Developmental plasticity, or the ability of early embryonic cells to contribute to multiple lineages, is traditionally considered equal among sister blastomeres during early cleavage. However, divergence may occur earlier than expected. We performed single-cell RNA sequencing of bovine embryos from the 2- to 8-cell stages to examine transcriptional asymmetry. While gene expression was uniform at the 2-cell stage, variability increased at the 4-cell stage and became pronounced by the 8-cell stage. At this stage, blastomeres showed heterogeneity in MAPK pathway genes (e.g., RAC1, MAPK14) and the trophectoderm marker CDX2. These differences were associated with blastomere size; as larger blastomeres exhibited molecular and functional features associated with an increased propensity to generate trophectoderm cells. Thus, sister blastomeres exhibited progressive transcriptional divergence prior to compaction in bovine embryos, and these early differences may influence subsequent lineage trajectories.

5-Aminolevulinic acid (5-ALA) is a naturally occurring amino acid, which plays an important role in the biosynthesis of heme. We investigated its immunomodulatory effect as a potential bioactive feed additive by evaluating the expression of immune-related genes and oxidative status in dairy cows. Using a crossover design, six lactating Holstein cows randomized to control and treatment groups received a total mixed ration (TMR) or TMR plus 10 mg 5-ALA/kg of dry matter for 2 weeks. In vitro, peripheral blood mononuclear cells (PBMC) obtained from untreated dairy cows were incubated with 5-ALA in the presence of Escherichia coli lipopolysaccharide (LPS). Tumor necrosis factor-α (TNF-α) mRNA expression significantly decreased in PBMC of dairy cows fed 5-ALA, and interleukin-1β (IL-1β) tended to decrease. 5-ALA had no effect on the cluster of differentiation 3, interferon-γ, and Toll-like receptors in PBMC and 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity, relative superoxide dismutase activity, and thiobarbituric acid reactive substances in the blood, milk, and ruminal fluid of dairy cows. In vitro, TNF-α expression decreased in PBMC treated with 5-ALA (10 μg/mL) in the presence of LPS compared with LPS alone. Thus, 5-ALA may optimize the inflammatory response by controlling the expression of TNF-α in PBMC and eventually improving immunity in dairy cows.

Early mammalian embryos can develop normally despite containing abnormal cells. Here, we show that compaction functions as an early quality checkpoint in bovine embryos. Blastomeres excluded during compaction were enriched for extensive chromosomal abnormalities and nuclear defects, including micronuclei, binucleation, and anucleation. Transcriptomic analyses further revealed downregulation of mitochondrial and metabolic programs and developmental delay in excluded cells. Notably, embryos remained developmentally competent with limited blastomere exclusion, whereas extensive exclusion reduced pregnancy outcomes after embryo transfer. Together, these findings indicate that compaction evaluates multiple aspects of cellular fitness to refine embryo composition and preserve developmental integrity, redefining compaction as a surveillance phase rather than a purely morphogenetic event.

Doxorubicin (DOX), an anthracycline chemotherapeutic agent, induces significant male reproductive toxicity, however, its molecular mechanisms remain incompletely defined. Although oxidative stress is implicated in DOX-induced testicular injury, the roles of iron dysregulation and antioxidant suppression have not been fully characterized in the testis. We investigated time-dependent effects of DOX on testicular morphology, iron homeostasis, and oxidative stress, and evaluated the potential protective effects of (4-(methylthio)phenylthio) methane bisphosphonate (MPMBP), an iron-chelating and antioxidant compound. Male ICR mice received a single intraperitoneal injection of DOX (5 mg/kg). In Experiment 1, testes were collected at 24 h, 48 h, and 1-5 weeks to assess morphological changes, germ cell counts, transcriptional alterations in apoptosis-, antioxidant-, and iron metabolism-related genes, and iron deposition using electron probe microanalysis (EPMA). In Experiment 2, MPMBP (10 mg/kg) was administered either concomitantly with DOX or starting 5 days later, and testicular endpoints were evaluated at 5 weeks. DOX induced progressive testicular atrophy and depletion of early-stage germ cells. Apoptosis-related genes exhibited biphasic upregulation, accompanied by iron accumulation and downregulation of Gpx4 and mitochondrial ferritin (Ftmt). EPMA confirmed iron deposition within seminiferous tubules and interstitial regions. MPMBP co-administration was associated with partial preservation of testicular architecture, reduced iron accumulation, and modest restoration of Gpx4 and Ftmt expression, although these effects were incomplete under the present experimental conditions. These findings associate iron dysregulation and impaired antioxidant defense with DOX-induced testicular injury and support further preclinical investigation of iron-chelating and antioxidant strategies aimed at mitigating chemotherapy-associated reproductive toxicity.

Developmental plasticity, or the ability of early embryonic cells to contribute to multiple lineages, is traditionally considered equal among sister blastomeres during early cleavage. However, divergence may occur earlier than expected. We performed single-cell RNA sequencing of bovine embryos from the 2- to 8-cell stages to examine transcriptional asymmetry. While gene expression was uniform at the 2-cell stage, variability increased at the 4-cell stage and became pronounced by the 8-cell stage. At this stage, blastomeres showed heterogeneity in MAPK pathway genes (e.g., RAC1, MAPK14) and the trophectoderm marker CDX2. These differences were associated with blastomere size; larger blastomeres more frequently initiated cavity formation, a functional marker of trophectoderm fate. Our findings suggest that both molecular and physical asymmetries contribute to early lineage bias, and that developmental plasticity may be lost in an asynchronous, cell-specific manner before visible morphological events such as compaction.

In assisted reproduction, many factors in the culture environment, including light, temperature, pH, and culture media, can reduce preimplantation embryo viability. Laboratory glassware is also a known risk factor for in vitro embryos; however, the underlying mechanisms that disrupt embryonic development remain unclear. We identified Zn eluted from glassware as an embryotoxic substance. In mouse embryos, Zn induced delayed development, abnormalities in chromosome segregation, cytokinesis, zygotic gene activation (e.g. Zscan4a and murine endogenous retrovirus with leucine, also known as MERVL), and aberrantly upregulated developmental gene expression (e.g. Hoxa1, Hoxb9, T, and Fgf8) that could be mediated through metal regulatory transcription factors (e.g. Mtf1). Subsequently, Zn exposure led to significantly reduced blastocyst formation. Post-implantation, Zn-exposed embryos were associated with normal birth rates, however, the birth weight increased by an average of 18% compared with embryos cultured without Zn. Furthermore, Zn exposure affected the development of bovine and human embryos, with species-based variation in the strength and timing of these effects. To mitigate these embryotoxic effects, we identified a method to prevent glass toxicity using chelating agents. This research not only highlights the importance of risk control in embryo culture but also facilitates the development of safe and effective methods for assisted reproduction.

Antioxidants are free radical scavengers that increase oocyte quality and improve female fertility by suppressing oxidative stress. However, the related mechanisms remain unclear. The present study was designed to examine whether a reduction of oxidative stress from using the antioxidant sericin led to expanded cumulus cell (CC)-oocyte communication and oocyte developmental acquisition in a bovine model. We found that cumulus-oocyte complexes (COCs) matured in the presence of sericin showed a significantly increased oocyte meiotic maturation rate (P < 0.01) and accelerated subsequent blastocyst formation, as more blastocysts were found at the hatched stage (P < 0.05) compared to that in the control group. In contrast to the control group, sericin suppressed H2O2 levels in COCs, resulting in a markedly enhanced CC-oocyte gap junction communication index and number of transzonal projections, which were preserved until 18 h of oocyte maturation. These findings indicate that sericin reduces disruption of oocyte-follicular cell communication induced by oxidative stress. Sericin consistently increased intra-oocyte glutathione (GSH) levels and reduced oocyte H2O2 levels (P < 0.05), both of which were ablated when GSH synthesis was inhibited by buthionine sulfoximide (an inhibitor of GSH synthesis). Furthermore, the inhibition of GSH synthesis counteracted the positive effects of sericin on subsequent embryo developmental competence (P < 0.01). Intra-oocyte GSH levels were positively associated with blastocyst development and quality. These outcomes demonstrate new perspectives for the improvement of oocyte quality in assisted reproductive technology and may contribute to developing treatment strategies for infertility and cancer.

OBJECTIVE:Livestock droppings cause some environmental problems, but they have the potential to be used as effective biomass resources. The black soldier fly (BSF), Hermetia illucens (Diptera: Stratiomyidae), is suitable for efficiently processing such resources. By using BSF larvae for the disposal of livestock droppings, we can obtain two valuable products: protein resources and organic fertilizer. However, there is insufficient research on the digestive enzymes suitable for processing this waste. Here, we aimed to construct an efficient BSF processing system using livestock droppings, and we explored the digestive enzymes involved in this process. RESULTS:First, we investigated the characteristics of transcripts expressed in the midgut of BSF larvae and found that immune response-related genes were expressed in the midgut. Then, we investigated digestive enzymes and identified a novel serine protease, HiBrachyurin, whose mRNA was highly expressed in the posterior midgut when BSF larvae fed on horse droppings. Despite the low protein content of horse droppings, larvae that fed on horse droppings accumulated more protein than those in the other groups. Therefore, HiBrachyurin may contribute to digestibility in the early stage of protein degradation in BSF larvae fed on horse droppings.

Embryo culture is crucial to achieve successful outcomes in in vitro production-embryo transfer for cattle. This study explored the innovative use of dry incubators for bovine embryo culture, building on their advantages in human medicine, such as reduced contamination risk, stable temperature control, and lower gas consumption. In this study, we examined changes in osmotic pressure, the in vitro developmental potential of IVP embryos including the cleavage rate, blastocyst development rate, blastocyst diameter, and blastocyst cell number, morphokinetics, and the transcriptional profile of the blastocysts between humidified and dry incubators. Our research demonstrates the feasibility of this approach, showing that although the osmotic pressure gradually increases over the culture period (on day 8: 271.7 vs. 299.0, respectively; P = 0.09), it did not negatively affect the blastocyst formation rate (62.4 % vs. 69.8 %) and the morphological quality of blastocysts (diameter: 237.4 vs. 242.8, total cell number: 189.2 vs. 242.8). Embryos cultured in dry incubators exhibited morphokinetics comparable to those cultured in conventional humidified incubators. Furthermore, RNA-seq revealed that while a few genes showed changes, the transcriptomic profiles of blastocysts cultured in dry incubators were largely similar to those of blastocysts cultured in humidified incubators. These findings highlight the considerable potential of dry incubators for the in vitro production of bovine embryos.

Conventionally, bovine in vitro-produced (IVP) embryos for transfer are morphologically evaluated at day 7–8 of embryo culture, as recommended by the International Embryo Technology Society (IETS). However, this method is subjective, relying on the percentage of degenerated cells and developmental stage, leading to variability depending on the operator. In contrast, we have implemented a novel selection system for bovine IVP blastocysts using time-lapse monitoring in specially developed microwell culture dishes (LinKID micro25). This approach allows for continuous tracking of individual embryo development. Additionally, we have advanced live-cell imaging technology to observe nuclear and chromosomal dynamics during early embryogenesis, identifying prognostic factors indicative of viability post-transfer. Integrating these factors has significantly improved conception rates compared to conventional morphological evaluation. This review discusses morphokinetics relevant to viability and our innovative selection system, which enable accurate prediction of embryo viability after transfer in cattle.

Direct cleavage, a type of abnormal cleavage in which one zygote divides into three or more blastomeres, has been reported in mammals. The incidence of direct cleavage increases in zygotes with three or more pronuclei (multi-PN) and those showing abnormal pronuclei migration. However, there are few reports on the relationship between pronuclei and direct cleavage, and the effects of these relationships on subsequent embryogenesis have not been clarified. It is difficult to observe pronuclei under visible light, especially in bovine zygotes, because of abundant dark lipid droplets in the cytoplasm. We visualized pronuclei by removing lipid droplets from bovine zygotes and analyzed the relationship between the number of pronuclei and direct cleavage using time-lapse cinematography. The direct cleavage rate of multi-PN zygotes was 78.6%, which was significantly higher than that of zygotes with one pronucleus (1 PN, 0.0%) and two pronuclei (2 PN, 8.2%). Observation of pronuclei migration in 2 PN zygotes showed that 3.1% of 2 PN zygotes had non-apposed pronuclei. The direct cleavage rate of zygotes with non-apposed pronuclei was 66.7%, which was significantly higher than that of zygotes with apposed pronuclei (6.4%). Among multi-PN zygotes, the proportions of zygotes with apposed pronuclei and non-apposed pronuclei were 37.5% and 64.3%, respectively. The direct cleavage rate of multi-PN zygotes with non-apposed pronuclei was 100.0%, which was significantly higher than that of zygotes with apposed pronuclei (40.0%). Three-dimensional live-cell imaging of bovine zygotes injected with the mRNA-encoding histone H2B-mCherry showed that the direct cleavage rates of 2 PN and multi-PN zygotes bypassing syngamy were 63.2% and 75.5%, respectively. These rates were significantly higher than that of 2 PN and multi-PN zygotes that underwent syngamy (5.6% and 20.0%, respectively). Regardless of the number of pronuclei, a high frequency of direct cleavage was observed in zygotes in which the pronuclei did not migrate inward the cytoplasm and bypassed syngamy. These results suggest that abnormal fertilization such as multi-PN and migration error of pronuclei in cattle is the primary reason for direct cleavage during the first mitosis. Assessment of direct cleavage during the first mitosis allows exclusion of embryos with abnormal fertilization and may contribute to in vitro produced embryo transfer success.

In assisted reproductive technology (ART)-derived embryos of non-rodent mammals, including humans and cattle, chromosome segregation errors are highly likely to occur during early cleavage division, resulting in aneuploidy, including mosaicism. However, the relationship between chromosomal segregation errors during early cleavage and subsequent embryonic development has not been detailed in these mammals. In the present study, we developed non-invasive live-cell imaging of chromosome segregation dynamics using a histone H2B-mCherry mRNA probe in bovine preimplantation embryos. Chromosome segregation errors in early cleavage affected blastocyst formation. Especially, embryos that underwent abnormal chromosome segregation (ACS) with multiple or large micronucleus formation rarely developed into blastocysts. Embryos with the severe ACS had prolonged cell cycle duration. After transfer of blastocysts with live-cell imaging of chromosome segregation to ten cows, six became pregnant and four of them gave full-term offspring. Interestingly, two of them were derived from blastocysts with ACS. Hence, chromosomal segregation errors with micronucleus formation during early cleavage can be a fatal hallmark of preimplantation embryogenesis in cattle. This technique has shown potential for understanding the relationship between chromosome segregation error and subsequent embryo development, and for selecting viable ART-derived embryos for medical and livestock production.

Enzymes excreted by rumen microbiome facilitate the conversion of ingested plant materials into major nutrients (e.g., volatile fatty acids (VFA) and microbial proteins) required for animal growth. Diet, animal age, and health affect the structure of the rumen microbial community. Pathogenic organisms in the rumen negatively affect fermentation processes in favor of energy loss and animal deprivation of nutrients in ingested feed. Drawing from the ban on antibiotic use during the last decade, the livestock industry has been focused on increasing rumen microbial nutrient supply to ruminants through the use of natural supplements that are capable of promoting the activity of beneficial rumen microflora. Selenium (Se) is a trace mineral commonly used as a supplement to regulate animal metabolism. However, a clear understanding of its effects on rumen microbial composition and rumen fermentation is not available. This review summarized the available literature for the effects of Se on specific rumen microorganisms along with consequences for rumen fermentation and digestibility. Some positive effects on total VFA, the molar proportion of propionate, acetate to propionate ratio, ruminal NH3-N, pH, enzymatic activity, ruminal microbiome composition, and digestibility were recorded. Because Se nanoparticles (SeNPs) were more effective than other forms of Se, more studies are needed to compare the effectiveness of synthetic SeNPs and lactic acid bacteria enriched with sodium selenite as a biological source of SeNPs and probiotics. Future studies also need to evaluate the effect of dietary Se on methane emissions.

Chromosome instability leading to aneuploidy during early cleavage is well known in humans and cattle. Partial compaction (PC), which occurs only in some blastomeres, is suggested as a self-correction mechanism through which human embryos avoid aneuploid mosaicism. Partially compacted embryos show abnormal cleavages more frequently during early development; however, the mechanism by which blastomeres are excluded has not been elucidated. Here, we confirmed PC in approximately half of the tested bovine embryos, similar to that in human embryos. DNA sequencing of single-cell and intact embryos revealed that the morulae that excluded some blastomeres had euploidy, but many of the excluded blastomeres had aneuploidy. Time-lapse imaging of zygotes without the zona pellucida revealed that the excluded blastomeres underwent reverse and direct cleavages, which are abnormal cleavages, more frequently than the blastomeres involved in compaction. These results suggest the potential role of abnormal cleavage in the self-correction mechanism during the development of mammalian preimplantation embryos.

The morphokinetics of pronuclei (PN) are considered crucial factors affecting embryogenesis in mammals. Whereas, since bovine zygotes contain a large number of cytosolic lipid droplets, detailed observation of PN has not been performed. In this study, we visualized PN using time-lapse cinematography (TLC) with light microscopy for the first time in delipidated bovine zygotes. The proportions of 0 PN, 1PN, 2PN, and multi-PN in delipidated bovine zygotes were 10.1%, 6.5%, 72.7%, and 10.8%, respectively. Abnormal fertilization, including 1 PN and multi-PN, was observed in 15.6% of blastocysts. The times from IVF to PN appearance, PN fading, and first cleavage in 2 PN bovine zygotes that developed into blastocysts were 10.4, 25.5, and 27.6 h, respectively, which were similar to PN morphokinetics in humans. The 2 PN zygotes showed that the prolonged time from IVF to the appearance of PN and from the fading of PN to the first cleavage negatively affected blastocyst formation. The time from appearance to fading of PN in multi-PN zygotes that developed into blastocysts was longer than that in multi-PN zygotes that did not develop into blastocysts. Besides, among zygotes that developed into blastocysts, the time from appearance to fading of PN in multi-PN zygotes was longer than that in 2 PN and 1 PN zygotes. These results suggest that PN morphokinetic abnormalities are associated with subsequent embryonic development. Observation of PN in bovine zygotes by using non-invasive visible light TLC by delipidation could be a powerful tool to clarify the relationship between PN morphokinetics and developmental competence.

Wild large Japanese field mice (Apodemus speciosus) responses to cyclic seasonal changes are associated with physiological and behavioral changes. However, the detailed regulation of oogenesis in the ovary during the seasonal reproductive cycle in wild large Japanese field mice has not been studied. We assessed the dynamics and changes in ovarian morphology and hormone concentrations associated with reproductive seasonality throughout the year. The stages of the ovarian morphological breeding cycle of wild large Japanese field mice were classified as breeding, transition, and non-breeding periods during the annual reproductive cycle. Measurement of blood estradiol concentrations throughout the year showed that the levels in September and October were higher than those in other months. It is presumed that follicle development starts from a blood estradiol concentration of 38.4 ± 27.1 pg/mL, which marks a shift from the transitional season to the breeding season, followed by the transition to the non-breeding season at 26.1 ± 11.6 pg/mL. These results suggest that seasonal follicle development in wild rodents is correlated with estradiol regulation. We consider this species to be an alternative animal model for studying seasonal reproductive changes and the effects of environmental changes.

Efforts directed toward enhancing animals' productivity are focused on evaluating the effects of non-traditional feed additives that are safer than antibiotics, which have been banned because of their health hazards. Many studies used an amino acid that contributes to heme biosynthesis, known as 5-aminolevulinic acid (5-ALA), to promote the productivity of farm animals. However, these studies demonstrate inconsistent results. In order to develop a clear understanding of the effects of 5-ALA in farm animals, we comprehensively searched PubMed and Web of Science for studies evaluating 5-ALA effects on the performance, iron status, and immune response of different farm animals. The search retrieved 1369 publications, out of which 16 trials were relevant. The 5-ALA-relevant data and methodological attributes of these trials were extracted/evaluated by two independent researchers, based on a set of defined criteria. Samples were comprised of pigs, chickens, and dairy cows. The 5-ALA doses ranged from 2 mg to 1 g/kg of feed, and treatment duration ranged from 10 to 142 days. Overall, 5-ALA improved iron status in most studies and increased white blood cells count in 3 out of 10 studies, in addition to improving animals' cell-mediated immune response following immune stimulation with lipopolysaccharide. Inconsistent findings were reported for growth performance and egg production; however, a combination of 10 mg/kg of 5-ALA with 500 mg/kg of vitamin C promoted the highest egg production. In addition, 5-ALA improved milk protein concentration. In conclusion, 5-ALA can enhance farm animals' iron status and immune response; however, the heterogeneity of the reviewed studies limits the generalizability of the findings. Standard procedures and outcome measures are needed to confirm the benefits of 5-ALA. Attention should also be paid to any adverse effects.

The Fukushima nuclear power plant (FNPP) accident raised worldwide attention to the health risk of radiation exposure and to its potential transgenerational effects. Here, we analysed transgenerational effects on calf spermatogenesis and on blood plasma metabolome in order to detect alterations associated with paternal exposure to low-dose-rate (LDR) radiation. Sperm was collected from a bull exposed to radiation for 2 years abandoned in the ex-evacuation zone of the FNPP accident (the abandoned bull) and was used for artificial insemination (AI) into a non-radiocontaminated cow. Haematoxylin and eosin stained sections of the testis of a 13-month-old calf revealed spermatogonia, spermatocytes, spermatids, and sperm in normal morphology. Nuclear and acrosomal morphology of sperm was generally normal. Metabolomic profiling of plasma using capillary electrophoresis-mass spectrometry resulted in 104 peaks of candidate compounds suggestive of paternal exposure. A calf was delivered by AI using sperm from the abandoned bull. Regarding glycolysis, the contents of nucleotide sugars tended to be lower in the delivered calf than in the control calf. Among energy carries, AMP and ATP showed different tendency between non-radiocontaminated and delivered calf. In conclusion, there were no apparent transgenerational effects on both spermatogenesis and blood plasma metabolome in a calf obtained by AI using sperm from the abandoned bull exposed to LDR in the ex-evacuation zone of the FNPP accident for about 2 years.

The aim of this study was to investigate the proliferation and apoptosis of male germ cells during the seasonal reproductive cycle of the large Japanese field mice (Apodemus speciosus). Male mice residing in their natural habitat were captured in Niigata, Japan. Testis sections were stained with haematoxylin and eosin, and mitotic male germ cells were identified using immunofluorescence staining for proliferating cell nuclear antigen (PCNA). Apoptosis was analysed using terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay. The phases of spermatogenesis during the seasonal reproductive cycle were classified as active, transitional, and inactive based on the diameter of the seminiferous tubules. The number of PCNA-positive germ cells was less during the inactive than other phases. The percentage of TUNEL-positive germ cells per seminiferous tubule was greater during the inactive than active and transitional phases. Spermatogenesis during the seasonal reproductive cycle is controlled by proliferation and apoptosis in male germ cells. This species of undomesticated mice could be used as an animal model to study spermatogenesis as a valuable indicator of the effects of ecological and anthropogenic factors on animal reproduction.