Broodiness is observed in most domestic fowls and influences egg production. The goose is one of the most important waterfowls, having strong broody

Broodiness is the primary factor influencing egg production in geese, in which several genes and miRNAs participate. Detailed spatiotemporal profiles of miRNAs encompassing follicle development levels, however, are lacking. In this study, we collected preovulatory follicles (classified as small white follicles, large white follicles, and small yellow follicles) from brooding and laying geese and aimed to analyze microRNA (miRNA or miR) during folliculogenesis. High-throughput sequencing and bioinformatics analysis were used to identify the miRNAs involved in follicle development. The let7 family, miR-10 family, and miR-143 family were abundant in these libraries, and they have been suggested to play a housekeeping role during folliculogenesis. Joint comparisons revealed 23 upregulated and 21 downregulated miRNAs (in at least two comparisons of follicles during brooding and laying, P < 0.1) in the laying stage. Unlike reproduction pathways reported for ovaries, GO and KEGG analysis suggested pathways for cell apoptosis and proliferation, such as the regulation of actin cytoskeleton, endocytosis, axon guidance, pathways in cancer, tight junctions, focal adhesion, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and the Wnt signaling pathway in folliculogenesis. This study revealed the miRNAs that were directly involved in follicular atresia, and our results added to the understanding of the functional involvement of miRNAs during specific stages of follicle development.

Duck egg production, like that of laying hens, follows a typical low–peak–low cycle, reflecting the dynamics of the reproductive system. Post-peak, some ducks undergo a cessation of egg laying, indicative of a regression process in the oviduct. Notably, the magnum, being the longest segment of the oviduct, plays a crucial role in protein secretion. Despite its significance, few studies have investigated the molecular mechanisms underlying oviduct regression in ducks that have ceased laying eggs. In this study, we conducted single-cell transcriptome sequencing on the magnum tissue of Shaoxing ducks at 467 days of age, utilizing the 10× Genomics platform. This approach allowed us to generate a detailed magnum transcriptome map of both egg-laying and ceased-laying ducks. We collected transcriptome data from 13,708 individual cells, which were then subjected to computational analysis, resulting in the identification of 27 distinct cell clusters. Marker genes were subsequently employed to categorize these clusters into specific cell types. Our analysis revealed notable heterogeneity in magnum cells between the egg-laying and ceased-laying ducks, primarily characterized by variations in cells involved in protein secretion and extracellular matrix (ECM)-producing fibroblasts. Specifically, cells engaged in protein secretion were predominantly observed in the egg-laying ducks, indicative of their role in functional albumen deposition within the magnum, a phenomenon not observed in the ceased-laying ducks. Moreover, the proportion of THY1⁺ cells within the ECM-producing fibroblasts was found to be significantly higher in the egg-laying ducks (59%) compared to the ceased-laying ducks (24%). Similarly, TIMP4⁺ fibroblasts constituted a greater proportion of the ECM-producing fibroblasts in the egg-laying ducks (83%) compared to the ceased-laying ducks (58%). These findings suggest a potential correlation between the expression of THY1 and TIMP4 in ECM-producing fibroblasts and oviduct activity during functional reproduction. Our study provides valuable single-cell insights that warrant further investigation into the biological implications of fibroblast subsets in the degeneration of the reproductive tract. Moreover, these insights hold promise for enhancing the production efficiency of laying ducks.

The aim of the study was to investigate the mechanisms of the N-acetyl-L-glutamic (NAG) diet in improving rooster semen quality after cryopreservation. A total of 60 individually housed chickens were randomly allocated to three groups: a basal diet group (C), a basal diet supplemented with 2 g/kg NAG group (NL), and a basal diet supplemented with 4 g/kg NAG group (NH). Semen quality was evaluated on days 15 and 30, including fresh sperm motility and cryopreservation efficacy. Serum biochemical parameters, hormone levels, immune indices, semen metabolites, and gut microbiota composition were assessed at 30 d. NAG supplementation significantly improved semen quality. At 15 and 30 d, both NL and NH groups showed a significant increase in the total motile sperm ratio (TM) in fresh semen compared to the control (P ≤ 0.05). At 30 d, group NH exhibited significantly higher percentage of sperm in a straight-line trajectory (LIN) in fresh semen than both the C and NL groups. After cryopreservation, the sperm TM and LIN from chickens with NAG dietary for 30 days were significantly better in the NL group compared to C and NH groups (P ≤ 0.05). Metabolomic analysis identified significant upregulation of metabolites, including melatonin and ferulic acid, in cryopreserved semen from NAG-supplemented groups, while 5-hydroxylysine and inosine-1-phosphate were significantly reduced. Serum antioxidant capacity, as measured by glutathione peroxidase (GSH-Px) was significantly higher, while malondialdehyde (MDA) levels lower in NAG groups compared to C (P ≤ 0.05). No significant differences in immune markers or reproductive hormones were observed across groups. Gut microbiota analysis revealed that NAG supplementation enhanced beneficial bacterial populations. In conclusion, NAG supplementation improved sperm motility and cryopreservation outcomes, potentially by increasing the levels of semen melatonin and ferulic acid and reducing the body oxidative stress. And it can improve the overall health level by regulating the gut microbiota through diet. These findings highlight NAG's potential as a dietary supplement to improve semen quality and overall reproductive efficiency in poultry.

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.