Jun-ichi SHIRAISHI

Summary When retroviruses infect germ cells and are transmitted to offspring, they become endogenous retroviruses (ERVs), whose insertions influence the expression of nearby genes. This study aimed to identify the genomic loci of ERVs in commercial broiler (Ross308), Tosa-Jidori, and Yakido chickens as well as to elucidate their impact on neighboring gene expression. Whole-genome data were obtained using next-generation sequencing, and candidate ERV loci were identified using the RetroSeq software. The Integrative Genomics Viewer tool was used to confirm target site duplications (TSDs) as evidence of ERV insertions. All reads within 200 bp of these TSDs were extracted to create contigs, confirming the presence of ERV sequences in the contigs using BLASTN. Gene expression levels were estimated by focusing on genes located near the 172 identified ERV loci. Among these, 119 loci were detected in broiler chickens, 80 in Tosa-Jidori chickens, and 86 in Yakido chickens, with 28 loci shared among them. Moreover, of these 172 loci, 75 were located within or near genes. Significant differences in gene expression were observed for N-acetylated alpha-linked acidic dipeptidase 2, glypican 6, and phospholipid scramblase family member 5 depending on the presence of ERV insertions. These results suggest that ERV insertions may influence the expression of certain genes, providing insights into the genetic diversity and evolutionary background of commercial and indigenous chickens. Understanding the effects of ERV insertions on gene expression can inform future genetic research and poultry breeding programs aimed at improving health and productivity. Author Summary Recently, endogenous retroviruses (ERVs) have gained significant attention as valuable markers for understanding genetic relationships and evolutionary processes among species. In this study, we investigated the loci and characteristics of ERVs in commercial and traditional Japanese chickens. ERVs are genetic remnants of ancient viral infections that can provide insights into avian evolution. We identified a total of 172 ERV loci in broiler, Tosa-Jidori, and Yakido chickens. Each chicken breed exhibited unique ERV insertion patterns. Notably, we found that ERV insertions near certain genes may influence gene expression. Our research enhances the understanding of how chickens have acquired traits, particularly through genetic mechanisms influenced by ERVs. These insights significantly contribute to our knowledge of biological evolution and the overall biodiversity of birds.

For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary storage on oocytes for the purpose of cryopreserving avian female gametes by using a chicken as a model of endangered avian species. After excision, the ovaries were stored at either a low temperature (4 °C) or room temperature for 1–3 days. Ovarian follicles stored under different conditions for each period were examined by neutral red staining, histology, and gene and protein expression analysis. In addition, the pH of the storage medium after preserving the ovaries was measured. Then, ovarian tissues were vitrified to determine the cryopreservation competence. Storing the ovarian tissues at 4 °C kept the follicles viable and morphologically normal for 3 days with slow decline. In contrast, although different storage temperature did not influence follicle viability and morphology after only 1 day of storage, ovarian tissues stored at room temperature rapidly declined in structurally normal follicles, and viable follicles were rarely seen after 3 days of storage. Gene and protein expression analysis showed that apoptosis had already started on the first day, as shown by the higher expression of CASP9 under room temperature conditions. Furthermore, high expression of SOD1 and a rapid decline of pH in the storage medium under room temperature storage suggested the influence of oxidative stress associated with low pH in this condition on the follicle survivability in hen ovarian tissues. Our cryopreservation study also showed that ovarian tissues stored at 4 °C could recover after cryopreservation even after 3 days of storage. The described storage conditions and cryopreservation methods, which preserve chicken follicle survival, will lay the foundation of ovarian tissue preservation to preserve the fertility of wild female birds.