Chicken semen cryopreservation is crucial for utilizing high-quality cockerel genetics, but semen is highly sensitive to cryoinjury, leading to poor preservation outcomes. This study aimed to establish a theoretical foundation for selecting cockerels for semen cryopreservation through serum testing and to improve semen quality via DNA methylation editing. Semen and serum samples were collected from 102 Xiaoshan cockerels, with semen cryopreserved and thawed following standardized protocols. Post-thaw semen quality and serum testosterone (T) levels were assessed. Eight cockerels were selected based on motile sperm quality, and whole-genome bisulfite sequencing (WGBS) was used to analyze sperm DNA methylation. The results showed a significant positive correlation between serum T levels and sperm motility. There were notable differences in sperm motility and serum T levels between high-quality and low-quality semen groups but no differences in estradiol (E₂), superoxide dismutase (SOD), or glutathione peroxidase (GSH-Px) levels. A total of 217 differentially methylated regions (DMRs) and 116 differentially methylated genes (DMGs) were identified. Key genes such as PRKACB (protein kinase, cAMP-dependent, catalytic, beta) and ACSL1 (long-chain-fatty-acid--CoA ligase 1) were associated with sperm motility. These findings provide important insights for improving semen cryopreservation and contribute to breeding practices and the development of cryoprotectants.

The gut microbiota plays a vital role in regulating the host’s physiological functions, including metabolism and immunity. The microbial composition and metabolism are modulated by multiple factors; host sex is an important yet under-explored determinant. To investigate the sex-dependent differences in the gut microbiota within the small and large intestine, sixteen somatic mature Jiangshan black pigs (eight males and eight females) were analyzed. The ileal and colonic microbial community and metabolites were profiled using 16S rRNA gene high-throughput sequencing and gas chromatography. Distinct sex-related discrepancies were observed in both the microbial composition and metabolism of the ileum and colon. In the ileum, compared with the male group, the female group exhibited higher abundances of Unclassified Chloroplast and Pseudomonas but a lower abundance of Romboutsia (adjusted p < 0.05). Functional prediction indicated enrichment in amino acid metabolism pathway in females, with more copy numbers of genes encoding key enzymes for propionate (mmdA) generation and elevated valerate levels (p < 0.05). In the colon, compared with the male group, the female group showed higher abundances of Streptococcus, Phascolarctobacterium, and Prevotella spp. and lower abundances of Eubacterium coprostano-ligenes group, Blautia, Christensenellaceae R-7 group, and Ruminococcus (adjusted p < 0.05). Additionally, the female group had more copies of genes mmdA and LcdA (associated with lactate production), along with higher concentrations of propionate and lactate (p < 0.05). Correlation analysis between microbial metabolites and sex-biased bacteria further revealed that the SCFA concentration positively correlated with Prevotella spp. and negatively correlated with Romboutsia, Christensenellaceae R-7 group, and Blautia. Collectively, these findings highlight the pronounced sex-dependent discrepancies in the microbial composition and metabolism within the small and large intestines of Jiangshan black pigs, providing new insights for precisely modulating the microbiota community and metabolism in pigs according to sex.

Trichomonas gallinae (T. gallinae) is a flagellated protozoan and the causative agent of trichomoniasis, or canker, in birds. In the current study, the prevalence of T. gallinae was firstly investigated in five breeds. According to the results of the prevalence study, White King pigeons were selected as the experimental animals. A total of 135 White King squabs at one day of age were randomly divided into two groups and raised in separate isolators. The challenged group (N = 100) was challenged intranasally with 5 × 10⁶ parasites/mL of the T. gallinae strain, and the control group (N = 35) was intranasally administered medium of equivalent volume. At 1, 2, 3 and 5 days post infection (DPIs), the crops and esophagi were collected for RNA extraction and formaldehyde fixation. The results showed that prevalence of T. gallinae in the five breeds ranged from 27.13% (White Carneau) to 43.14% (White King). After the challenge, mild microscopic lesions were observed in both tissues. Apoptosis rates were higher in the challenged group than in the control group at 2 and 5 DPIs in the crop and at 1, 2 and 7 DPIs in the esophagus. For both tissues, relative expression of IL-1β increased dramatically at the beginning and decreased at 5 DPIs, and TGF-β increased stably in the challenged group.