石原 慎矢

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.

Abstract Endogenous retroviruses (ERVs) are genetic elements present in the genome that retain traces of past viral infections. Characterization of ERVs can provide crucial insights into avian evolution. This study aimed to identify novel long terminal repeat (LTR) loci derived from ERVs (ERV-LTRs) absent in the reference genome using whole-genome sequencing data of red junglefowl, gray junglefowl, Ceylon junglefowl, and green junglefowl. In total, 835 ERV-LTR loci were identified across the four Gallus species. The numbers of ERV-LTRs loci detected in red junglefowl and its subspecies gray junglefowl, Ceylon junglefowl, and green junglefowl were 362, 216, 193, and 128, respectively. The phylogenetic tree was congruent with previously reported trees, suggesting the potential for inferring relationships among past junglefowl populations from the identified ERV-LTR loci. Of the detected loci, 306 ERV-LTRs were identified near or within the genes, and some were associated with cell adhesion. The detected ERV-LTR sequences were classified as endogenous avian retrovirus family, avian leukosis virus subgroup E, Ovex-1, and murine leukemia virus-related ERVs. In addition, the sequence of the EAV family was divided into four patterns by combining the U3, R, and U5 regions. These findings contribute to a more comprehensive understanding of the characteristics of junglefowl ERVs.

The Vietnamese native pig (VnP)-a porcine breed with a small body-has proven suitable as a biomedical animal model. Here, we demonstrate that, compared to other breeds, VnPs have fewer copies of porcine endogenous retroviruses (PERVs), which pose a risk for xenotransplantation of pig organs to humans. More specifically, we sought to characterize non-reference PERVs (nrPERVs) that were previously unidentified in the reference genome. To this end, we used whole-genome sequencing data to identify nrPERV loci with long terminal repeat (LTR) sequences in VnPs. RetroSeq was used to estimate nrPERV loci based on the most current porcine reference genome (Sscrofa11.1). LTRs were detected using de novo sequencing read assembly near the loci containing the target site duplication sequences in the inferred regions. A total of 21 non-reference LTR loci were identified and separated into two subtypes based on phylogenetic analysis. Moreover, PERVs within the detected LTR loci were identified, the presence of which was confirmed using conventional PCR and Sanger sequencing. These novel loci represent previously unknown PERVs as they have not been identified in the porcine reference genome. Thus, our RetroSeq method accurately detects novel PERV loci, and can be applied for development of a useful biomedical model.

We aimed to clarify the genomic characteristics of porcine endogenous retroviruses (PERVs) in Vietnamese native pig (VnP) breeds. First, we investigated genetic polymorphisms in β- and γ-like PERVs, and we then measured the copy numbers of infectious γ-like PERVs (PERV-A, B, and C). We purified genomic DNA from 15 VnP breeds from 12 regions all over the country and three Western pig breeds as controls, and investigated genetic polymorphisms in all known PERVs, including the beta (β)1-4 and gamma (γ)1-5 groups. PERVs of β1, β2, β3, and γ4 were highly polymorphic with VnP-specific haplotypes. We did not identify genetic polymorphisms in β4, γ1, or γ2 PERVs. We then applied a real-time polymerase chain reaction-based method to estimate copy numbers of the gag, pol, and env genes of γ1 PERVs (defined as A, B, and C). VnP breeds showed significantly lower copy number of the PERV genes compared with the Western pig breeds (on average, 16.2 and 35.7 copies, respectively, p < .05). Two VnP breeds showed significantly higher copy number compared with the other VnPs (p < .05). Our results elucidated that VnPs have specific haplotypes and a low copy number of PERV genes.

We have elucidated genetic relationships of Vietnamese native pigs (VNP) using preliminarily collected samples by a single-nucleotide polymorphism (SNP) array. In order to confirm our previous results and compare with the results of a previous study using microsatellite (MS) markers, we aimed to characterize genetic diversity and population structure in wider varieties (24 breeds from 21 Provinces) of VNP across the country using 20 polymorphic MS markers recommended by ISAG/FAO (International Society for Animal Genetics/Food and Agriculture Organization) for diversity study. In this study, we collected 1,136 DNA samples of the VNPs and three exotic breeds. Our results revealed that the average number of alleles and allelic richness across the loci in VNPs were 10.0 and 7.6, which were higher than those of exotic breeds. Genomic components among VNPs were subjected to the sampling locations. Interestingly, Co Binh Thuan showed remarkable genetic feature compared to the other VNPs, because the habitation of Co Binh Thuan was relatively far from the other breeds. The results of this study provided useful information for exploitation, conservation, and development trends of the VNP breeds. More recently, African swine fever caused significant damage to most of the VNP populations. Therefore, our findings will help a reconstruction scheme of the VNP genetic resources.

In the present study, we propose an alternative technique called cytoplast fusion to improve the maturation rate and developmental competence of growing oocytes collected from early antral follicles in pigs. We examined whether the fusion of a growing oocyte with the cytoplast from a fully-grown oocyte (CFR group) could better promote maturation and developmental competence of the growing oocyte compared to germinal vesicle (GV) transfer (GVTR group). After 44 h of in vitro maturation (IVM), most growing oocytes (GR group) were still arrested at the GV stage (64.0 ± 5.1%); this number was significantly higher (P < 0.01) than that of the other groups. No matured oocyte was observed in the GR group. The maturation rate of GVTR oocytes was significantly improved (18.8 ± 3.5%) compared with that of growing oocytes. The proportion of oocytes that reached the metaphase-II (M-II) stage in the CFR group (37.8 ± 2.0%) was significantly higher (P < 0.05) than that in the GVTR group, although still lower than that in the control group (75.2 ± 4.4%). No blastocyst was derived from growing oocytes. Among in vitro fertilized GVTR oocytes, 3.0 ± 1.9% developed into blastocysts; however, this percentage showed an insignificant increase compared with the GR group. On the other hand, the percentage of CFR embryos that developed into blastocysts (12.0 ± 4.3%) was significantly higher than that of GR embryos (0.0%), although still lower than that of control embryos (27.0 ± 5.5%). Total cell number in blastocysts in the GVTR group (23.3 ± 6.9) was significantly lower (P < 0.05) than that in the control group (50.4 ± 5.0). Meanwhile, the total cell number in blastocysts derived from CFR oocytes (36.3 ± 4.8) was comparable to that of the control group. In summary, cytoplast fusion significantly improves maturation rate and developmental competence of growing oocytes compared with GV transfer.

The Critically Endangered tamaraw Bubalus mindorensis is endemic to Mindoro Island, Philippines, and little is known of its ecology. During 2006-2011 we used community-based monitoring to examine the population status and ecology of tamaraw in the species' core habitat of Mount Iglit-Baco National Park. Each year, for 5 consecutive days at the end of the dry season, trained local volunteers and rangers or project staff were allocated to 18 vantage points in the study area (c. 160 km(2)). Tamaraw were categorized as adult (> 5 years), juvenile (2-5 years) or calf (< 2 years), and sexed when possible. During the study period the population was 239-314 (mean 271), with no significant fluctuations in age structure (percentage of adults, juveniles and calves: 57.8, 21.0 and 21.3%) or estimated adult female reproductive rates (29.1%). In adults, but not in juveniles, the sex ratio was biased towards females (1 : 1.86, P < 0.01). Bulls were often solitary (32.2% of sightings), whereas the majority of cows (94.7%) formed small groups of 2-12 individuals of different ages, with or without bulls (53.4 and 46.6%, respectively). These results demonstrate that the population remained relatively stable, maintaining a constant age structure and reproductive rate, and that long-term community-based monitoring was effective for quantitative characterization of the tamaraw's social behaviour, which is critical for conservation and management of the species.

Fecal DNA analysis is a useful tool for the investigation of endangered species. Tamaraw (Bubalus mindorensis) is endemic to the Philippine island of Mindoro but knowledge of its genetic and ecological information is limited. In this study, we developed a species identification method for tamaraw by fecal DNA analysis. Eighteen feces presumed to be from tamaraw were collected in Mount Iglit-Baco National Park and species-known feces from domestic buffaloes and cattle were obtained from a farm. Additionally, one species-unknown fecal sample was obtained in Mount Aruyan Preserve, where the sighting of tamaraw has not been reported in recent years. Based on DNA sequence data previously reported, the genus Bubalus- and tamaraw-specific primers for PCR of cytochrome b gene were newly designed. The Bubalus-specific primer yielded a 976 bp fragment of cytochrome b for all fecal samples from tamaraw and domestic buffaloes, but not for cattle, whereas the tamaraw-specific primer yielded a 582 bp fragment for all tamaraw fecal samples and for one of the four domestic buffalo samples. PCR-RFLP (restriction fragment length polymorphism) analysis of the 976 bp PCR fragment with AvrII or BsaXI provided distinct differences between tamaraw and domestic buffalo. PCR-RFLP analysis also showed that the species-unknown sample obtained in Mount Aruyan Preserve, originates from tamaraw.

Tamaraw (Bubalus mindorensis) is a wild water buffalo endemic to the island of Mindoro, Philippines. It is one of the world&rsquo;s critically endangered species, and only three mountainous areas have confirmed tamaraw populations. One of the habitats, Mt. Aruyan, is inhabited by hundreds number of indigenous peoples (Mangyan) who live in clusters of villages. To understand the present status of the tamaraw in Mt. Aruyan, especially human-tamaraw conflicts, we conducted a field survey by camera trap, route censuses, and interviews with the Mangyan. One adult male was identified by camera trap and some individuals were identified by signs. Tamaraw habitat and the domiciles of the Mangyan completely overlap, and slash-and-burn farming by the Mangyan has significantly reduced tamaraw habitat. Moreover, outsiders poaching with guns directly contributed to the decline of tamaraw population. These results indicate that the conservation of tamaraw in the Mt. Aruyan habitat is extremely difficult. Therefore, conservation in other habitats is considered a higher priority.

The tamaraw (Bubalus mindorensis) is a wild buffalo endemic to Island of Mindoro, Philippines and one of the world's critically endangered animals. In the early 1900s the tamaraw could be seen all over the island of Mindoro, but its distribution is now restricted to only three protected areas, with a total estimated population of about 250. The main reasons for its population decline have been unrelenting habitat destruction and illegal hunting. Recent field surveys of the tamaraw have revealed that survival of the tamaraw in the Aruyan Preserve is critical, and the subpopulation in the Mount Iglit-Baco National Park seems to be the only core herd that can be subjected to a practical conservation program. Identification of the tamaraw by fecal DNA analysis would be useful in further studies to verify the animal's ecological behavior, including its existence, range, population dynamics, and genetic diversity.

Tamaraw, an endemic species on the Philippine island of Mindoro, is a critically endangered animal listed by IUCN. Although the population size of tamaraw has been monitored by the Department of Environment and Natural Resources of the Philippines annually from 1999, there is no academic report on the wild tamaraw population. Therefore, we investigated the present tamaraw population size and herd behavior in their natural habitat. The study area covered about 4,000 ha of natural grassland located in Mts. Iglit-Baco National Park, with 16 strategically located observation sites. The tamaraw population was counted using the Intensive Concentration Count Method or Simultaneous Multi Vantage Point Counts for five consecutive days in April, 2006. Fresh fecal samples of tamaraws were also collected from seven observation sites to determine prevalence of endoparasites. A total of 263 individuals were observed, consisting of 162 adult (62%), 52 juveniles (estimated ages of 1-4 years: 20%) and 49 yearlings (19%). Out of the 263 individuals, 29 (11%) were observed solitary, of which the majority were adult males (15/29). On the other hand, the remaining 234 tamaraws formed 71 groups consisting of 2-7 head per group. Out of 65 groups successfully sexed for adult animals, 63 (93%) had one or two adult cows with or without calves and 36 (55%) had one adult bull. From 15 fresh fecal samples, Coccidia eggs were detected in 10 cases and Fasciola eggs in 3 cases. These results demonstrated that 1) the tamaraw population is still in the critical ranges and there is a considerable deviation in the sex ratio of adult animals and in the proportion of juveniles to yearlings, 2) the tamaraw usually form families consisting of one bull with one or two cows, with the consequence of some solitary bulls, and 3) further analysis is required on the prevalence of endoparasites in the tamaraw.