Due to the strong demand for embryo production from young and genotyped superior animals using ovum-pick up (OPU) combined with in vitro fertilization (IVF), the number of in vitro-produced embryos has exceeded that of in vivo-derived embryos globally since 2016. One of the merits of OPU-IVF is that the administration of follicle-stimulating hormone (FSH) is not essential, while FSH treatment prior to OPU promotes oocyte developmental competence. Thus, investigations are needed to optimize OPU-IVF protocols with and without FSH. In addition, OPU enables oocyte collection from antral follicles in living animals. However, there are numerous immature oocytes in follicles at earlier stages, which are potentially destined to degenerate in ovaries. The technology used to foster acquisition of maturational and developmental competences in these immature oocytes is called in vitro growth (IVG). IVG is expected to contribute to assisted reproductive technologies for livestock, humans, and endangered species. However, no offspring from preantral follicles has been reported using IVG in animals other than in mice. Furthermore, IVG can be used to investigate factors affecting the fertility and developmental competence of oocytes by reconstituting follicle growth at each stage in vitro, which cannot be evaluated in vivo. Here, the technological progress of the optimization of immature bovine oocyte utilization is reviewed alongside findings from a variety of other ruminants.

In the future, human beings will surely expand into space. But given its unique risks, will humanity thrive in space environments? For example, when humans begin living and reproducing in space habitats or on other planets in the solar system, are there risks that future generations may suffer from adverse mutations induced by space radiation, or that embryos and fetuses will develop abnormally in gravitational environments that differ from that of Earth? Moreover, human expansion to other stellar systems requires that for each breed of animal, thousands of individuals must be transported to destination planets to prevent populations from experiencing inbreeding-related degeneration. In even more distant future, when humans have spread throughout the galaxy, all genetic resources on Earth, the planet where humans originated, must be permanently and safely stored— but is this even possible? Such issues with future space colonization may not be an urgent research priority, but research and technological development accompanying advancements in spaceflight will excite many people and contribute to technological improvements that can improve living standards in the present day (e.g., more effective treatments for infertility, etc.). This review will therefore focus primarily on issues related to mammalian reproduction in space environments.

Urovagina and purulent vaginal discharge (PVD) are usually diagnosed using a speculum, Metricheck device, or gloved hand. In periestrous dairy cows, a comparative study of these vaginal examinations for diagnosing urovagina or PVD has not yet been conducted. This study aimed to identify an effective vaginal examination method for periestrous dairy cows to ensure successful artificial insemination. Data were collected from 227 Holstein-Friesian cows during 300 occurrences of spontaneous estrus. Urovagina was evaluated using the speculum and gloved-hand methods and classified as mild, moderate, or severe. Vaginal discharge was evaluated using speculum, Metricheck, and gloved-hand methods and classified as vaginal discharge score (VDS) 0–5, with 2–5 defined as PVD-positive. Sensitivity and specificity of the gloved-hand versus speculum method in diagnosing urovagina, speculum versus Metricheck method, and gloved-hand versus Metricheck method in diagnosing PVD positivity were equivalent. The incidence of urovagina tended to be higher with the gloved-hand than with the speculum method. The incidence of PVD positivity tended to be higher with the gloved-hand than with the speculum and Metricheck methods. To analyze the correlation between pregnancy outcomes and mucus characteristics diagnosed using each method, logistic regression analysis was conducted, and the final models were compared. In this model, urovagina was selected as the explanatory variable and was associated with poor pregnancy. The results indicate that the gloved-hand method would be useful for managing fertility by detecting urovagina and PVD in periestrous dairy cows.

Artificial insemination (AI) in cattle involves introducing frozen-thawed sperm, a minimal amount of seminal plasma, and a significant volume of semen extender (SE) into the uterus. Previous studies have demonstrated that sperm interact with bovine endometrial epithelia via TLR 2/1, triggering a weak inflammatory response to clear the endometrium. This study investigated the impact of the major component of the insemination dose, egg yolk-based SE, on the uterine immune response in vitro. The results showed that SE did not affect sperm kinetic parameters or the entry of sperm into the uterine glands. SE alone significantly upregulated the mRNA expression of inflammatory cytokines (NFKB2, TNF, IL1B, CXCL8), TLR2/1, and the inflammasome NLRP3, while downregulating NOD1. Immunofluorescence analyses confirmed the upregulation of the strong inflammatory marker TNF alongside TLR2 and the downregulation of NOD1 in the uterine epithelium, similar to the effects observed with sperm. When combined with sperm, SE did not enhance the protein or mRNA expression of these markers, except for IL1B and CXCL8. In silico analyses revealed a strong affinity between triglycerides (the primary components of egg yolk) and TLR2/1, suggesting a potential role in stabilizing heterodimerization. These findings demonstrate that egg yolk-based SE, independent of sperm, triggers a mild physiological inflammatory response mediated by the TLR2/1 and NOD1 signaling pathways. The suppression of NOD1 by sperm and SE ensures a controlled and weak immune response in the uterus. Notably, despite the SE-induced inflammation, the sperm-uterine immune crosstalk was not disrupted, suggesting that SE does not negatively impact the physiological interactions between sperm and the uterus during AI.

The objective of this study was to examine the relationship between the insulin-like peptide 3 (INSL3) receptor (RXFP2) expression levels on spermatozoa and INSL3 concentrations in the seminal plasma of fresh semen from beef bulls with different levels of sperm morphological normality. Ejaculates (n = 44) were collected from 21 yearling Japanese Black beef bulls and categorized into three groups based on the levels of sperm morphological normality: High (normal morphology ≥ 80%; n = 23), Mid (< 80% & ≥ 65%; n = 10) and Low (< 65%; n = 11). Immunofluorescence was used to determine the localization and expression levels of RXFP2 in spermatozoa. Sperm RXFP2 was detected in the principal and equatorial segments of the acrosomal region, postacrosomal region, and neck in all groups. The levels of RXFP2 in the acrosomal principal segment, postacrosomal region, and neck in the Low group were significantly lower than those in the High and Mid groups, and those in the equatorial segment tended to be lower than those in the High group. The total level of RXFP2 in the Low group was also significantly reduced compared with that in the other two groups. Seminal plasma INSL3 concentrations were significantly higher in the Low group than in the other two groups, whereas testosterone levels did not differ significantly between the groups. In conclusion, RXFP2 levels were reduced in the sperm head and neck in bovine semen, with a lower level of sperm morphological normality, suggesting possible associations between increased sperm deformity and INSL3 receptor reduction. Higher seminal INSL3 concentrations in abnormal semen are probably related to fewer INSL3 receptors in spermatozoa.

Insulin-like growth factor 2 (IGF2) is essential for cell growth and differentiation and functions through the IGF2 receptor (IGF2R) to regulate embryonic and placental development. Exosomes that are synthesized and released from cells and play important roles in embryogenesis and placental development rely on the IGF2R for sorting and transport. However, the role of the imprinted Igf2-Igr2r axis and exosomes in the co-regulation of early placental development remains unknown. Cotyledon villi were collected from bovine placentas at different gestational ages, and the localization and expression of IGF2, IGF2R, and exosomal marker proteins were detected. Furthermore, the expression of exosomal marker factors was detected after the expression of IGF2R or IGF2 was inhibited through RNA interference or the addition of inhibitors, respectively. Our results demonstrated that IGF2, IGF2R, and the exosomal markers CD63, CD9, TSG101, and Rab11 are mainly located on the cell membrane of mononuclear trophoblast cells and binuclear trophoblast cells, which make up the cotyledon villi of the bovine placenta. The expressions of IGF2, IGF2R, and the exosomal marker proteins CD63, CD9, TSG101, and Rab11 showed a significant upward trend with increased gestation duration. Additionally, both Igf2r-knockdown and suppressing the expression of IGF2 with chromeceptin (IGF2 inhibitor) led to the downregulation of exosomal marker proteins in both bovine placental trophoblast cells (BTCs) and BTC-derived exosomes. Our study confirmed that the imprinted Igf2-Igf2r axis participates in the early development of cotyledon villi in the bovine placenta by manipulating exosome biogenesis, providing evidence for improving disorders during placental development.

Calcium release from the endoplasmic reticulum via sperm-derived phospholipase C zeta is crucial for oocyte activation during fertilization. Chloroquine (CQ) inhibits the increase in cytoplasmic calcium. This study investigated the effects of CQ on fertilization and oocyte activation. Oocytes were collected from ICR mice, and in vitro fertilization and artificial oocyte activation with strontium ions (Sr²⁺) and ethanol were performed in the presence of 50 µM CQ. Pronuclear formation was assessed via Hoechst33242 nuclear staining, cortical granule release was evaluated using lens culinaris agglutinin-fluorescein isothiocyanate staining, and cytosolic calcium levels were measured using fluorescence microscopy with Cal-520 AM. In the presence of CQ, no pronuclei were formed even 8 h after Sr²⁺-induced oocyte activation. Furthermore, cortical granule release in CQ-treated oocytes was significantly suppressed, although not completely inhibited, and no increase in cytosolic calcium was detected. CQ also inhibited pronuclear formation during ethanol-induced oocyte activation. In in vitro fertilization, although the fertilization rate was decreased in the CQ-treated group, in which CQ treatment was continuously applied during insemination, pronuclear formation and cortical granule release were observed. The decrease in the fertilization rate was likely attributable to reduced sperm motility and decreased penetration of the zona pellucida. The findings indicate that the oocyte activation pathways triggered by ethanol/Sr²⁺ and sperm are distinguishable by CQ, and that CQ can be used as a selective inhibitor of oocyte activation induced by Sr²⁺ or ethanol treatment.

To produce an embryo, a high conception rate must be complied along with four evaluation criteria based on the timing of early cleavage and proper embryo morphology (hereafter, these blastocysts will be referred to as “four-criteria-compliant blastocysts”). Therefore, it is necessary to construct a culture system for high efficiency production of embryos meeting these four criteria. Non-essential amino acids (NEAAs) are widely used for the culture of bovine embryos fertilized in vitro; however, the necessity and optimal concentration of individual NEAA must be verified to produce four-criteria-compliant blastocysts. DNA methylation is a critical event for blastocyst formation in bovines, and serine is a common NEAA that serves as a methyl donor and participates in DNA methylation. Serine is generally added at a concentration of 100 µM in bovine embryo culture medium. However, the rate of formation of four-criteria-compliant blastocysts was significantly improved when 1000 µM of serine was added. Analysis of endogenous serine synthases gene expression in oocytes and embryos revealed that phosphoglycerate dehydrogenase, the rate-limiting enzyme in the serine synthesis pathway, is expressed at the morula stage and beyond. The addition of serine at 1000 µM increased the amount of methyl donors; moreover, the addition of an inhibitor of serine-metabolizing enzymes decreased the number of methyl donors and markedly inhibited blastocyst formation. These results indicate that the addition of serine at an optimal concentration of 1000 µM favors production of four-criteria-compliant blastocysts, and that methyl donor synthesis may be involved in this effect.

Herein, we report a case of pregnancy of a female bottlenose dolphin (Tursiops truncatus) that was subjected to artificial insemination (AI) in water based on its estrous behavior using simple instruments. AI was performed on this female dolphin once or twice daily for 4 days at the detection of estrous behavior, such as floating horizontally and showing reduced responsiveness, likely indicating the appropriate timing for AI. The female was placed in supine a position in the water to position the genital slit above the water surface. A Nélaton catheter (Fr. 10, 40 cm length), with its tip modified, was inserted approximately 20 cm into the vagina through the genital slit, and 1–2 ml of fresh semen was injected. The AI procedure was performed within 1 min by two technicians. Thus, this AI method may be a new choice for artificial reproduction, as pregnancy success can be achieved with relatively less cost, less difficulty, and less invasive treatments of cetaceans.