The study aimed to screen candidate genes associated with litter size in goats and investigate the molecular mechanism by which the RHOU gene regulates goat litter size. The GenoBaits^® Goat 40K liquid chip was used to perform SNP genotyping on a hybrid population of 337 Boer goats and Haimen goats under selection， and a genome-wide association study was conducted in combination with the first-parity litter size phenotype. The coding region of the goat RHOU gene was cloned by the PCR method， and its sequence and structural characteristics were analyzed. The large and small follicles and ovarian granulosa cells of goats were isolated， and methods such as RT-qPCR， CCK-8， and ELISA were used to detect gene expression levels， cell proliferation， and estrogen levels. A C/T mutation on chromosome 28 （position 44 302 456， 177 365 bp upstream of RHOU） was significantly associated with first-parity litter size. The cloned RHOU coding region spanned 768 bp， encoding 255 amino acids with high homology to other mammals. The RHOU gene was widely expressed in various tissues of goats， and its expression was extremely significantly higher in ovarian tissue （P<0.01）， and the expression level in small follicles was extremely significantly higher than that in large follicles （P<0.01）. Overexpression of the RHOU gene significantly reduced cell proliferation ability （P<0.05） and the mRNA expression levels of cell proliferation marker genes PCNA （P<0.05） and MKI67 （P<0.01）， and significantly down-regulated the expression level of the CYP19A1 gene and the E2 concentration in the culture medium of goat ovarian granulosa cells （P<0.05）. This study identified that the RHOU gene was significantly associated with goat litter size based on the goat 40K liquid chip. The study found that it regulates goat litter performance by negatively regulating the proliferation of goat ovarian granulosa cells， reducing the expression of CYP19A1 in granulosa cells， and inhibiting the synthesis of E2.

This study aimed to analyze the metabolic mechanisms of testicular tissue associated with semen quality in ducks from a metabolomics perspective. Semen quality was evaluated in a duck population. Twelve drakes （6 with significantly high semen quality and 6 with significantly low semen quality） were selected， and testicular tissues were collected. Ultra-performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） was used to analyze differences in testicular metabolites between groups. Enrichment analysis of differential metabolites was performed to screen potential key pathways related to testicular semen quality. A total of 98 differentially abundant metabolites was identified in testicular tissues of ducks with varying semen quality， including 15 significantly upregulated and 83 significantly downregulated metabolites. These metabolites primarily belonged to categories such as fatty acyls， amino acids and their metabolites， glycerophospholipids， nucleotides and their metabolites， benzene derivatives， coenzymes and vitamins， and hormones. Correlation analysis revealed the strongest positive correlation between fatty acyls and benzene derivatives， while the strongest negative correlation was observed between carbohydrates/their metabolites and aldehydes/ketones/esters. Semen quality parameters （e.g.， sperm motility and kinematic parameters） showed significant positive/negative correlations （|R|≥0.80） with fatty acyls， glycerophospholipids， and other metabolites. Enrichment analysis highlighted two critical metabolic pathways： fatty acid degradation （P=0.000 59） and glycerophospholipid metabolism （P=0.012 76）. This study elucidated differential metabolites and potential metabolic mechanisms associated with semen quality in duck testicular tissues through metabolomics. It identifies 98 differential metabolites dominated by lipids （fatty acyls and glycerophospholipids）， characterized by significant downregulation of carnitines and enrichment of hexadecanoic acid （FFA16：0） and maltitol in the high semen quality group， and identifies fatty acid degradation and glycerophospholipid metabolism as core pathways significantly associated with semen quality. These findings provide key metabolites and pathway targets for research on avian testicular function.

The objective of this study was to investigate the effects of starch with different digestion rates on dietary energy and protein utilization in broilers. Eight dietary treatments from each of 4 starches with different digestion rates including cassava starch （k_d=4.29 h^-1）， wheat starch （k_d=2.51 h^-1）， corn starch （k_d=2.55 h^-1）， and pea starch （k_d=1.03 h^-1） combined with soybean meal or corn gluten meal were arranged in a single-factor completely randomized experiment. In the study on the effects of starch with different digestion rates on nitrogen retention and metabolizable energy of diets， a total of 192 Arbor Acres plus broilers of 22 days of age with similar body weight （BW=1 050±6 g） were selected and randomly assigned to 8 diets. Each treatment contained 6 replicates of 4 broilers in each replicate. The energy metabolizability and nitrogen retention rates were determined for diets fed to broilers aged 25 to 28 days. In the study on the effects of starch with different digestion rates on apparent ileal digestibility of starch and standardized ileal digestibility of amino acid （SIDAA） of diets， a total of 432 Arbor Acres plus broilers of 25 days of age with similar body weight （BW=1 270±10 g） were selected and randomly assigned to 8 diets and a nitrogen-free diet. Each treatment contained 6 replicates of 8 broilers in each replicate. The apparent ileal digestibility of starch and SIDAA were determined for diets fed to broilers at 28 days of age. The results showed as follows： 1） In the starch-soybean meal diets， the apparent ileal digestibility of starch in diets of moderately digestible starch （wheat starch and corn starch） were greater than these in diet of rapidly or slowly digestible starch （cassava starch or pea starch） （P<0.05）. The apparent ileal digestibility of starch in rapidly digestible starch （cassava starch） diet was greater than that in slowly digestible starch （pea starch） diet （P<0.05）. The diets of rapidly or moderately digestible starch （cassava starch， corn starch and wheat starch） had greater energy metabolizability than slowly digestible starch （pea starch） diet （P<0.05）. Starch with different digestion rates didn’t significantly affect the SIDAA of soybean meal. The nitrogen retention rate of corn starch diet was higher than other 3 starch diets （P<0.05）. 2） In the starch-corn gluten meal diets， the apparent ileal digestibility of starch in diets of moderately digestible starch （wheat starch and corn starch） were greater than these in diet of rapidly or slowly digestible starch （cassava starch or pea starch） （P<0.05）. The moderately digestible starch （corn starch） diet had greater energy metabolizability than diet of rapidly or slowly digestible starch （cassava starch or pea starch） （P<0.05）. Compared with rapidly digestible starch （cassava starch）， moderately digestible starch （wheat starch and corn starch） enhanced the standardized ileal digestibility of 13 amino acids and total amino acids in corn gluten meal （P<0.05）. However， no statistically significant difference was observed in nitrogen retention rate across diets with different digestion rates of starch. 3） A significant linear regression relationship between apparent metabolizable energy or nitrogen-corrected apparent metabolizable energy and digestible starch and metabolizable protein was observed in the 8 experimental diets （P<0.05）. The dietary energy metabilizablility and total amino acid digestibility decreased as the increase of the ratio of starch digestibility to total amino acid digestibility （P<0.05）. In conclusion， starches with moderate digestion rates （wheat starch， corn starch） have higher ileal digestibility of starch and dietary energy metabolizability compared to slowly digestible starch （pea starch）. However， when rapidly digestible starch （cassava starch） is combined with rapidly digestible protein （corn gluten meal）， the ileal digestibility of starch， dietary energy metabolizability and amino acid digestibility in broilers is reduced. Generally， the ratio of starch digestibility to total amino acid digestibility is negatively related with the energy metabolizability and amino acid digestibility.

The objective of this experiment was to investigate the effects of different calcium （Ca）， non-phytic phosphorus （NPP） levels and electrolyte balance （DEB） value in a low protein diet on growth performance， serum biochemical indices and metabolic rate of nutrients in broilers. A 3×3 two-factor completely randomized design was adopted. Three dietary calcium and non-phytate phosphorus levels were 0.70% Ca and 0.35% NPP， 0.80% Ca and 0.40% NPP， 0.90% Ca and 0.45%NPP， respectively. And three levels of dietary electrolyte balance （Na⁺+K⁺-Cl^-） were 200， 250 and 300 mmol·kg^-1， respectively. In addition， dietary crude protein level was 19.5%. A total of 324 1-day-old Arbor Acres （AA） male broilers were randomly allotted to 1 of 9 treatments with 6 replicate cages per treatment and 6 birds per cage. The experimental period was 21 days. The results showed that： 1） Dietary Ca， NPP and DEB value and their interaction had no significant effect （P>0.05） on growth performance of broilers from 1 to 21 d of age； 2） The interaction of dietary Ca， NPP and DEB value had significant effect （P<0.05） on the alanine aminotransferase （ALT） activity in serum. As dietary Ca and NPP levels were 0.70% and 0.35%， the serum ALT activity from broilers in the 300 mmol·kg^-1 DEB value group was significantly higher （P<0.05） than that of the 200 and 250 mmol·kg^-1 DEB value groups. As the levels of Ca and NPP were 0.80% and 0.40%， the serum ALT activity from broilers in the 250 or 300 mmol·kg^-1 DEB value groups was significantly lower （P<0.05） than that in the 200 mmol·kg^-1 DEB value group. As the levels of Ca and NPP were 0.90% and 0.45%， DEB levels had no significant effect （P>0.05） on serum ALT activity； 3） As dietary Ca and NPP levels increased， the apparent metabolic rate of Ca decreased significantly （P<0.05）. The apparent metabolic rates of dry matter， crude protein and Ca in broilers from 300 mmol·kg^-1 DEB value group were significantly higher （P<0.05） than those in 250 and 200 mmol·kg^-1 DEB value groups. As the levels of dietary Ca and NPP were 0.70% and 0.35%， 300 mmol·kg^-1 DEB value could significantly increase （P<0.05） the apparent metabolic rate of P compared with the 200 and 250 mmol·kg^-1 DEB value. And as the levels of Ca and NPP were 0.90% and 0.45%， the apparent metabolic rate of P in 300 mmol·kg^-1 DEB value group was significantly higher （P<0.05） than that in the 200 mmol·kg^-1 group. These results indicated that the Ca and NPP levels of 0.80% and 0.40% and DEB value of 300 mmol·kg^-1 in a low protein diet （Crude protein： 19.5%） were optimal for the growth and nutrient utilization in broilers from 1 to 21 d of age.

The aim of this experiment is to investigate the effect of 18β-glycyrrhetinic acid （18β-GA） on oxidative stress induced lung injury in weaned piglets induced by D-galactose （D-gal）.In total， 24 weaned piglets of similar body weight at 28 days old were randomly divided into 3 groups， with 8 replicates in each group and 1 piglet in each replicate. The experimental groups were as follows： the control group （CK） was fed with basal diet， the model group （D-gal） was fed with basal diet supplemented with 10 g·kg^-1 D-gal， and the experimental group （GA+gal） was fed with basal diet supplemented with 10 g.kg^-1 D-gal and 100 mg·kg^-1 18 β-GA. The pre trial period is 7 days， and the main trial period is 28 days. The results showed that compared with the CK group， the D-gal group showed an increase trend in malondialdehyde （MDA） content， 8-hydroxy-2-deoxyguanosine （8-OHdG） content， and catalase （CAT） activity in the lung tissue of weaned piglets （0.05≤P<0.10）， and a significant increase in superoxide dismutase （SOD） activity （P<0.05）； The expression levels of Nrf2， CAT， and SOD1 genes significantly increased （P<0.05）； The levels of inflammatory factors interleukin-8 （IL-8） and transforming growth factor-β （TGF-β） increased significantly （P<0.05）， and the mean linear intercept （MLI） and apoptosis rate of lung tissue cells increased significantly （P<0.05）； Compared with the D-gal group， the GA+gal group showed a significant decrease in the activities of CAT and SOD， content of MDA and 8-OHdG， as well as the expression levels of antioxidant related genes Nrf2， CAT， and SOD1 in the lungs （P<0.05）. The levels of inflammatory factors IL-8， interleukin-18 （IL-18）， and TGF-β were also reduced significantly （P<0.05）， and the apoptosis rate was also reduced significantly （P<0.05）. Transcriptomic analysis showed that oxidative stress has an impact on the cell cycle， PI3K/AKT signaling pathway， and P53 signaling pathway in piglet lung cells. 18β-GA can alleviate lung injury by regulating pathways such as PI3K/AKT signaling pathway and retinol metabolism. Further verification using fluorescence quantitative PCR and Western blot techniques revealed that the expression levels of key genes PI3K， AKT， TP53， as well as proteins P53 in the oxidative stress pathway of piglets increased significantly （P<0.05）. After adding 18β-GA， the expression levels of pathway genes PI3K and TP53 decreased significantly （P<0.05）， while the expression levels of gene AKT and protein P53 showed a downward trend （0.05≤P<0.1）.In summary， the addition of 18β-GA can alleviate D-gal induced lipid peroxidation， DNA oxidative damage， increased inflammatory factors， and cell apoptosis in weaned piglet lungs. The underlying mechanism might involve the alleviation of lung injury through the modulation of the P53 signaling pathways.

Since 2011，the new outbreak of pseudorabies virus（PRV）variant strain in China has greatly increased the detection workload of the virus. At present， the widely use of blocking ELISA kits has raised the greater demand for monclonal antibodies. However， hybridoma cells often lose antibody secretion capacity during passages. This study aimed to prepare single-chain fragment variable with good binding activity on the basis of monoclonal antibody and achieve large-scale production via prokaryotic expression system， providing a foundation for novel detection methods. The prokaryotically expressed PRV gE protein was used to immunize mice. Positive hybridoma cells stably secreting anti-PRV gE mabs were obtained through cell fusion and screening. The cDNA of the positive hybridoma cells was used as a template to amplify the variable region sequence of antibody，and the scFv gene was constructed by linking the variable regions of heavy and light chain with a Linker peptide. The scFv gene was cloned into pET-28a plasmid for prokaryotic expression to produce the recombinant antibody. A monoclonal antibody 7B7 specific to the gE protein of PRV was successfully screened. Using the cDNA of 7B7 hybridoma cells as a template，a single chain fragment variable capable of specifically reacting with PRV gE was successfully prepared which can be expressed by prokaryotic expression system， providing a basis for the development of new detection methods for PRV.

In this study， we aimed to develop recombinant PRV rPRV-AH-gI^-/gE^-/gM^-/gD⁺ with double gD and evaluate its immune efficacy. The recombinant virus rPRV-AH-gI^-/gE^-/gM^-/gD⁺ was constructed by deleting gM gene and inserting gD expression cassette at the deficient region. Four-week-old Kunming mice were immunized twice with 10^7.0 TCID₅₀ inactivated rPRV-AH-gI^-/gE^-， rPRV-AH-gI^-/gE^-/gM^-， rPRV-AH-gI^-/gE^-/gM^-/gD⁺ at an interval of 3 weeks. Commercial vaccine and DMEM were as the control group. After immunization， regular blood was collected， neutralizing antibody levels in sera were detected by neutralization test， and total antibody levels were detected by ELISA. At 6 weeks of first immunization， mice were challenged with 10^5.5TCID₅₀ PRV-AH. The inserted gD gene was expressed with the molecular weight of 53 ku in the rPRV-AH-gI^-/gE^-/gM^-/gD⁺. The neutralizing and total IgG antibody levels of rPRV-AH-gI^-/gE^-/gM^-/gD⁺ group were significantly higher than those of rPRV-AH-gI^-/gE and rPRV-AH-gI^-/gE^-/gM^- at 4 and 6 weeks after first immunization （P<0.01）. However， compared with the commercial vaccine group， the difference was not significant （P>0.05）. After challenged with PRV-AH， the protection rates were 100%， 70%， 60% and 90% of the rPRV-AH-gI^-/gE^-/gM^-/gD⁺， rPRV-AH-gI^-/gE^-/gM^-， rPRV-AH-gI^-/gE^- and commercial vaccine groups respectively. The results showed that the insertion of gD gene can improve the immune protection effect of the recombinant virus which is helpful in developing vaccine candidate for PRV control.

A microdroplet digital polymerase chain reaction （ddPCR） method for the detection of fowlpox virus （FWPV） was established. In this study， a more complete ddPCR reaction system was established through the optimization of the reaction system， including the optimization of primer and probe concentrations， the optimization of annealing temperature and other steps， and reproducibility， sensitivity， and specificity experiments. In this method， the optimal primer concentration was determined to be 900 nmol·L^-1， the optimal probe concentration was 200 nmol·L^-1， and the results of ddPCR were optimized under the reaction condition of 55 ℃. The method showed no cross-reactivity with other common avian viruses and is highly specific. The reproducibility of the method is good， and the coefficients of variation between and within batches are less than 10%. The lowest detection limit of this ddPCR method was 2.37 copies per reaction. Using the established ddPCR detection method and the industry standard （SN/T 1226—2015） detection method for 80 samples， the coincidence rate was 100%， which was higher than the coincidence rate of 75% of the industry standard nested PCR method. In 25 artificially inoculated samples， the detection rate of ddPCR for positive samples reached 100%， while the detection rate of the nested PCR method specified in the industry standard for positive samples was 33.3%. The establishment of this method helps the relevant inspection and quarantine departments at ports to detect the FWPV in a timely manner and reduces the risk of the domestic poultry breeding industry by providing a more accurate and efficient detection method.

This study aimed to express a nanobody targeting H7-subtype avian influenza virus using an E. coli system. Nanobody and antigen expression vectors were constructed via homologous recombination. Recombinant vectors were amplified using E. coli DH5α， followed by transformation into E. coli BL21（DE3） and BEVS for high-yield protein production. Nanobodies and antigens were purified using streptavidin affinity chromatography and IMAC purification， respectively. Finally， immunofluorescence assay （IFA）， enzyme-linked immunosorbent assay （ELISA）， hemagglutination inhibition （HI） and surface plasmon resonance （SPR） were employed to analyze nanobody titer， specificity， immunoreactivity， and antigen-nanobody interaction dynamics. Expression vectors were successfully constructed， followed by the production of a 15 ku nanobody （D67）， a 42 ku fluorescent nanobody （D67-GFP） and a 55 ku avian influenza antigen （H7-HA1）. In IFA， H7-HA1-positive cells incubated with the nanobody exhibited significantly stronger green fluorescence signals compared to the control group. ELISA revealed significant OD₄₅₀ signals exclusively in the H7-HA1 antigen group， with P/N ratios decreasing as D67 concentrations diminished. HI assays demonstrated specific hemagglutination inhibition only for the H7 antigen group， achieving a titer of 8log2. SPR analysis quantified the interaction kinetics， yielding an association rate of 3.99×10³ L·（mol·s）⁻¹， dissociation rate of 3.23×10^-3 s^-1， and equilibrium dissociation constant of 8.1×10^-7 mol·L⁻¹， confirming strong antigen-nanobody affinity.The H7-subtype-targeting nanobody D67 was successfully prepared and validated through a multidimensional evaluation system， demonstrating its high-efficiency and specific binding to H7-HA1. This work provides a technical foundation for developing rapid detection methods for H7-subtype avian influenza.

In order to understand the drug resistance and virulence gene distribution of Riemerella anatipestifer isolates in some areas of China， K-B method was used to determine the sensitivity of 15 serotypes of R. anatipestifer to 10 commonly used antibiotics， and PCR technology was used to detect 16 drug resistance genes and 16 virulence genes of different serotypes of R. anatipestifer isolates. The results showed that the resistance rates of amoxicillin and ampicillin were 93.33% and 80%， respectively. The detection results of drug-resistant genes showed that the detection rates of tet（X） gene of tetracycline， floR gene of amide alcohol and aph（3'）-Ⅱ gene of aminoglycoside were 93.33%， 80% and 73.33% respectively. The results of virulence gene detection showed that tctR， TbdR1， TR， CAMP and AS87-0405 had the highest detection rates， which were 86.67%， 86.67%， 80%， 73.33% and 73.33% respectively. These isolates are sensitive to tetracyclines， quinolones and amide alcohols， and have high resistance to β-lactams. At the same time， these isolates carry a variety of virulence genes， which may indicate that they have strong virulence. The above results provide some reference for the prevention and control of R. anatipestifer in some areas of China.