronger biofilm-forming ability than standard strain and could cause liver and spleen enlargement， lung congestion， intestinal wall thinning， intestinal bleeding， and villus shedding in BALB/c mice， eventually leading to death. The isolated bacteria were sensitive to levofloxacin， norfloxacin and so on， but resistant to tetracycline， streptomycin and other drugs， and carried six resistance genes tetA， ampC， bla_TEM， Sul1， Sul2 and strA-strB. Conclusion In this study， the pathogenic E. coli srtain was isolated and identified form Pavo cristatus. The results of drug sensitivity test showed that this E. coli strain was sensitive to levofloxacin， norfloxacin， and so on， but resistant to tetracycline， streptomycin， and so on. This study provided a valuable reference for the diagnosis and clinical medication of E. coli-related diseases in Pavo cristatus.

Objective This study aimed to explore the etiology of fungal dermatitis in Sichuan cattle and to provide reference for prevention and control of the disease. Method Skin scabs from animals suspected of having dermatophytosis were collected from various locations in Sichuan province， and the collected samples were inoculated into the dermatophyte identification medium by three-point inoculation method. The isolated strains were amplified by internal transcribed spacer （ITS） sequence， and the genetic similarity was analyzed by NCBI BLAST and the phylogenetic tree was constructed. Immunosuppressed mice were infected with 10⁷ CFU/mL spore suspension of the isolates to observe the clinical symptoms and pathological features， and the drug resistance phenotype of the isolates was explored by micro-double dilution method. Result Ten fungal strains （rntv01 to rntv10） were isolated from 24 samples. PCR amplification results showed that all the isolates obtained amplification fragments of 681 bp in size. The BLAST comparison results showed that the similarities between the isolates and Trichophyton verrucosum were 97% to 100%. The results of animal experiments showed that after immunosuppressed mice were infected with the isolates， their skin presented symptoms such as hair loss， scabbing， and increased dandruff. After HE staining， incomplete keratinization of the epidermal stratum corneum and hyperplasia of inflammatory cells were observed， and after PAS staining， a large amount of mycelial infection was observed. The results of the drug sensitivity test showed that， except for rntv10， the other strains were intermediate to amphotericin B. The isolated strains had different sensitivities to itraconazole， rntv09 and rntv10 were sensitive， rntv01 and rntv04-rntv08 were intermediate， and rntv02 and rntv03 were resistant. rntv10 was sensitive to ketoconazole and intermediate to 5-flucytosine， while the remaining strains were drug-resistant. All strains were intermediate to ciclopirox olamine and resistant to fluconazole and terbinafine. Conclusion The main pathogen of fungal dermatitis in Sichuan beef cattle was Trichophyton verrucosum. The fungus caused invasive skin infections in mice. Most of the diseased cattle could be treated with amphotericin B， itraconazole and ciclopirox olamine.

Objective Cathelicidins are a family of endogenous antimicrobial peptides unique to vertebrates， which have potent antibacterial and anti-inflammatory activities. This study identified a novel Cathelicidin from Quasipaa spinosa and investigated its gene structure and function， thereby providing a basis for future research on the antimicrobial peptide gene family. Method The complete sequence of Cathelicidin gene was obtained through gene cloning. The physicochemical properties， domains， signal peptides， secondary and tertiary structures of the encoded protein were analyzed by bioinformatics methods， and the phylogenetic tree was constructed by Mega X software. The expression of Cathelicidin gene in various tissues of Quasipaa spinosa was detected by Real-time quantitative PCR， as well as the expression changes of Cathelicidin gene under the stress of Citrobacter freundii （C. freundii） and Citrobacter braakii （C.braakii）. Result The full length of the Cathelicidin gene sequence was 730 bp， the open reading frame was 465 bp， encoded 154 amino acids. The molecular mass of Cathelicidin was 137 254.64 u， and its isoelectric point （pI） was 5.20. The 1-20 amino acids were signal peptides， and there was a structural domain between the 21-116 amino acids， which contained four conserved cysteine residues and a mature peptide containing 18 amino acids at the terminal. The prediction of secondary structure showed that the proportions of alpha helix， beta sheet and random coil of the Cathelicidin protein were 34.42%， 21.43% and 44.16%， respectively. The homology modeling of the tertiary structure showed that it was formed by the winding of a single peptide chain， and its functional domain was composed of alpha helix and beta sheet. Phylogenetic analysis revealed that the Cathelicidin from Quasipaa spinosa was most closely related to that of Nanorana yunnanensis （family Dicroglossidae）， with the two forming a distinct clade. The results of Real-time quantitative PCR showed that Cathelicidin gene was expressed in all tissues of Quasipaa spinosa， with higher expression in liver and kidney. After infection with Citrobacter， the expression of Cathelicidin gene in the liver of Quasipaa spinosa showed a temporal expression change characteristic. After infection with C. freundii， compared with 0 h， its expression reached an extremely significant peak at 72 h. However， C.braakii infection caused a continuous downregulation of its expression. Conclusion The Cathelicidin gene exhibited a well-defined structure and a broad expression profile across tissues in Quasipaa spinosa. Its mRNA expression displayed distinct temporal patterns in response to different pathogenic bacteria. These findings laid a theoretical foundation for further exploration of the Cathelicidin potential applications in the aquaculture industry.

Objective This study aimed to screen differentially expressed proteins between canine mammary tumor cells CHMM and CHMP， so as to provide scientific basis for the search of effective biomarkers for the diagnosis and treatment of canine mammary tumors. Method Using canine mammary tumor thoracic metastatic cells CHMM and primary cells CHMP as research objects， the morphologies， cloning ability， and migration ability of the two cell lines were compared through microscopic observation， cloning experiment， and Transwell experiment. Then， differentially expressed proteins in cells were screened using high-performance liquid chromatography-mass spectrometry analysis technology. GO function and KEGG pathway enrichment analysis on differentially expressed proteins was performed. Protein-protein interaction analysis was performed on differentially expressed proteins using STRING database. The proteomics results were validated through Western blotting experiment. Result CHMP cells exhibited a long spindle shape with single-cell growth， while CHMM cells grew in clusters with relatively small cells. CHMP cells had stronger migration ability， while CHMM cells had stronger cloning ability. A total of 2 049 proteins with differential expression were screened by sequencing （FoldChange>1.2 and P<0.05）， including 1 161 down regulated proteins and 888 upregulated proteins. GO functional enrichment analysis results showed that the differentially expressed proteins in cells involvement in biological processes mainly included cellular process， single-organism process， metabolic process，regulation of biological process， response to stimulus， etc. The cellular components included cell， cell part， organelle， organelle part， etc. Molecular functions mainly included catalytic activity， binding， and transporter activity. KEGG pathway enrichment analysis showed that the differentially expressed proteins in cells mainly participated in metabolic pathway signaling pathways. Differentially expressed proteins interaction analysis showed that CD44， MMP14 and DPYSL3 proteins had an interaction relationship. Western blotting showed that compared with CHMM cells， the expression level of GSDMD in CHMP cells significantly increased and the expression level of CD44 significantly decreased （P<0.05）， which was consistent with proteomic results，confirming the accuracy of sequencing. Conclusion Through proteomics screening， 1 161 downregulated proteins such as GSDMD and 888 upregulated proteins such as CD44 were identified in canine mammary tumor cells CHMM and CHMP， involving multiple biological processes such as metabolic pathways. The results of this study could provide scientific basis for the diagnosis and treatment of canine mammary tumors.

Objective The purpose of this experiment was to clarify the cause of the death of raccoon dogs in a raccoon dog farm in Hebei province， and provide a scientific basis for the effective prevention and control of the disease. Method Sterile collection of diseased tissues and respiratory secretions from raccoon dogs was collected， and the target strains were obtained through pathogen isolation and culture. The morphology of the isolate was identified by Gram staining. The classification of isolate was clarified by using 16S rRNA gene sequence alignment. The virulence genes and drug resistance genes of the isolate were detected by PCR amplification， and further drug sensitivity tests were carried out to evaluate the drug resistance phenotypes. The pathogenicity of the isolate was detected through the mouse pathogenicity test. Result The strain isolated from raccoon dogs formed round， neatly edged grayish-white colonies on TSB agar plates and smooth， moist， transparent colonies on MAC agar plates. Gram staining revealed the strain as Gram-negative coccobacilli with bipolar staining. 16S rRNA sequence alignment analysis showed that the similarity of the isolate to other Bordetella bronchiseptica in GenBank was 97.0%-100%， and this isolate was Bordetella bronchiseptica.Detection of virulence and resistance genes demonstrated the presence of six virulence genes （prn， bvgs， fla， fhaB， cyaA and dnt） and five resistance genes （bla_TEM， bla_SHV， parC， tetC and gyrA）. The results of the drug sensitivity test showed that the isolate was resistant to penicillins， macrolides， glycopeptides， sulfonamides and nitrofurans， but sensitive to aminoglycosides and quinolones. Pathogenicity tests showed that the median lethal dose of the isolate to mice was 3.67×10⁷ CFU. Conclusion In this experiment， one strain of Bordetella bronchiseptica was isolated from dead raccoon dogs， which carried multiple virulence genes and drug resistance genes. The isolate showed resistance to penicillins， macrolides， glycopeptides， sulfonamides and nitrofurans， and was sensitive to aminoglycosides and quinolones， demonstrating multidrug resistance and strong pathogenicity. The results of this study provided a scientific basis for the diagnosis， clinical medication， and vaccine development of Bordetella bronchiseptica disease in raccoon dogs.

Objective This experiment aimed to study the biological characteristics of Actinobacillus pleuropneumoniae from pigs in Yunnan. Method The bacteria was isolated from the lung of pigs suffering from respiratory diseases in a pig farm located at Honghe， Yunnan. The bacterial isolate was identified through biochemical tests， 16S rDNA sequence analysis and PCR amplification. The biological characteristics of the isolate， including serotypes， pathogenicity， drug sensitivity and resistance genes， were analyzed. Result A Gram-negative coccobacillus was isolated from the lung samples of the diseased pig and was numbered YN240724. The biochemical identification results showed that the isolate decomposed glucose， fructose， sucrose， xylose and mannitol to produce acid， and the oxidase， urease and nitrate reduction tests were positive. These biochemical characteristics were the same as those of the reference strain of Actinobacillus pleuropneumoniae. The results of the 16S rDNA gene sequence analysis showed that the 16S rDNA sequence similarity between the isolate and the type strain ATCC 27088^T of Actinobacillus pleuropneumoniae and other reference strains of Actinobacilluspleuropneumoniae was 100% and 99.6%-100%， respectively. The isolate formed the same evolutionary branch with all the reference strains of Actinobacillus pleuropneumoniae. Based on these characteristics， the isolate YN24074 was identified as Actinobacillus pleuropneumoniae. The serotype identification results showed that the isolate was Actinobacillus pleuropneumoniae serotype 7. The results of the drug resistance gene detection showed that the isolate carried the β-lactam resistance genes bla_CIT and bla_TEM， as well as the tetracycline resistance gene tetM. The results of the antimicrobial susceptibility test showed that the isolate was sensitive to aminoglycosides （gentamicin and amikacin）， quinolones （ofloxacin）， chloramphenicols （florfenicol）， tetracyclines （tetracycline）， sulfonamides （sulfamethoxazole）， and cephalosporins （cefotaxime and cefuroxime）， but resistant to penicillins （penicillin， ampicillin and piperacillin） and glycopeptides （vancomycin）. The pathogenicity test results showed that the median lethal dose （LD₅₀） of the isolate for mice was 7.5×10⁴ CFU， indicating a relatively strong pathogenicity. Conclusion In this study， a strain of porcine Actinobacillus pleuropneumoniae serotype 7 from Yunnan was isolated. This strain carried the resistance genes bla_CIT， bla_TEM and tetM， and showed resistance to various antibacterial agents， with strong pathogenicity. This test results could provide important references for the treatment and immunization prevention and control of porcine contagious pleuropneumonia in pig farms of Yunnan.

Objective Deer antler exhibit significant efficacy in promoting cartilage injury repair. This study aimed to identify the underlying key factor responsible for the cartilage-repairing effects of deer antler. Method Tissue from the apical growth center of fresh antlers was extracted using ultrasonic and cold-water extraction methods， respectively， with the optimal method identified by high-performance liquid chromatography （HPLC）. Preparative liquid chromatography was employed to isolate different peak fractions from the extract. Rat articular chondrocytes were isolated and cultured in vitro. The proliferative effects of the different peak fractions on these chondrocytes were compared using the CCK-8 assay. The compositional differences among the peak fractions were analyzed by untargeted metabolomics. Further validation of the effects of key substances in the extract on chondrocyte proliferation was performed using the CCK-8 assay. Result Ultrasonic extraction was identified via HPLC as an effective method for deer antler extract preparation， producing well-resolved peaks with convenient operation. Using preparative liquid chromatography， this study successfully isolated four distinct peak fractions from the extract， all exhibiting stable baselines， well-defined peaks， and minimal or no extraneous signals. Compared with other concentrations extract，0.1 mg/mL extract significantly promoted proliferation in rat articular chondrocytes （P<0.01）. Compared with other peak components， peak 1 exhibited an extremely significant promoting effect on chondrocyte proliferation （P<0.01）. Untargeted metabolomics analysis demonstrated that lipid-like metabolites comprised the majority of the extract （43.74%）， and principal component analysis （PCA） clearly separated peaks 1-4. The relative spermine content in peak 1 was extremely significantly higher than in other fractions （P<0.01）. Compared to other concentrations of spermine， 0.25 mg/mL of spermine extremely markedly enhanced chondrocyte proliferation （P<0.01）. Conclusion Antler extract significantly promotes the proliferation of rat articular chondrocytes. Preparative liquid chromatography successfully isolated four distinct peak components of the extract， and spermine in peak 1 was identified as the key factor responsible for significantly enhancing chondrocyte proliferation of deer antler.