Journal of Reproduction and Development最新文献

Reliable pre-implantation sex determination and genetic screening enables informed embryo transfer decisions in equine breeding while avoiding later interventions. We developed a streamlined workflow that couples rapid whole genome amplification (WGA) with a multiplex real-time PCR targeting ETSTY5 as a Y-specific marker and UBC as an autosomal control. On purified equine DNA, sex was correctly assigned down to 10 pg gDNA and to a single fibroblast cell. Direct testing of embryo biopsies without WGA yielded inconsistent results, whereas introducing a short WGA step produced tight allelic-discrimination clusters and 100% diagnostic calls, including in cloned embryos of known sex. The same WGA product supported targeted genotyping for inherited disease screening of hyperkalemic periodic paralysis (HYPP) and hereditary equine regional dermal asthenia (HERDA) alleles. This WGA plus real-time PCR pipeline supports robust and practical embryo sexing and targeted pre-implantation genetic diagnostics within in vitro produced (IVP) equine embryo and embryo transfer workflows.

In vitro experiments have become a powerful alternative to in vivo approaches for analyzing gene functions and molecular mechanisms. However, successful in vitro oogenesis using the C57BL/6 strain-widely used as a standard mouse model in mammalian studies-has not yet been reported. In this study, we established a 30-day culture system combining ovarian and follicular culture, and successfully produced fully competent oocytes from neonatal C57BL/6N ovaries, which contain only non-growing oocytes prior to primordial follicle formation. A key contributing factor to this success was the supplementation of dibutyryl cyclic AMP, a membrane-permeable analogue of endogenous cyclic AMP, which promoted follicular growth. This culture system provides a valuable tool for investigating the mechanisms underlying oogenesis.

N-Myristoyltransferase 1 (NMT1), the predominant enzyme catalyzing myristoylation of proteins, is involved in various biological processes, including early embryonic development, immune responses, apoptosis, cellular homeostasis, tumorigenesis, and infection, with therapeutic potential in viral and parasitic infections as well as cancer. Despite the critical functions of NMT1, there have been no reports to date regarding its role in reproduction, especially in spermatogenesis. To investigate the function of NMT1 in this context, we utilized CRISPR/Cas9 technology to create a germ cell-specific Nmt1 knockout mice model for the first time. Surprisingly, male mice lacking NMT1 maintained fertility, exhibiting normal testicular structure and sperm morphology, with no significant differences in spermatogenic tubule structure or germ cell distribution compared to wild-type mice. Additionally, the Nmt1^f/f; Stra8-Cre male mice showed no notable defects in meiosis. These findings suggest that NMT1 is not critical for spermatogenesis or male fertility in mice. However, further studies have shown that the compensatory role of NMT2 may play an unexpected role in maintaining myristoylation levels, which provides a new perspective for understanding the role of myristoylation in spermatogenesis.

Our previous study in hamsters indicated that progesterone (P) induces enhancement of spermatozoal hyperactivation, while 17β-estradiol (Eβ) and γ-aminobutyric acid (GABA) suppress this enhancement. Since P has also been shown to enhance hyperactivation of rat spermatozoa in another study conducted by our team, we examined the effects of Eβ and GABA on the P-enhanced hyperactivation of rat spermatozoa in the present study. Our results showed that Eβ suppressed P-enhanced hyperactivation in a dose-dependent manner through an estrogen receptor. In contrast, GABA did not affect P-enhanced hyperactivation. Instead, 5-500 fM GABA significantly enhanced spermatozoal hyperactivation, and this GABA-induced enhancement of hyperactivation was associated with a GABA_A receptor. In conclusion, P-enhanced hyperactivation of rat spermatozoa was suppressed by Eβ although it was not suppressed by GABA. Moreover, low concentrations of GABA enhanced rat spermatozoal hyperactivation.

Reduced glutathione (GSH), a thiol compound, plays an important role in protecting sperm from excessive levels of reactive oxygen species. Our previous study demonstrated that GSH supplementation was associated with improved in vitro fertilization (IVF) outcomes. However, GSH supplementation also increased the proportion of DNA-damaged sperm detected in the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, indicating involvement of its thiol components. In this study, we investigated the effects of several thiol compounds, including l-cysteine (Cys), N-acetyl-l-cysteine (NAC), and dithiothreitol (DTT), on bovine sperm DNA integrity. The addition of Cys or NAC (0.5, 1, or 5 mM) to the medium caused an increase in DNA damage, which was observed either throughout the sperm head or specifically in the post-acrosome (PA) region. DTT treatment increased DNA damage in both the PA and acrosomal regions, and it induced progressive sperm head lysis from the acrosome to the entire head. These treatments caused sperm DNA-damage rates that exceeded the normal range, suggesting that thiol groups enhance the detection of DNA damage. The effects of these thiols on IVF outcomes were further examined. Short-term exposure to 1 mM Cys before insemination improved cleavage rates, whereas prolonged exposure at concentrations ≥ 1 mM during insemination reduced cleavage rates. NAC supplementation had no significant effects. A mild negative correlation was observed between TUNEL-positive sperm rates and both cleavage and blastocyst formation rates following short-term treatment with 0.5 mM Cys. These findings suggest that thiol supplementation, when optimally dosed and timed, may enhance the sensitivity of DNA-damage detection rather than induce additional damage.

Improving and sustaining the health and productivity of dairy cows require integrated strategies that combine management and appropriate nutrition. This review adopts two complementary perspectives: optimizing periparturient management to support high productivity without compromising animal health and improving gestational nutrition to produce healthy replacement heifers. Several studies have examined how maternal nutritional and metabolic status before and after calving influence postpartum reproduction, productivity, placental development, and offspring health. Prepartum energy status is a key determinant of early postpartum ovarian function, with a timely first ovulation associated with adequate insulin-like growth factor-I exposure during follicular development. Insufficient β-carotene intake in energy-prioritized feeding systems adversely affects ovarian resumption. Uterine recovery is also nutrition-sensitive; cows with endometritis exhibit signs of impaired liver function, whereas delayed uterine involution is associated with inefficient ruminal nitrogen utilization. Supplementation with lysine, the first limiting amino acid in corn-based diets, further promotes uterine recovery. From a developmental perspective, maternal insulin resistance is associated with low birth weight and altered endocrine profiles in calves, which are indicative of long-term metabolic vulnerability. In contrast, lysine supplementation during the dry period supports neonatal protein synthesis and growth. Furthermore, in growing primiparous cows, greater milk yield during early pregnancy was associated with reduced anti-Müllerian hormone concentrations in female calves, suggesting a lower ovarian reserve. Collectively, these findings highlight the importance of gestational and peripartum nutritional management not only for improving dairy cow fertility and productivity but also for optimizing the health and reproductive potential of the next generation.

Artificial insemination (AI) is the predominant reproductive technology in Japanese cattle breeding, particularly for genetic improvement of dairy and beef cattle. This review outlines the historical development, current status, and recent advances in semen production technologies for AI in Japan and addresses the remaining challenges and future directions. The adoption of AI accelerated after the Act on Improvement and Increased Production of Livestock was implemented in 1950, with frozen semen replacing liquid semen by the mid-1960s. Advances in cryobiology and genomic selection have improved breeding efficiency; however, fertility issues persist. In dairy cattle, conception rates have declined primarily due to high milk yield, negative energy balance, and heat stress. In beef cattle, particularly Japanese Black cattle, conception rates remain stable overall, but subfertile sires still occur despite normal post-thaw semen quality. Studies on sperm motility, acrosomal integrity, and genomic variants have identified a single nucleotide polymorphism (SNP) marker linked to extreme subfertility in Japanese Black bulls. Japan has developed innovative technologies such as the two-layer semen straw (FCMax), which enhances post-thaw sperm function through in-straw dilution and supplementation. Field trials have suggested potential improvements in conception rates; however, large-scale validation studies are still ongoing. Furthermore, sexed semen technology has been widely adopted in Japan and achieved conception rates comparable to those of conventional semen. Emerging challenges include labor efficiency in large-scale farms, prompting interest in improved thawing protocols or liquid semen alternatives. Future priorities include integrating genomic tools, refining cryobiological techniques, and implementing practical innovations to sustain cattle reproduction in evolving production systems.

We examined the effect of niacin, a water-soluble vitamin essential for the metabolism of carbohydrates, lipids, and other substrates, on the developmental potential of oocytes grown in vitro, as well as the number and activity of mitochondria and reduced glutathione (GSH) content in oocytes. Porcine oocytes were cultured in medium containing 0-10 mM niacin for 12 days. Subsequently, in vitro maturation was performed, followed by in vitro fertilization using intracytoplasmic sperm injection, and the ability to develop into blastocysts was examined. Niacin treatment (10 mM) increased the mitochondrial, GSH, and glutamate-cysteine ligase catalytic subunit contents in porcine oocytes grown in vitro. Furthermore, niacin treatment enhanced male pronuclear formation and blastocyst development rates in porcine oocytes grown in vitro. Niacin increased the mitochondrial content and GSH levels in porcine oocytes and enhanced the ability of oocytes to form male pronuclei, which in turn promoted embryonic development.

This study aimed to improve estrus management in Japanese Black cows by investigating the effects of controlled internal drug release (CIDR) treatment on luteal function including luteolysis and estrous characteristics. Cows with normal estrous cycles were divided into three groups, and CIDR treatment was initiated on day 5 (Group 1), 10 (Group 2), or 16 (Group 3) of the estrous cycle and continued for 12 days. Plasma progesterone (P₄) levels, luteal diameter, and luteal blood flow (LBF) were monitored during treatment, and correlations among these parameters were analyzed. Additionally, the interval from CIDR removal to estrus, and the incidence of standing (ST) behavior during estrus were evaluated. All luteal parameters changed in a cycle-dependent manner, irrespective of CIDR treatment. Significant correlations were observed between P₄ levels and both luteal diameter and LBF. The interval from treatment withdrawal to estrus was significantly longer in Group 1 (4.6 ± 2.2 days) than in Groups 2 (2.1 ± 0.4 days) and 3 (2.2 ± 0.7 days). Silent heat, defined as estrus without ST behavior, occurred in 27.3% of cows in group 1 and 10.0% in Group 2, but not in Group 3. These findings suggest that initiating CIDR treatment in the early luteal stage and removing it just before or during luteolysis, causes variability in estrus timing after treatment and increases the risk of silent heat. When implementing estrus synchronization using CIDR in the field, both the timing of treatment initiation and removal should be carefully considered.

To efficiently produce high-quality bovine calves by transferring embryos obtained from in vitro fertilization (IVF), it is important to evaluate their ability to conceive and their ability to develop into normal litters. In this study, we aimed to clarify the effects of timing and morphology of blastomere cleavage on the gene expression status of bovine IVF embryos. Bovine IVF embryos were classified in four categories, which were divided according to the time of the first blastomere cleavage and the presence or absence of direct cleavage. In addition, the gene expression profiles of these embryos were examined. The timing and morphology of the blastomere cleavage was involved in pre-implantation development and gene expression status of bovine IVF embryos. Our results indicate the possibility of multiple evaluations for bovine IVF embryos and the selection of the most suitable embryos for embryo transfer.