This study aims to investigate the histological changes, sperm parameters, and their impact on embryo development rates and offspring numbers in advanced-age male repro57 heterozygous mice, corresponding to approximately 40 years of age in humans. Sperm parameters were assessed in both young and advanced-age repro57 heterozygous mice, as well as in young and advanced-age wild-type mice. Additionally, testis weight and histological analysis of seminiferous tubules were conducted to identify degenerative changes. Male mice from each group were mated with young wild-type females to compare offspring numbers, and in vitro fertilization (IVF) was used to evaluate fertilization and blastocyst formation rates. No significant differences in sperm concentration and motility were observed between young and aged wild-type mice or between young wild-type and young repro57 heterozygous mice. However, advanced-age repro57 heterozygous mice exhibited significantly lower sperm parameters and testis weight compared to advanced-age wild-type mice. Histological analysis revealed increased Sertoli cell vacuolation in the seminiferous tubules of advanced-age repro57 heterozygous mice. Additionally, these advanced-age mice exhibited significantly lower blastocyst formation rates and produced fewer offspring compared to advanced-age wild-type mice. Advanced reproductive aging in repro57 heterozygous male mice is associated with marked senescence-like degenerative changes, leading to a decline in offspring numbers, attributed to increased Sertoli cell vacuolation and diminished sperm quality.

Mutations in the bovine myostatin (MSTN) gene have been identified as the causative factor for the double-muscling phenotype in several European cattle breeds, including Belgian Blue, Piedmontese, and Shorthorn. In Japan, following the Meiji Restoration, several European breeds, including Shorthorn, Brown Swiss, Devon, Simmental, and Ayrshire, were introduced and crossbred with native cattle to develop modern Japanese beef cattle breeds, such as Japanese Black cattle. Historical records regarding the breeding of Japanese Black cattle indicate that the double-muscling phenotype, referred to as "Butajiri," occasionally appeared in Japanese Black cattle population. These historical observations suggest the potential presence of MSTN gene mutation in the Japanese Black cattle population. The aim of this study was, therefore, to investigate the presence of MSTN gene mutation in the current Japanese Black cattle population. Through screening 400 reproductive females, we identified one cow carrying an 11-bp deletion in the MSTN gene. While further investigation of the animals in the pedigree of this cow could not reveal any living animals with this mutation, this is the first report demonstrating the presence of the MSTN mutation in the Japanese Black cattle population.

STUDY QUESTION:At which arrest stage can spermatocytes be rescued by injection into meiotic oocytes? SUMMARY ANSWER:In mice, spermatocytes arrested at the diplotene stage, but not at the pachytene stage, can resume meiosis within immature oocytes and support full-term embryonic development. WHAT IS KNOWN ALREADY:In mice, at least some of the spermatocyte arrest mutations can be overcome by injecting spermatocytes into immature oocytes. STUDY DESIGN SIZE DURATION:The study was carried out from October 2019 to April 2025. Adult azoospermic mice (at 4-26 weeks of age) from nine strains carrying spermatocyte arrest mutations were used as spermatocyte donors. Adult B6D2F1 females at 9-12 weeks of age were used as oocyte donors for spermatocyte injection. Adult ICR strain pseudopregnant females at 9-12 weeks of age were used as recipients for embryo transfer experiments. PARTICIPANTS/MATERIALS SETTING METHODS:The most advanced stage of spermatocytes from each mutant strain was assessed by chromosome spread analysis. These most advanced spermatocytes of each strain were injected into metaphase I (MI) oocytes. About half a volume of the ooplasm had been removed from the recipient oocytes to ensure more stable chromosome behaviours during meiosis. The spermatocyte-injected oocytes were allowed to mature in vitro to the metaphase II (MII) stage, and their ooplasm was refreshed with the ooplasm from intact MII oocytes. After activation with SrCl2, the reconstructed oocytes that reached the 2-cell stage were transferred into the oviducts of pseudopregnant females. On Day 19.5, recipient females were euthanized and their uteri were examined for live foetuses. MAIN RESULTS AND THE ROLE OF CHANCE:Based on spermatocyte spread analysis, sperm mutants were categorized into three classes: Class 1, arrest at mid-diplotene or later stage; Class 2, arrest at early diplotene stage; and Class 3, arrest at pachytene stage. All four Class 1 mutants could resume normal meiosis following injection into MI oocytes, as evidenced by births of normal offspring. Similarly, one of two Class 2 mutants could be rescued, but the other could not. By contrast, three Class 3 mutants did not support embryo development to term because of complete implantation failure, indicating that reconstructed embryos carried severe chromosomal aberrations. LARGE-SCALE DATA:N/A. LIMITATIONS REASONS FOR CAUTION:The number of mutant strains examined was limited. Nevertheless, the findings were consistent: the more advanced the arrest stage of spermatocytes, the higher the likelihood of a successful rescue. WIDER IMPLICATIONS OF THE FINDINGS:In humans, a considerable proportion of spermatogenic arrest occurs at the primary spermatocyte stage. Spermatocyte injection might be an option to treat human male-factor infertility due to azoospermia in the future. However, numerous ethical and technical challenges remain to be addressed, and the reproductive physiological differences between mice and humans must be carefully taken into account. STUDY FUNDING/COMPETING INTERESTS:This study was supported by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science to A.O. (grant number: JP19H05758), K.I. (grant number: 23H04956), M.I. (grant number: JP23K20043), and N.O. (grant number: 25H01372), and 2023 and 2025 grants of the University of Castilla-La Mancha for stays in foreign universities and research centres to E.C.-E. The authors declare that they have no conflicts of interest.

We evaluated metabolic abnormalities in six neonatal Noma foals (Nos. 54-57, 62, and 66) that died shortly after birth, using laboratory tests, pathological examinations, serum amino acid (AA) analyses, gas chromatography/mass spectrometry (GC/MS), and genetic analyses. Nonspecific clinical symptoms, such as poor suckling and weakness, were commonly observed at birth. Sepsis caused by various bacterial infections was detected in foal Nos. 54, 62, and 66, while a heart malformation was identified in foal No. 57. Laboratory tests showed high aspartate transaminase, lactate dehydrogenase, and creatine kinase levels and low globulin and glucose levels in dead foals. The AA and GC/MS analyses revealed elevated levels of ammonia, orotic acid, and uracil in foal Nos. 54 and 55, while citrulline, arginine, and ornithine levels were low or within normal ranges, suggesting accelerated pyrimidine synthesis and suppressed urea cycle activity. Foal No. 56 had high uric acid and tyrosine levels, hypoglycemia, and liver dysfunction, suggesting glycogen storage disease. In foal No. 57, hypertyrosinemia was suggested because of high phenylalanine and tyrosine levels. We conducted a sequencing analysis of the ornithine transcarbamylase, argininosuccinatelyase, argininosuccinate synthase 1, uridine monophosphate synthase, G6PC1, and G6PT1/SLC37A4 genes associated with metabolic disorders. However, no mutations were detected. In conclusion, although metabolic pathways abnormalities resembling certain hereditary metabolic disorders were observed in neonatal foals that died in Noma horses, no specific mutations were identified in candidate genes, making hereditary disorders less likely.

This study aims to investigate the role of the RNASE9 gene mutation (c.1 + 1G > A) in male infertility, specifically focusing on its impact on sperm motility in patients with asthenozoospermia. Despite known genetic causes for azoospermia, the genetic basis of asthenozoospermia remains poorly understood. Given that RNASE9 is specifically expressed in the epididymis, where sperm maturation occurs, we hypothesized that mutations in RNASE9 might influence post-testicular sperm maturation. Whole-exome sequencing (WES) was performed on 20 Japanese patients with abnormal semen parameters to identify high-impact genetic variants. Variants were filtered based on allele frequencies in the 1000 Genomes Phase 3 East Asian (EAS) population and the Japanese 38KJPN database. RNASE9 was selected for further analysis, and the presence of the c.1 + 1G > A variant was confirmed in a cohort of 71 patients via Sanger sequencing. Sperm motility and other semen parameters were evaluated, and allele frequencies in asthenozoospermic, azoospermic, and normospermic groups were compared. The allele frequency of the RNASE9 (c.1 + 1G > A) variant was significantly higher in asthenozoospermic patients than in individuals with normal semen parameters (p = 0.022), while no significant difference was found in azoospermic patients. This suggests that the RNASE9 variant primarily affects sperm motility rather than sperm production. The RNASE9 (c.1 + 1G > A) mutation may contribute to reduced sperm motility in humans, likely impacting post-testicular sperm maturation. The findings provide new insights into the genetic factors affecting sperm motility, particularly in asthenozoospermia.

The SURF1 gene encodes a mitochondrial protein critical for cytochrome c oxidase (COX) assembly. The c.604G>C (p.D202H) variant, identified in the homozygous state, has conflicting pathogenicity classifications in ClinVar and was found in 6.7 % of individuals with recurrent ART failure or pregnancy loss. This study included 30 Japanese women with infertility, 25 with recurrent ART failure and 5 with recurrent pregnancy loss, and 23 Japanese women with normal fertility, including 12 with successful ART outcomes and 11 with natural conception histories. The median ages of the infertility and control groups were 41.5 and 36 years, respectively. Participants were recruited from Hanabusa Women's Clinic and Okayama University Hospital. Whole-exome sequencing (WES) was performed using DNA extracted from peripheral blood mononuclear cells. Variant prioritization focused on 1136 mitochondrial-related nuclear genes listed in the MitoCarta3.0 database. Analysis was restricted to homozygous missense variants absent in the control group and with minor allele frequency < 0.05 in Japanese databases. SURF1 (p.D202H) was significantly enriched in the infertility group compared to population databases. Structural modeling with AlphaFold2 and ChimeraX revealed local hydrogen bonding alterations caused by the substitution of aspartate with histidine at position 202. These findings suggest that while p.D202H is not pathogenic for Leigh syndrome, it may contribute to mitochondrial dysfunction in reproductive tissues. Further investigation into its tissue-specific effects is warranted.

In brief Repro57 mice, bearing an Rnf212 gene mutation, exhibit infertility in both homozygous mutant males and females, revealing arrested spermatogenesis in males and investigating unclear mechanisms in females. The study highlights aneuploidy and altered kinetochore patterns in repro57 homozygous mutant oocytes, which impact later stages of embryo development. Abstract Repro57 mice, induced with N-ethyl-N-nitrosourea and harboring a mutation in the Rnf212 gene, exhibit infertility in both homozygous mutant males and females. Rnf212 plays a crucial role in recombination and crossover designation. In male repro57 homozygous mutants, spermatocytes often degenerate during late prophase, and mature spermatozoa are absent in the seminiferous epithelium, indicating arrested spermatogenesis as the cause of infertility. Despite reports of infertility in Rnf212-knockout female mice, the specific mechanisms underlying infertility in female repro57 homozygous mutants remain elusive. This study investigates the chromosomal and kinetochore patterns of mature oocytes and their developmental potential following in vitro fertilization in female repro57 homozygous mutant mice. While all wild-type oocytes progress to metaphase II and exhibit euploidy, all repro57 homozygous mutant mouse oocytes display aneuploidy. Additionally, kinetochore distances in repro57 homozygous mutant oocytes exceed those observed in wild-type counterparts. Although no significant differences are noted in fertilization and early embryo development rates between wild-type and repro57 homozygous mutant mice, embryos derived from repro57 homozygous mutants exhibit significantly lower morula and blastocyst rates, accompanied by frequent cytokinesis failure and vacuole formation. These findings suggest that the premature segregation of sister chromatids in repro57 homozygous mutant mice adversely impacts the later stages of embryo development.

Plasma or serum amino acids are used to evaluate nutritional status and metabolic disorders. In this study, we aimed to set reference values of serum amino acid concentrations in the Noma horse, a Japanese native horse. Thirty-one horses were classified into six age groups: neonatal foal (0-4 days), foal (0.5-1 years), youth (5 years), middle age (10 years), old (15 years), and extra-old (>20 years). Horses >5 years of age were analyzed together as the adult group. In the adult horses, there were no significant differences among the serum amino acid concentrations of each age group. The foal group had higher concentrations of alanine, aspartic acid, glutamic acid, α-aminoadipic acid, and 3-methyl-histidine than the adult group. The neonatal foal group had higher serum concentrations of phenylalanine, lysine, alanine, proline, aspartic acid, glutamic acid, β-alanine, and β-amino-iso-butyric acid and lower tryptophan concentrations and Fischer's ratios than the adult group. The neonatal foal group had higher β-amino-iso-butyric acid concentrations and lower tryptophan and 3-methyl-histidine concentrations than the foal group. Therefore, reference values might be set separately in neonatal foals, foals, and adult horses. The data for the serum amino acid concentrations can be used for health care through physiological and pathological evaluations in Noma horses.

Context Conception rate, which is an important parameter to evaluate female fertility, has been gradually decreasing in Japanese Black cattle during the past decades. Meiosis is an essential biological process in gamete formation and meiotic failure could be a cause of infertility or reduced fertility in mammals. Of note, single nucleotide polymorphisms (SNPs) in the MLH3 gene involved in meiotic recombination were reported to affect the genome-wide recombination rate of meiosis in cattle.Aims The present study was conducted to investigate the association of SNPs in the MLH3 gene with conception rate in Japanese Black cattle.Methods On the basis of the reproductive data of 2045 Japanese Black cattle born from 1990 to 2009, we selected two groups of the reproductive females with high conception rate (n = 103) and low conception rate (n = 109). Then, we genotyped the SNPs in MLH3 gene in both reproductive groups by sequencing and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The significant association of SNPs with conception rate in Japanese Black cattle was estimated by Fisher's exact test, by using R commander.Key results We found the presence of five SNPs of MLH3 gene in Japanese Black cattle, including non-synonymous variants of MLH3 N408S that has been reported to be involved in meiotic recombination rate and MLH3 S591G newly identified in the present study. Comparison of genotype distributions and allele frequencies of the MLH3 N408S between high and low conception-rate groups indicated a significant difference between these groups, suggesting that this SNP is associated with conception rate. However, there is no significant difference between MLH3 S591G with conception rate in Japanese Black cattle.Conclusions We found a significant association between a non-synonymous variant of MLH3 N408S and conception rate, suggesting that this SNP can be a potential marker for selecting the Japanese Black cattle for improving fertility. We also identified a novel non-synonymous variant of MLH3 S591G in Japanese Black cattle.Implications These findings will be informative for future marker-assisted selection to improve the fertility of Japanese Black cattle. This is the first report of a genetic variant implicated in meiotic recombination being linked to female fertility in cattle.

The Y chromosome carries information about the demography of paternal lineages, and thus, can prove invaluable for retracing both the evolutionary trajectory of wild animals and the breeding history of domesticates. In horses, the Y chromosome shows a limited, but highly informative, sequence diversity, supporting the increasing breeding influence of Oriental lineages during the last 1500 years. Here, we augment the primary horse Y-phylogeny, which is currently mainly based on modern horse breeds of economic interest, with haplotypes (HT) segregating in remote horse populations around the world. We analyze target enriched sequencing data of 5 Mb of the Y chromosome from 76 domestic males, together with 89 whole genome sequenced domestic males and five Przewalski’s horses from previous studies. The resulting phylogeny comprises 153 HTs defined by 2966 variants and offers unprecedented resolution into the history of horse paternal lineages. It reveals the presence of a remarkable number of previously unknown haplogroups in Mongolian horses and insular populations. Phylogenetic placement of HTs retrieved from 163 archaeological specimens further indicates that most of the present-day Y-chromosomal variation evolved after the domestication process that started around 4200 years ago in the Western Eurasian steppes. Our comprehensive phylogeny significantly reduces ascertainment bias and constitutes a robust evolutionary framework for analyzing horse population dynamics and diversity.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leucosis. Our previous study showed the BLV existence in cattle kept in the Red River Delta Region of Vietnam. However, no positive samples were identified in beef cattle. Besides, information related to the BLV circulation in the remained parts of Vietnam is limited. Therefore, we tested the existence of BLV in 48 beef cattle kept in the Central Coast Regions. Nested PCR targeting the BLV-env-gp51 confirmed the prevalence of 14.6% in investigated regions. Phylogenetic analysis suggested the co-existence of genotypes 1 and 10. The close relationship between strains found in Vietnam, Thailand, Myanmar, and China was revealed suggesting the possibility of BLV transmission through the movement of live cattle.

Microinjection of spermatozoa or spermatids into oocytes is a major choice for infertility treatment. However, the use of premeiotic spermatocytes has never been considered because of its technical problems. Here, we show that the efficiency of spermatocyte injection in mice can be improved greatly by reducing the size of the recipient oocytes. Live imaging showed that the underlying mechanism involves reduced premature separation of the spermatocyte's meiotic chromosomes, which produced much greater (19% vs. 1%) birth rates in smaller oocytes. Application of this technique to spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans, resulted in the production of live offspring. Thus, the microinjection of primary spermatocytes into oocytes may be a potential treatment for overcoming a form of nonobstructive azoospermia caused by meiotic failure.

The complex geographic and temporal origins of chicken domestication have attracted wide interest in molecular phylogeny and phylogeographic studies as they continue to be debated up to this day. In particular, the population dynamics and lineage-specific divergence time estimates of chickens in Southeast Asia (SEA) and the Pacific region are not well studied. Here, we analyzed 519 complete mitochondrial DNA control region sequences and identified 133 haplotypes with 70 variable sites. We documented 82.7% geographically unique haplotypes distributed across major haplogroups except for haplogroup C, suggesting high polymorphism among studied individuals. Mainland SEA (MSEA) chickens have higher overall genetic diversity than island SEA (ISEA) chickens. Phylogenetic trees and median-joining network revealed evidence of a new divergent matrilineage (i.e., haplogroup V) as a sister-clade of haplogroup C. The maximum clade credibility tree estimated the earlier coalescence age of ancestral D-lineage (i.e., sub-haplogroup D2) of continental chickens (3.7 kya; 95% HPD 1985–4835 years) while island populations diverged later at 2.1 kya (95% HPD 1467–2815 years). This evidence of earlier coalescence age of haplogroup D ancestral matriline exemplified dispersal patterns to the ISEA, and thereafter the island clade diversified as a distinct group.

Several studies have reported the gene polymorphisms associated with high-altitude adaptation in goats. The FGF5 gene is a regulator in the hair-growth and a SNP c.-253G>A located within 5'UTR has been reported to cause long-haired phenotype. The EPAS1 gene is a transcription factor for various genes that have hypoxia-adaptive functions and a nonsynonymous SNP (Q579L) located in exon 5 has been reported to be associated with the mean corpuscular hemoglobin concentration. Nepal has large difference in altitudes in the north-south direction and four indigenous goat breeds are bred depending on the altitude. We used a total of 130 animals in Nepal, Chyangra (n = 37), Sinhal (n = 24), Khari (n = 33), and Terai (n = 36), and genotyped these two gene polymorphisms to compare the gene frequencies among the breeds and investigate the associations between breeding altitudes and allele frequencies. The genotyping results revealed that the mutant allele frequency in both polymorphisms tended to increase, as the breeding altitude of each population increased. In addition, correlation coefficients showed a relatively strong positive correlation between the breeding altitude and the mutant allele frequencies (r = 0.87 in FGF5 and r = 0.68 in EPAS1). These results suggested that both polymorphisms would significantly contribute to the high-altitude adaptation in Nepalese goat breeds.

Objective The biological importance for the signaling of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) has been recognized. However, the details remain unclear and are debatable. The Npr2 is a gene of NPR-B, and we previously reported a unique phenotype of a spontaneous mutant mouse lacking Npr2 (Npr2(slw/slw)), such as severe ileus-like disorder with bloodless blood vessels. In this study, we analyzed the bloodless mesenteric vascular morphology of Npr2(slw/slw) by histological observation to clarify the effects of the CNP/NPR-B signal deficiency. Results Blood vessels in the mesentery were clearly dilated in the preweaning Npr2(slw/slw) mice. Additionally, in the Npr2(slw/slw) mice, the lacteals were partially dilation or randomly direction mucosal epithelial cells in villi, and mesenteric adipocytes were undeveloped. These findings provide important information for understanding the role of CNP/NPR-B signals on intestine with mesentery.

al . 2012; Kadri et al . 2016; Kong et. al . 2008), and Kong et al . (2004) also reported that the genome-wide recombination rate correlate positively with reproductive success of females in human. RNF212 (ring finger protein 212) acts as a putative regulator of crossover (Reynolds et al . 2013; Qiao et al . 2014). The studies on mice and human showed that mutations of Rnf212/RNF212 caused the diminishing of crossover and sterility in both sexes (Reynolds et al . 2013; Fujiwara et al . 2015; Riera-Escamilla et al . 2019). Sandor et al . (2012) and Kong et al . (2008) reported that genetic varianst of RNF212 showed significant association with genome-wide recombination rate in cattle and human, respectively. These lines of evidences suggested that the variants of RNF212 could associate with female fertility via

While the large volume of mammalian oocytes is necessary for embryo development, it can lead to error-prone chromosomal segregation during meiosis. Conversely, we hypothesized that smaller oocytes would have a great unidentified potential to stabilize unstable meiosis and improve the development of the resultant embryos. Here, we show that reducing ooplasmic volume can rescue highly error-prone fertilization using primary spermatocytes by preventing segregation errors of chromosomes during biparental meiosis. High-resolution live-imaging analysis revealed that erroneous chromosome segregation occurred in most (90%) spermatocyte-injected oocytes of normal size, but could be ameliorated to 40% in halved oocytes. The birth rate improved remarkably from 1% to 19% (P < 0.0001). Importantly, this technique enabled the production of offspring from azoospermic mice with spermatocyte arrest caused by STX2 deficiency, an azoospermia factor also found in humans. Thus, contrary to popular opinion, oocytes inherently have a strong potential for precise meiotic divisions, which can be evoked by reduction of the ooplasmic volume. Their potential might help rescue cases of untreatable human azoospermia with spermatocyte arrest.