Precise and early pregnancy detection is crucial for better breeding management and enhancing the overall production of ruminant livestock. Throughout the years, numerous methods have evolved for pregnancy detection in ruminants, each possessing specific advantages and limitations. This review thoroughly discusses both traditional and emergent diagnostic methods, emphasizing their principles, implementation, merits and challenges. Behavioral observation, rectal palpation and ultrasonography are the traditional approaches widely used because of their accessibility and direct detection of pregnancy conditions. Progesterone measurement, pregnancy-associated glycoprotein detection, and estrone sulfate examination are the hormonal assays that provide biochemical proof at specific phases of gestation. Recently, the analysis of interferon-stimulated gene expression and circulating microRNAs has shown promising roles in early pregnancy detection at the genetic and transcriptomic levels. The investigation of volatile organic compounds is a novel approach in pregnancy diagnosis, though it is non-invasive, and further confirmation is required for regular application. This review highlighted the importance of incorporating multiple examination strategies to enhance the accuracy and reliability of pregnancy detection in ruminants. Future research should center on the refinement and field application of advanced technologies to ensure their proper implementation in diverse ruminant production systems.

Immature zebrafish oocytes are sensitive to chilling, and their survival is markedly reduced by exposure to 0°C. In the present study, we investigated the involvement of cold-sensitive channels and lipid mediators in chilling injury in immature zebrafish oocytes. The oocytes were injected with inhibitors of a cold-sensitive channel (TRPA1), cytosolic phospholipase A₂α (cPLA₂α), cyclooxygenases (COXs), arachidonate 5-lipoxygenase (ALOX5), and lysophosphatidylcholine acyltransferase 2 (LPCAT2). The cells were then chilled at 0–12°C for 5–30 min, incubated at 25°C for 2 h, and stained with propidium iodide. Oocytes were damaged when exposed to temperatures below 12°C. When oocytes were chilled at 0°C for 15 min, the survival rate was very low (9%). However, when the oocytes were injected with a TRPA1-specific inhibitor, their survival markedly improved (70%). This strongly suggests that activation of the cold-sensitive TRPA1 channel triggers chilling injury in oocytes. When a cPLA₂α-specific inhibitor was injected, the survival of chilled oocytes markedly improved (60%). This strongly suggests that lipid mediators are involved in chilling injury in oocytes. When oocytes were injected with specific inhibitors of COXs, ALOX5, and LPCAT2, the survival of chilled oocytes significantly improved by 47%, 28%, and 43%, respectively. These results strongly suggest that eicosanoids and platelet-activating factor are involved in the chilling injury in oocytes. The results of this study may facilitate advancements in successful cryopreservation of fish oocytes.

L-tryptophan (Trp), an essential amino acid, is a precursor of 5-hydroxytryptamine (5-HT; also known as serotonin) that promotes mammalian sperm hyperactivation. Since mammalian sperm contain Trp hydroxylase (TPH), they may contribute to 5‑HT biosynthesis. Therefore, this study aimed to examine the effect of Trp on hamster sperm hyperactivation and determine whether sperm are involved in 5-HT biosynthesis. Trp significantly enhanced sperm hyperactivation via the 5-HT₄ receptor and its associated signals. In contrast, D-tryptophan did not affect sperm hyperactivation. Furthermore, hamster sperm contained the 5-HT biosynthesis enzymes TPH and aromatic L-amino acid decarboxylase (AADC). Additionally, hamster sperm secreted 5-HT. Trp-enhanced hyperactivation and 5-HT secretion were significantly inhibited by TPH and AADC inhibitors. Overall, our findings suggest that Trp enhanced sperm hyperactivation through the biosynthesis of 5-HT within the sperm.

Immature zebrafish oocytes are highly susceptible to high temperatures, making it difficult to warm cryopreserved oocytes rapidly. In the present study, we aimed to investigate whether thermosensitive channels, lipid mediators, and ferroptosis are involved in heat stress-induced injury in immature zebrafish oocytes. Oocytes were injected with inhibitors of a heat-sensitive channel (TRPV1) and multiple enzymes—cytosolic phospholipase A₂α (cPLA₂α), cyclooxygenases (COXs), arachidonate 5-lipoxygenase (ALOX5), and lysophosphatidylcholine acyltransferase 2 (LPCAT2). In addition, a ferroptosis-specific inhibitor was administered. The oocytes were then warmed at 45°C for 15 min, incubated at 25°C for 2 h, and then stained with propidium iodide. When the control oocytes were warmed at 45°C for 15 min, their survival was low (1%–8%). However, the survival of oocytes injected with the TRPV1-specific inhibitor markedly improved (40%), suggesting that TRPV1 activation triggers heat stress injury in oocytes. When a cPLA₂α-specific inhibitor was injected, survival of oocytes after warming significantly improved (30%), suggesting that lipid mediators or ferroptosis are involved in heat stress-induced injury in oocytes. In contrast, survival either slightly improved or did not improve when oocytes were injected with specific inhibitors of COXs, ALOX5, and LPCAT2 (16%, 8%, and 3%, respectively). Notably, the ferroptosis-specific inhibitor markedly improved oocyte survival (60%). These results may facilitate methodological advancements in fish oocyte cryopreservation. Additionally, they suggest that ferroptosis is involved in heat stress-induced injury in immature zebrafish oocytes, following TRPV1 activation and subsequent cPLA₂α activation.

Luteal blood flow (LBF) is essential for progesterone (P₄) biosynthesis in the corpus luteum (CL) and affects bovine fertility. However, the mechanism by which LBF affects fertility remains unclear. This study was conducted to investigate the effects of LBF on endometrial P₄ concentrations and gene expression. Endometrial biopsies and blood samples were collected from 13 Japanese Black cows after ultrasound examination on Day 7 (Day 0 = day of estrus). Based on LBF, the cows were divided into low- (LV; n = 5), medium- (MV; n = 2), and high-vascularity (HV; n = 6) groups. Plasma and endometrial P₄ concentrations were measured using enzyme immunoassays. RNA sequencing was performed to compare the endometrial gene expression profiles from three cows in each of the LV and HV groups. Reverse transcription-quantitative PCR was performed for genes selected from the differentially expressed genes (DEGs), P₄ receptors (PGR, PGRMC1, and PGRMC2), and P₄-regulated genes (ANPEP, DGAT2, DKK1, and LTF). No differences were observed in plasma or endometrial P₄ concentrations between the HV and LV groups. CCN3 was identified as a DEG between the HV and LV groups and was upregulated in the HV group. Compared to those of the LV group, the HV group exhibited higher CCN3 and PGR mRNA expression levels and lower ANPEP, DGAT2, and DKK1 mRNA expression levels. In conclusion, LBF affects endometrial gene expression without changing plasma or endometrial P₄ concentrations on Day 7.

Assisted reproductive technologies (ART) to generate developmentally competent oocytes necessitates light exposure due to the use of microscopes. Previous studies in several species have reported that the wavelength of light during the light exposure period is a critical factor in embryo development. However, the effects of different light wavelengths on embryo development in pigs remain unexplored. This study aimed to identify the optimal light conditions to enhance oocyte maturation, parthenogenetic activation of mature oocytes, and pre-implantation development of parthenogenetic embryos in pigs. Conducted irradiation experiments during in vitro maturation (IVM), parthenogenesis (PG), and in vitro culture (IVC) using visible (390–750 nm), blue (445–500 nm), green (500–575 nm), yellow (575–585 nm), and red (620–750 nm) light. Variation in light wavelengths during IVM, PG, or IVC did not significantly influence oocyte maturation of cumulus–oocyte complexes (COCs) derived from median antral follicles (MAFs), developmental competence of in vitro-culture COCs after PG, and the production of blastocysts. However, continuous irradiation with green light throughout the entire process (IVM, PG, and IVC) significantly enhanced in vitro blastocyst production, and the resulting blastocysts showed significantly elevated HSP70 expression and a numerical increase in PCNA expression. We recommend conducting all in vitro procedures (IVM, PG, and IVC) for producing blastocysts from MAFs in porcine models under green light. This study will contribute to yielding higher success rates of porcine ART and reduce artificial stress to oocytes and embryos caused by in vitro manipulation under strong light exposure.

Golden hamsters (Mesocricetus auratus) have been extensively used in biomedical research. With the advent of genome-editing technology, it is now possible to generate gene-knockout hamsters, providing unique research models that cannot be achieved with mice or rats. Therefore, the development of cryopreservation techniques for hamster embryos is in high demand. In this study, we present a simplified vitrification protocol for hamster embryo preservation. In vivo-derived 8-cell or morula embryos (Day 3) were vitrified using Cryotop in modified HECM-3 medium containing ethylene glycol, DMSO, and sucrose. After warming, the embryos were transferred into the uteri of Day 3-pregnant females with a different coat color. The results showed that 21–26% of the transferred embryos developed to the term. The experiments were conducted in a conventional laboratory setting, avoiding direct light exposure. Given the reproducibility of our vitrification protocol, it has broad applicability in laboratories that use hamsters.

Correlative light-electron microscopy (CLEM) combines fluorescence microscopy and scanning electron microscopy (SEM) to achieve nanoscale resolution while highlighting regions of interest identified by fluorescence microscopy. CLEM is becoming increasingly important in life sciences but traditionally requires highly dried samples to withstand the high vacuum of SEM. The NanoSuit method, which mimics native extracellular substances, was developed to address this limitation by encasing samples in a thin, vacuum-proof membrane, allowing SEM observation of live or wet multicellular organisms. While previous NanoSuit CLEM studies focused on formalin-fixed paraffin-embedded sections and cultured cells, cryosections had not yet been explored. In this study, NanoSuit CLEM with diluted NanoSuit solution was applied to cryosections of bovine anterior pituitary tissue. Secretory granules in gonadotrophs, which constitute less than 12% of anterior pituitary cells, were successfully visualized. However, other organelles remained unobserved due to fixation conditions. Therefore, NanoSuit CLEM enabled visualization of the ultrastructure of important cells in cryosections, even from large animals.