This study aimed to evaluate the effects of linseed supplementation on the in vitro production of embryos subjected to vitrification. Pantaneira cows supplemented with linseed or not (control) were evaluated. The best-quality embryos produced in vitro from both groups were vitrified. Oocyte quality and blastocyst rate did not differ between the groups. However, the rates of vitrifiable embryos and re-expansion at 3 h were higher in the linseed-supplemented group. In conclusion, linseed supplementation in Pantaneira cows improved the quality of embryos produced in vitro.

Spermatogonial stem cells (SSCs) constitute the foundation of male sperm production and fertility. SSCs are characterised by their capacity for self-renewal and differentiation into spermatozoa throughout a male’s reproductive life, thereby transmitting genetic information to subsequent generations. During embryonic development, SSCs are derived from postnatal testicular gonocytes, which originate from long-lived primordial germ cells. A large proportion of men’s infertility is detected by sperm analysis. Unusual sperm parameters include sperm concentration, morphology and motility. Male infertility is mainly due to hormone deficits, physical causes, sexually transmitted diseases, environment, lifestyle and hereditary factors. The most severe form of male infertility is non-obstructive azoospermia (NOA), which is defined as no sperm in the ejaculate because of spermatogenesis failure. The first-line treatment for these individuals has been testicular sperm extraction under an operating microscope (micro-TESE). SSCs are also considered a promising pathway for regenerating impaired or damaged spermatogenesis. This review addresses SSC transplantation as a potential method for treating male infertility and restoring spermatogenesis in patients with degenerative diseases such as cancer, radiotherapy and chemotherapy.

Infertility aetiology has been shown to be related to reproductive outcomes in the wide reproductive age range. The aim of this study was to examine young women in vitro fertilization (IVF) cycle outcomes according to age and infertility cause. This retrospective cohort study was comprised of 305 couples with a female partner aged 17–25 years with either male or female infertility that underwent a fresh IVF cycle between 2003 and 2020. Comparison according to age groups 17–22 years (n = 117) and 23–25 years (n = 188) showed similar outcomes, except for higher rates of 3 pronuclei (PN) embryos in the younger group. However, allocation to groups by infertility aetiology - male factor (MF) (n = 241) vs. female factor (FF) (n = 64) demonstrated lower fertilization rate in the FF vs. MF group (58% vs. 63%, p = 0.049) and higher percent of 1PN and 3PN embryos (0.7% vs. 0.2% and 0.7% vs. 0.1%, p < 0.001 for both). Chemical pregnancy and live-birth rate were significantly higher in the MF vs. FF group (48.9% vs. 29.5%, p = 0.02 and 41.9% vs. 11.0%, p = 0.038). The only parameter associated with live birth rate in a logistic regression analysis was male infertility aetiology (OR = 2.17, p = 0.038). Young couples undergoing IVF cycles due to female factor infertility have poorer reproductive outcomes compared to male factor-related infertility and higher rates of 1PN and 3PN embryos.

The role of Polyvinylpyrrolidone (PVP) on human gametogenesis and embryonic development has been investigated in clinical setting. PVP is routinely used in sperm handling during intracytoplasmic sperm injection (ICSI) procedure. However, the controversies about its harmful effects on embryonic cells has been debated in recent years. Some reported that PVP decreases the membrane integrity and chromatin stability and strongly affects the fine structure of spermatozoa, such as axonemal tubules, fibrous sheath, and accessory fibres. However, others claimed that PVP delayed the onset of calcium oscillations in the oocyte in ICSI setting. Therefore, PVP affects the decondensation of male pronuclear formation after sperm injection. Also, it has been reported that after injection of conventional 10%PVP to embryos, PVP solution remained in the embryos and suppressed development of these embryos. Recent studies indicated that with a decrease in PVP concentration, embryo development increased and morphokinetics improved in ICSI cycles. Also, low concentration of PVP decreased the rate of apoptosis when compared to 10%PVP. In this review, the irreversible effects of PVP on sperm and embryonic chromosomal aberrant were reported. In conclusion, it is recommended to use the low concentration of PVP in conventional ICSI programme to limit its damage on fertilization processes as well as embryo development.

This study investigated how hormonal induction, female presence, and production system affect sperm quality in Astyanax lacustris across three experiments. In Experiment 1, males and females were kept together in the same recirculating aquaculture system (RAS) before testing. Hormonal induction consistently boosted initial motility and prolonged sperm activity, while female presence offered only a modest benefit to non-induced males and no measurable effect when males were induced. Sperm concentration remained similar across treatments. Experiment 2 evaluated the same factors using broodstock originating from different rearing environments. When males and females came from separate RAS units, hormonal induction again sustained higher motility, and induced males paired with females showed higher sperm concentration than some non-induced groups. In the RAS–biofloc technology (BFT) pairing, hormonal induction maintained motility regardless of female presence; however, in the absence of hormonal induction, both sperm motility and concentration were modulated by the rearing system of females, with non-induced males paired with BFT females exhibiting lower reproductive performance. Overall, hormonal induction proved to be the most reliable strategy for ensuring high semen quality in A. lacustris. Nonetheless, when induction was not applied, male reproductive performance became more sensitive to female origin and rearing environment, highlighting the importance of broodstock compatibility and production system history in reproductive management protocols.

This study investigated the expression profile of the Toll-like receptor 2 (TLR2) gene in goats across three reproductive groups – twin-bearing (n = 12), single-bearing (n = 10) and non-pregnant (n = 8) – using quantitative real-time PCR (qRT-PCR). Descriptive statistics revealed that twin-bearing goats were significantly heavier at service and kidding, with higher litter weights compared to other groups (p < 0.05). No breed-wise differences were observed, allowing pooled analysis for gene expression. Melt curve and amplification plot analyses confirmed primer specificity and efficient amplification. TLR2 expression was significantly upregulated in twin-bearing goats (ΔCt = −2.14 ± 0.89), with a mean 4.5-fold increase (p < 0.001) compared to non-pregnant controls, suggesting enhanced immune activation. Single-bearing goats showed negligible and non-significant changes in expression (ΔCt = −0.01 ± 1.45; p = 0.984). Statistical comparisons showed a significant difference in TLR2 expression between twin- and single-bearing groups (mean difference: 3.39; p < 0.001). These results reveals a significant association of twin pregnancies with elevated expression level of TLR2 gene in resource population. While the functional implications require further investigation, this upregulation may be associated with immune adaptations to increased gestational load. Overall, this study identifies TLR2 as a candidate gene expression marker strongly correlated with twin pregnancy in goats. Further validation through longitudinal studies on larger cohorts and investigation of tissue-specific expression is necessary to assess its potential predictive value for reproductive performance in small ruminants.

The piracanjuba (Brycon orbignyanus) is a species threatened with extinction due to human activities, highlighting the need for genebanking and alternative propagation methods for its conservation. The objective of this study was to obtain a completely sterile yellowtail tetra (Astyanax altiparanae) and to transplant the stem spermatogonia (SSCs) of B. orbignyanus. Complete depletion of spermatogenesis in adult diploid males was successfully achieved through five applications of busulfan, using dosages of 15 mg kg−1 and 40 mg kg−1 at a high temperature of 35°C. One month after completing the busulfan treatment, spermatogenesis returned, occurring more rapidly in the 15 mg kg−1 treatment, with spermatozoa visible in the testicular lumen. In the treatment with 40 mg kg−1, only SSCs and some spermatid cysts were observed. Depletion of spermatogenesis in triploid juveniles was observed after two applications of busulfan (50 mg kg−1). One month after the end of the treatment, only a few SSCs cells were observed in the testes. The B. orbignyanus SSCs were isolated using a Percoll density gradient and identified by alkaline phosphatase activity, resulting in a cell suspension of 2.6 × 106 SSCs/mL with 93% cell viability. The cells were labeled with PKH26 and transplanted via the urogenital papilla into recipients (triploid juvenile males) previously treated with busulfan. PKH26-positive cells were visualized up to 80 days after transplantation. The combination of two sterilization methods appears to be more effective for cell transplantation after the endogenous germ cells have been eliminated from the recipient testes of triploid juveniles.

Recurrent pregnancy loss (RPL) is a clinically important condition in women undergoing assisted reproductive technologies (ART). This study evaluated embryo morphology and morphokinetic parameters using time-lapse monitoring (TLM) and assessed embryo ploidy by preimplantation genetic testing for aneuploidy (PGT-A) in women with RPL compared with unexplained infertility (UEI) controls. A total of 190 patients (100 RPL, 90 UEI) and 1169 embryos (634 RPL, 535 UEI) cultured under TLM were analyzed. Clinical characteristics, embryo morphology, morphokinetics and ploidy status were compared between groups, and logistic regression was used to identify predictors of aneuploidy. The euploidy rate was significantly lower in the RPL group than in controls (43.5% vs. 52.3%, p = 0.018). Group-wise analysis of all embryos revealed differences in selected morphokinetic parameters (t9 and tSC; p < 0.05). However, morphokinetic timing of euploid embryos did not differ between groups, suggesting that accelerated development alone was not associated with chromosomal abnormality. Embryo morphology was the strongest predictor of aneuploidy in both cohorts. Notably, direct uneven cleavage was associated with a 3.4-fold increased risk of aneuploidy specifically in the RPL group. Although embryos from RPL patients showed relatively faster developmental kinetics, morphokinetic speed alone did not predict chromosomal competence. Instead, embryo quality remained the key determinant of aneuploidy, while the association between direct cleavage and aneuploidy highlights the potential clinical value of TLM when combined with PGT-A. These findings support a complementary role for TLM in embryo assessment in RPL patients.

Histone lysine-specific demethylase 4A (KDM4A) plays a critical role in the embryonic development of mammals such as mouse and goat, however its function in zebrafish (Danio rerio) embryogenesis remains poorly understood, due to the existence of two kdm4a paralogs (kdm4aa and kdm4ab) in zebrafish. The current study revealed that kdm4aa–/– embryos exhibited dramatically increased mortality during gastrulation. RT-qPCR showed that RNA-binding motif protein 46 (rbm46) was downregulated after kdm4aa knockout. CUT&Tag-qPCR revealed that kdm4aa knockout significantly decreased H3K4me3, while increasing H3K9me3 and H3K36me3 at rbm46 promoter. kdm4aa–/– embryos displayed elevated reactive oxygen species (ROS) and reduced adenosine triphosphate (ATP). And rbm46 mRNA injection alleviated ROS accumulation and increased ATP level, thereby rescuing the lethal phenotype in kdm4aa–/– embryos. Our findings demonstrate that kdm4aa knockout disturbs zebrafish embryogenesis by suppressing rbm46 mRNA expression.

Oocyte maturation is regulated by well-orchestrated molecular mechanisms and any abnormalities during these events could result in an oocyte that cannot be fertilized or develop into normal embryos. Recently, MOS gene variants were reported to result in oocytes with large polar body (PB) or early embryonic arrest in humans leading to infertility. A thirty-year-old woman with 12 years of infertility underwent two in vitro fertilization cycles, and in both she had oocytes with large PBs and parthenogenetic activation. Clinical exome sequence was performed and revealed a novel homozygous variant in MOS (NM_005372.1):c.573C > A;p.(Phe191Leu). These findings might indicate that MOS mutation results in oocytes with a large PB and parthenogenesis in this patient. In conclusion, our data further confirms that MOS is important in proper oocyte maturation, and variant c 573 C > A results in the formation of a large PB and parthenogenetic activation as reported in animal models earlier. Patients with MOS variants need to be counselled about their fertility status.

The objective of this study was to characterize macroscopically the female gametes, evaluate the survival rates, as well as describe in detail the embryonic development of Prosomyleus rhomboidalis. Three adult females from the Xingu River, presenting promptly ovulation were fertilized by the dry method using sperm from adult males. Egg incubation was set at 27.5°C, and oocyte characteristics and the main developmental stages were described. The results showed that the non-hydrated oocyte has an average diameter of 2.12 mm, yellow colour, spherical shape, is non-adhesive and the diameter increased after hydration at 0.36 mm, referring to the perivitelline space(PS). The fertilization rates (96.79 ± 9.52%) and hatching of normal larvae (96.21 ± 2.40%) were analysed, with hatching above 96%. After hydration of the eggs the PS arose and formed a visible animal pole ending with the hatching of the larvae 69 h and 39 min after fertilization. The description of developmental stages following fertilization was described for the first time in this species. The information presented in this work will serve as a basic support for new possibilities in conservation strategies, re-stocking and genebanking.

Long-term trophectoderm (TE) cell culture provides a powerful model to investigate placenta-specific factors to better understand mechanisms relevant to pregnancy establishment and placental development. However, current TE culture systems rely on costly commercial media and extracellular matrix (ECM) components, which limit their scalability and accessibility. This study evaluated cost-effective alternatives to established conditions by testing modified DMEM/F-12 and a biphasic TE culture system as substitutes for commercial Advanced DMEM/F-12 and for continuous TE culture, and by assessing 0.1% gelatine as an ECM alternative to collagen IV. Trophectoderm outgrowths cultured on collagen IV or gelatine did not differ in attachment timing (P = 0.78) or in expression of placental and differentiation markers PLAC8 (P = 0.78), GATA2 (P = 0.18), and PAG10 (P = 0.39). Similarly, blastocysts cultured in commercial Advanced DMEM/F-12 or base DMEM/F-12 exhibited no differences in attachment day (P = 0.98), TE growth area from days 10–20 (P > 0.05), or expression of PLAC8 (P = 0.35) and PAG10 (P = 0.08), although PAG7 differed between treatments (P < 0.05). Embryos cultured in continuous TE attached later (P = 0.01) than those cultured in TE media, but no differences were observed in TE growth area or expression of PLAC8, PAG7, or PAG10 (P > 0.05). Collectively, these results indicate that affordable media formulations and gelatine-coated cultureware support TE attachment, proliferation, and differentiation. This cost-effective culture framework enables broader application of TE models and supports extended studies of trophoblast function, placental signalling, and early conceptus development.