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Background: In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear. Results: Critical biochemical events involved in ovulation in domestic chickens (Gallus gallus) were evaluated by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory follicle (F1) and postovulatory follicle (POF1) in continuous laying (CL) and intermittent laying (IL) chickens indicated the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes (DEGs), and the smallest difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin A, and lactate dehydrogenase A (LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro. Conclusions: This study reveals several important biochemical events involved in the process of ovulation, as well as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian species. [ABSTRACT FROM AUTHOR]

Background: Hepatic steatosis is a prevalent manifestation of fatty liver, that has detrimental effect on the health and productivity of laying hens, resulting in economic losses to the poultry industry. Here, we aimed to systematically investigate the genetic regulatory mechanisms of hepatic steatosis in laying hens. Methods: Ninety individuals with the most prominent characteristics were selected from 686 laying hens according to the accumulation of lipid droplets in the liver, and were graded into three groups, including the control, mild hepatic steatosis and severe hepatic steatosis groups. A combination of transcriptome, proteome, acetylome and lipidome analyses, along with bioinformatics analysis were used to screen the key biological processes, modifications and lipids associated with hepatic steatosis. Results: The rationality of the hepatic steatosis grouping was verified through liver biochemical assays and RNA-seq. Hepatic steatosis was characterized by increased lipid deposition and multiple metabolic abnormalities. Integration of proteome and acetylome revealed that differentially expressed proteins (DEPs) interacted with differentially acetylated proteins (DAPs) and were involved in maintaining the metabolic balance in the liver. Acetylation alterations mainly occurred in the progression from mild to severe hepatic steatosis, i.e., the enzymes in the fatty acid oxidation and bile acid synthesis pathways were significantly less acetylated in severe hepatic steatosis group than that in mild group (P < 0.05). Lipidomics detected a variety of sphingolipids (SPs) and glycerophospholipids (GPs) were negatively correlated with hepatic steatosis (r ≤ −0.5, P < 0.05). Furthermore, the severity of hepatic steatosis was associated with a decrease in cholesterol and bile acid synthesis and an increase in exogenous cholesterol transport. Conclusions: In addition to acquiring a global and thorough picture of hepatic steatosis in laying hens, we were able to reveal the role of acetylation in hepatic steatosis and depict the changes in hepatic cholesterol metabolism. The findings provides a wealth of information to facilitate a deeper understanding of the pathophysiology of fatty liver and contributes to the development of therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: As a ubiquitous reversible epigenetic RNA modification, N6-methyladenosine (m6A) plays crucial regulatory roles in multiple biological pathways. However, its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear. Therefore, we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7 (E7) by methylated RNA immunoprecipitation sequencing (MeRIP-seq) to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation. Results: The chicken embryonic gonadal transcriptome was extensively methylated. We found 15,191 and 16,111 m6A peaks in the female and male left gonads, respectively, which were mainly enriched in the coding sequence (CDS) and stop codon. Among these m6A peaks, we identified that 1013 and 751 were hypermethylated in females and males, respectively. These differential peaks covered 281 and 327 genes, such as BMP2, SMAD2, SOX9 and CYP19A1, which were primarily associated with development, morphogenesis and sex differentiation by functional enrichment. Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance. Furthermore, we found that YTHDC2 could regulate the expression of sex-related genes, especially HEMGN and SOX9, in male mesonephros/gonad mingle cells, which was verified by in vitro experiments, suggesting a regulatory role of m6A methylation in chicken gonad differentiation. Conclusions: This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation. Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry. [ABSTRACT FROM AUTHOR]

Background: Female sperm storage (FSS), the maintenance of sperm inside the female reproductive tract for an extended period of time, is pervasive among organisms with internal fertilization. Because FSS enables asynchronous mating and fertilization, it could be extremely important to reproduction. However, the physiological mechanisms underlying prolonged preservation and maintenance are poorly understood. Here, we used chicken, a typical oviparous animal, to determine the mechanisms ensuring sperm functionality in sperm storage tubules (SSTs). Results: We performed an insemination experiment on over two thousand hens at two periods, and found that the FSS capabilities varied widely among individuals. Except for the differences in the SST density between the two groups with distinct FSS abilities, we quantitatively profiled small-molecule metabolites derived from SST cells, and identified 28 metabolites with differential expression. In particular, high levels of lipids, fatty acids and lipid peroxidation product were observed in hens with low FSS capability. Pathway analysis showed that these differential metabolites were significantly enriched in the biosynthesis of unsaturated fatty acids. Moreover, we detected the total antioxidant capacity and lipid peroxidation level of SSTs, and found that chickens with a lower FSS ability had a significantly higher content of lipid peroxidation end-product, which was 2.4-fold greater than chickens with a higher FSS capability, and no significant difference was found in the total antioxidant capacity between these two groups. Conclusions: Our findings reveal that the long-term storage of sperm and the maintenance of their function in the female reproductive tract require an adequate microenvironment. The superabundance of fatty acids secreted by SST cells had detrimental effects on sperm storage in the female reproductive tract. Lipid peroxidation produces toxic biological substances that may cause irreversible damage to resident spermatozoa, resulting in short-term sperm retention and decreased fertility. Our findings provide new avenues for studying sperm storage and sustaining fertility. [ABSTRACT FROM AUTHOR]