Selenium (Se) is an essential micronutrient involved in male reproductive physiology through its integration into selenoproteins that orchestrate antioxidant defense and spermatogenesis. This study compared the effects of inorganic and yeast- and bacteria-derived Se on the bioavailability, testicular development, sperm functional traits, epididymal plasma biochemistry, and testicular glycogen reserves in growing lambs. Twenty-four lambs (6-7 months old) were randomly assigned to four dietary groups (n = 6) for 90 days: control and sodium selenite (0.5 mg/kg), Se-yeast (0.5 mg/kg), and bacteria-derived Se (0.5 mg/kg) supplemented groups. Se supplementation significantly increased Se concentrations in serum, epididymal plasma, and testicular tissue (P < 0.0001), with bacteria-derived Se exhibiting the highest tissue deposition. Improved bioavailability enhanced testicular development, as evidenced by paired testicular weight, gonadosomatic index, and other morphometric parameters (P < 0.0001). Se also fortified the antioxidant architecture, upregulating glutathione peroxidase, superoxide dismutase, and catalase activities, while markedly reducing malondialdehyde levels (P ≤ 0.0004). Epididymal sperm quality improved significantly, including motility, concentration, membrane integrity, viability, and reduced abnormalities (P < 0.0001), without affecting the recovered fluid volume (P = 0.6306). Additionally, Se enhanced epididymal plasma biochemical profiles and increased testicular glycogen reserves (P < 0.0001). Overall, bacteria-derived Se resulted in superior tissue Se deposition and improved reproductive parameters compared to that from other sources. While these responses are likely associated with an enhanced antioxidant status, the specific Se species and underlying molecular mechanisms require further investigation. Consequently, bacteria-derived Se represents a promising alternative source for supporting male reproductive development in small ruminants.

The Peruvian Paso horse, a breed of national significance in Peru known for its unique gait, has been selectively bred for nearly 500 years and now faces potential risks due to reduced genetic diversity. To investigate the potential of somatic cell nuclear transfer to enhance genetic conservation strategies, a program was initiated to produce cloned Peruvian Paso foals. Skin biopsies from a highly awarded stallion with exceptional breed-defining traits were used as the source of donor somatic cells. Of the 15 cloned embryos reconstructed, seven blastocysts were produced, and five were transferred to five recipient mares. Three pregnancies ensued, two of which resulted in the birth of healthy foals at 333 and 347 days of gestation. Cloned foals were genetically and phenotypically identical to the donor animal, demonstrating the successful application of this approach to support genetic conservation in a protected horse breed.

In vitro maturation (IVM) of germinal vesicle oocytes may broaden assisted reproductive technologies; however, developmental competence remains limited by the lack of standardized follicular somatic support. We generated an endogenous FOXL2-P2A-tdTomato reporter human induced pluripotent stem cell (hiPSC) line using CRISPR/Cas9 knock-in and reporter-guided transcription factor (TF) programming to produce fetal granulosa-like cells (FGLCs). A flow cytometric TF screen identified TCF21, WT1-KTS, and NR1H4 as the strongest FOXL2 inducers, and combinatorial optimization showed that removing TOX3/ETV5 and adding NR2F2 substantially increased the FOXL2-positive fraction. Bulk RNA-seq, principal component analysis, and TF activity inference positioned the induced cells close to early-gestational human fetal granulosa cell profiles. In a mouse IVM, in vitro fertilization (IVF), blastocyst culture, and embryo transfer pipeline, FGLC supplementation did not change nuclear maturation rates but improved downstream development, most clearly increasing offspring production from cumulus-oocyte complexes. A comprehensive safety assessment of F0 offspring derived from FGLC-treated oocytes revealed a normal sex ratio, postnatal growth, gross anatomy, gonadal histology, and modified SHIRPA neurobehavioral profiles. Next-generation natural mating and germ cell assays demonstrated preserved fertility, normal IVF outcomes, and normal sperm parameters in the F1 generation. These results establish a scalable strategy for generating fetal granulosa-like supporting cells from hiPSCs and provide an effective and multi-generational safety framework for oocyte-contact IVM interventions.

Malnutrition often suppresses reproductive function by inhibiting the pulsatile secretion of gonadotropin-releasing hormone (GnRH)/gonadotropins in mammals. This study aimed to determine whether cocaine- and amphetamine-regulated transcript (CART) neurons mediate the suppression of GnRH/gonadotropin pulses under malnutrition, since CART neurons have been reported to project to kisspeptin/neurokinin B/dynorphin A neurons (also known as the GnRH pulse generator) and GnRH neurons in rats. Wistar-Imamichi female rats were ovariectomized (OVX), and some of these rats were immediately implanted with subcutaneous Silastic tubing containing estradiol-17β (OVX + low E2) to maintain a diestrous level of estrogen. Free-moving conscious OVX and OVX + low E2 rats were subjected to 1-h frequent blood sampling followed by brain sampling to examine whether acute glucoprivation by peripheral administration of 2-deoxy-D-glucose (2DG), a glucose utilization inhibitor, suppressed the secretion of luteinizing hormone (LH) and activated hypothalamic CART neurons. The other cohort of animals was subjected to 3-h frequent blood sampling to examine whether central CART administration suppresses pulsatile LH secretion. Peripheral 2DG administration suppressed LH secretion and activated CART neurons in the hypothalamic supraoptic nucleus (SON) and parvocellular paraventricular nucleus (PVN), but not in the magnocellular PVN-zona incerta-dorsomedial nucleus continuum and arcuate nucleus, in OVX and OVX + low E2 rats. In addition, central CART administration suppressed pulsatile LH secretion in OVX and OVX + low E2 rats. These results suggest the possible involvement of CART neurons in the SON and parvocellular PVN in malnutrition-induced suppression of pulsatile LH secretion in female rats.

Glucose (GLU) is an essential energy source for mammalian cells, and its depletion induces various cellular responses, including apoptosis. The present study examined whether a decrease in GLU levels induces apoptotic cell death, which typically occurs during regression of the bovine corpus luteum (CL). To investigate the possible mechanisms of luteal cell death induced by low GLU levels, we examined the effect of GLU-free medium on the expression of apoptosis-regulating factors (FAS, BAX, BCL2, CASP8, and CASP3). GLU depletion induced cell death in bovine mid-luteal cells. Luteal cells cultured in GLU-free medium showed reduced viability, which was rescued by GLU supplementation in a dose-dependent manner. Culturing without GLU increased the expression of FAS and CASP3 mRNAs. Inhibiting glucose transporter 1 (GLUT1) with cytochalasin B or STF-31 in the presence of GLU also induced cell death. Based on these findings, a decrease in GLU supply, which is induced by a decrease in blood flow to the CL during luteolysis, may promote apoptosis by stimulating the expression of FAS and CASP3 in the bovine CL.

Gangliosides play important roles in membrane organization and cellular signaling. Although their involvement in spermatogenesis has been suggested, the specific functions of individual ganglioside species during each stage of germ cell development remain poorly understood. In this study, we generated beta-1,3-galactosyltransferase 4(B3galt4) knockout (KO) mice to investigate the role of B3GALT4-dependent ganglioside in male germ cell development. Immunostaining analyses demonstrated that B3galt4 deficiency caused selective alterations in ganglioside distribution. Although testis size did not differ significantly between KO and control mice, KO males were completely infertile. Histological examination revealed that spermatogenesis in KO mice proceeded to the elongating spermatid stage; however, abnormalities were observed during the late stages of spermatogenesis, including a reduced number of elongating/elongated spermatids and multinucleated giant cells in subsets of seminiferous tubules. Consistent with these observations, the number of sperm recovered from the cauda epididymis was markedly decreased, accompanied by morphological abnormalities and impaired motility. Collectively, these findings demonstrate that B3galt4 deficiency compromises late spermatogenesis and sperm function, resulting in male infertility. Our results further suggest that B3GALT4-dependent gangliosides have stage-dependent roles during late spermatogenesis and sperm development.

The preservation of chicken genetic resources is important to both backup commercial breeds that support egg and meat production worldwide and conserve the genetic diversity of indigenous breeds, which is essential for managing breeding according to future demands. Because chicken embryos are attached to a large yolk, cryopreservation is technically impossible. In chickens, a unique developmental engineering approach based on the manipulation of primordial germ cells (PGCs), the embryonic precursor cells of gametes, has been developed. Cryobanking of PGCs is an innovative strategy for efficiently maintaining chicken genetic resources without breeding live birds. Cryomedia containing dimethyl sulfoxide or propylene glycol (PG) as permeable cryoprotectants, combined with serum as a non-permeable cryoprotectant, have been widely used for the cryopreservation of chicken PGCs. However, because the serum composition varies greatly, performance differences arise between lots. Consequently, there is a demand for serum-free cryomedia with specific known compositions. Here, we investigated the cryoprotective effects of ovalbumin and sericin, as alternatives to serum, on chicken PGCs. Two cryomedia were designed: PO comprising 7.5% PG and 5% ovalbumin and PS comprising 7.5% PG and 2% sericin. Following the culture of PGCs cryopreserved in these cryomedia, viability and cell doubling times recovered to levels comparable to those of an unfrozen control group at 6 and 2 d post-culture, respectively. When frozen-thawed PGCs were transplanted, their gonadal migration ability was significantly lower immediately after thawing, but recovered to levels comparable to those of the unfrozen control group after 4 d of culture. We successfully revived viable offspring from Hinai-dori, designated as a natural monument of Japan, from PGCs cryopreserved in these cryomedia. In conclusion, we developed two serum-free cryomedia that achieved > 60% recovery of viable PGCs after thawing while maintaining germline competency.

Uterine inflammatory diseases are a major cause of subfertility in both humans and domestic animals. Lipopolysaccharide (LPS) is a key mediator of uterine inflammation and reproductive dysfunction. Although the acute suppressive effects of LPS on the hypothalamic-pituitary-gonadal axis are well documented, its sustained effects under persistent inflammation remain unclear. This study investigated the effects of systemic LPS exposure on reproductive and hepatic function, immune activation, and stress responses in estradiol-treated ovariectomized goats. Animals received either a single intravenous (i.v.) bolus injection of LPS or a continuous i.v. infusion. Serial blood samples were collected for 8 h to evaluate plasma concentrations of LPS, interleukin (IL)-1β, cortisol, and liver enzymes, as well as pulsatile luteinizing hormone (LH) secretion. Rectal temperature increased during the early phase after LPS administration. Plasma LPS concentrations increased several hours after LPS administration, with a biphasic elevation observed during continuous LPS infusion. IL-1β and cortisol increased within 1 h of LPS administration, coinciding with LH pulse suppression and preceding the rise in plasma LPS. Gamma-glutamyltransferase (GGT) increased within 3 h and remained elevated after LPS administration. Aspartate transaminase (AST) increased at the end of the continuous LPS infusion experiment. Systemic LPS administration significantly suppressed pulsatile LH secretion compared with saline treatment. These findings suggest that LPS-driven systemic immune activation and/or stress-related endocrine responses may contribute to suppression of pulsatile LH secretion, and that progressive hepatic dysfunction during continuous LPS exposure may contribute to sustained circulating LPS and prolonged reproductive dysfunction.

We evaluated the utility of vaginal electrical resistance (VER) as an indicator of the optimal mating time in alpacas. Three parous female alpacas were used in a total of five trials. The alpacas received an intramuscular injection of human chorionic gonadotropin to induce ovulation and synchronize the follicular waves on Day 0. VER was negatively correlated with plasma estradiol-17β and positively correlated with plasma progesterone (P4). VER on Days 11-12 was significantly lower than that on Day 7. The dominant follicles capable of ovulation were observed by transrectal ultrasonography in four of the five trials on Days 11 and 12. Elevated P4 levels on Day 7 and their subsequent reduction by Day 12 indicated that Days 11-12 were optimal for mating. Thus, VER measurements appear to be useful for estrus prediction. However, repeated VER measurements in each alpaca are necessary to predict the optimal timing of mating because of the significant interindividual variation in VER.

The quality of oocytes is crucial for embryonic development. The morphology of cumulus-oocyte complexes (COCs) and cumulus expansion are widely used indicators of oocyte quality; however, quantitative evidence is limited. We investigated the potential of assessing the quality of COCs based on their objective morphological characteristics and the impact of cumulus expansion on bovine in vitro-produced embryo development. COCs were graded before in vitro maturation (IVM) according to the International Embryo Technology Society (IETS) standards, and their morphological characteristics (area, perimeter, circularity, and solidity) were measured. The cumulus expansion rate and nuclear maturation were then evaluated after IVM. Decision tree models were developed based on COC morphology to classify COC grades and predict the cumulus expansion rate. In another experiment, COCs were divided into two groups based on a 100% cumulus expansion threshold, and embryonic development was assessed. The results revealed that the morphological characteristics of COCs were associated with the IETS grades and cumulus expansion rate but not with nuclear maturation. The predictive models for cumulus expansion capacity achieved an accuracy of 75.5%. COCs with greater cumulus expansion exhibited a significantly higher proportion of ≥ eight-cell-stage embryos on day 2 (14.0 ± 5.9%, P < 0.05). However, the blastocyst formation rate did not differ according to the extent of cumulus expansion. These findings indicate that an objective morphological assessment can predict cumulus expansion capacity and may provide a non-invasive and reproducible approach for selecting high-quality COCs before IVM, potentially indicating an association with the speed of early embryonic development.

Mouse embryonic stem cell (ESC) cloning via nuclear transfer is a potential platform for direct generation of genetically modified mice. However, the low G1 cell population in ESCs hampers their broader use in nuclear transfer, as S-phase donors induce 1-cell arrest in reconstructed oocytes. This study aimed to enrich the G1-phase population of ESCs to improve ESC-based cloning efficiency. We demonstrated that EB3 ESCs derived from the 129/Ola strain and C57BL/6×129/Sv hybrid ESCs exhibited superior traits for nuclear transfer in terms of cleavage rate and blastocyst formation but showed reduced efficiency in development to term. Transcriptomic analyses revealed that this failure was likely due to aberrant imprinted gene expression in critical clusters, such as H19-Igf2 or Dlk1-Dio3. In contrast, although cloned offspring were successfully generated at a promising rate (approximately 6%) using EGR-R01 (BDF1×129/Sv) ESCs, early cleavage to the 2-cell embryo was inefficient. To address this, we induced cell cycle synchronization by culturing ESCs to overconfluency, which markedly increased the G1 proportion, as confirmed via FACS analysis. This alteration led to a significant improvement in first cleavage efficiency without compromising the developmental potential of ESC-derived cloned embryos to live birth. Overall, our study highlights the importance of both cell cycle synchronization and imprinting maintenance in improving ESC cloning efficiency to facilitate broad applications, including the direct generation of genetically modified mice.

Mammalian spermatozoa begin swimming after ejaculation. After spermatozoa enter the oviduct, they undergo capacitation, penetrate the oocyte envelope, and fertilize the oocytes. These processes occur spontaneously but are influenced by hormones and neurotransmitters. Serotonin [5-hydroxytryptamine (5-HT)] is a well-known neurotransmitter found in reproductive organs. In the female reproductive system, serotonin is synthesized in the cumulus cells and affects steroidogenesis, oocyte maturation, fertilization, and embryo development. Recent studies have suggested that serotonin is also synthesized in spermatozoa and can affect motility, hyperactivation, acrosome reaction, and the success of in vitro fertilization (IVF) in hamsters, humans, mice, and rats. Importantly, the effects of serotonin on sperm function are dose-dependent, at least in hamsters, and serotonin concentrations in the female reproductive tract fluctuate during ovulation. The effects of serotonin on sperm function are mediated by 5-HT receptors (mainly 5-HT2 and 5-HT4), which stimulate Ca[2+] and cyclic AMP signaling, respectively. The 5-HT2 receptor is involved in regulating motility and hyperactivation in hamsters, humans, and mice, whereas the 5-HT4 receptor is involved in regulating hyperactivation, the acrosome reaction, and IVF success in hamsters, mice, and rats. Because the 5-HT4 receptor is a common receptor in rodents associated with capacitation and IVF success and is stimulated by serotonin synthesized in spermatozoa, current evidence indicates that the 5-HT4 receptor is especially important for the regulation of rodent spermatozoal functions regulated by serotonin.

The beta-nerve growth factor (β-NGF), a seminal plasma protein with established luteotrophic effects in camelids and cattle, has been proposed as a modulator of periovulatory follicular function. The optimization of the steroidogenic environment of the preovulatory follicle is critical for oocyte maturation, corpus luteum formation, and embryo quality. However, the molecular actions of β-NGF on granulosa cells (GCs) during the periovulatory period in dairy heifers remain poorly defined. This study evaluated the effects of systemic administration of heterologous β-NGF (purified from llama seminal plasma) on the expression of steroidogenic enzymes in GCs from preovulatory follicles in estrus-synchronized Holstein heifers. Animals received 1 mg of β-NGF intramuscularly on Day 9 (estradiol benzoate administration) or Day 10 (fixed-time artificial insemination) of a progesterone(P4)/estradiol (E2)-based synchronization protocol. The follicular fluid and GCs were collected via transvaginal ultrasound-guided aspiration for hormone quantification and gene expression analysis. β-NGF treatment on Day 9 significantly upregulated Lutenizing hormone/choriogonadotropin receptor (LHCGR) and key steroidogenic enzymes, including 3β-hydroxysteroid dehydrogenase (HSD3B), Cytochrome P450 family 11 subfamily A member 1 (CYP11A1), 17β-hydroxysteroid dehydrogenase (HSD17B), and Cytochrome P450 family 19 subfamily A member 1 (CYP19A1), whereas no transcriptional effects were observed when NGF was administered on Day 10, indicating a timing-dependent regulation of GCs differentiation. In contrast, Growth hormone receptor GHR and Steroidogenic acute regulatory protein expression levels remained unaffected, supporting the selectivity of NGF-induced transcriptional modulation. The follicular fluid P4/E2 ratios were not significantly altered by NGF treatment. These findings indicate that β-NGF increased steroidogenic and LHCGR gene abundance of GCs in a timing-dependent manner, suggesting that NGF may represent a potential modulator of the preovulatory follicle function in dairy cattle.

There is limited consensus among embryologists regarding the decision to freeze or discard borderline-quality blastocysts. This study aims to investigate the intrinsic developmental potential of blastocysts with poor inner cell mass (ICM) or trophectoderm (TE) quality using an in vitro three-dimensional (3D) culture system that excludes maternal factors. A total of 51 one-pronucleus (1PN)-derived blastocysts were obtained from couples undergoing assisted reproduction technology (ART). The embryos were categorized into three groups: 20 high-quality embryos, 17 embryos with poor ICM, and 14 embryos with poor TE. A 3D culture system was utilized to assess the post-implantation developmental potential of blastocysts with varying grades. Embryonic morphology and diameter were measured to evaluate growth characteristics, and immunofluorescence staining was performed to assess the differentiation of embryonic lineages. 35% of high-quality 1PN embryos developed into OCT4 + epiblast (EPI), GATA6 + hypoblast (HYP), and CK7 + TE, along with an amniotic cavity and yolk sac. Blastocysts with poor TE quality demonstrate reasonable developmental potential, whereas those with poor ICM quality are already compromised during implantation. This study provides valuable insights for ART and clinical management of low-quality blastocysts.

Oocytes develop through bidirectional communication with surrounding granulosa cells (GCs), and differences in the GC environment represent a major distinction between in vivo and in vitro oocyte growth systems. However, the influence of GC number and organization on oocyte growth and quality during in vitro culture remains unclear. In this study, oocyte-granulosa cell complexes (OGCs) derived from early antral follicles (0.5-0.7 mm) of cows were supplemented with spheroid granulosa cells (GC-S) derived from antral follicles (3-5 mm). GC-S were incorporated into OGCs during 14 days of incubation. Oocyte characteristics, maturation, and fertilization competence were evaluated, and the transcriptomic profiles of surrounding GCs were analyzed by RNA sequencing. GC-S supplementation increased transzonal projections and improved GC survival within OGCs, indicating enhanced GC-oocyte interactions. It also increased ATP content, oocyte diameter, and DNA methylation levels, reaching levels comparable to those observed in in vivo-grown oocytes. Furthermore, GC-S supplementation significantly improved oocyte maturation and fertilization rates, although these remained lower than those of in vivo counterparts. In contrast, lipid content, mitochondrial DNA copy number, and mitochondrial membrane potential were not affected and remained lower than those of in vivo-grown oocytes. RNA-seq analysis revealed that GC-S supplementation altered the expression of genes associated with focal adhesion and cytoskeleton-related pathways toward those observed in vivo. These results demonstrate that spheroid GC supplementation improves GC-oocyte interactions and partially restores in vivo-like characteristics of oocytes during in vitro growth.