Equine oocyte vitrification would benefit the growing in vitro embryo production programs, but further optimization of the protocol is necessary to reach clinical efficiency. Therefore, we aimed to perform a direct comparison of non-permeating and permeating cryoprotective agents (CPAs) during the vitrification and warming of equine immature oocytes. In the first experiment, cumulus oocytes complexes (COCs) were vitrified comparing sucrose, trehalose, and galactose in combination with ethylene glycol (EG) and dimethyl sulfoxide (DMSO). In the second experiment, the COCs were vitrified using three mixtures of permeating CPAs in a 50:50 volume ratio (ethylene glycol-dimethyl sulfoxide (ED), propylene glycol-ethylene glycol (PE), and propylene glycol-dimethyl sulfoxide (PD)) with galactose and warmed in different galactose concentrations (0.3 or 0.5 mol/L). Overall, all the treatments supported blastocyst formation, but the developmental rates were lower for all the vitrified groups in the first (4.3 to 7.6%) and the second (3.5 to 9.4%) experiment compared to the control (26.5 and 34.2%, respectively; p < 0.01). In the first experiment, the maturation was not affected by vitrification. The sucrose exhibited lower cleavage than the control (p = 0.02). Although the galactose tended to have lower maturation than trehalose (p = 0.060) and control (p = 0.069), the highest numerical cleavage and blastocyst rates were obtained with this CPA. In the second experiment, the maturation, cleavage, and blastocyst rates were similar between the treatments. Compared to the control, only the ED reached similar maturation (p = 0.02) and PE similar cleavage (p = 0.1). The galactose concentration during warming did not affect the maturation, cleavage, or blastocyst rates (p > 0.1), but the PE-0.3 exhibited the highest blastocyst rate (15.1%) among the treatments, being the only one comparable to the control (34.2%). As such, PE–galactose provides a valuable option for equine immature oocyte vitrification and should be considered for the future optimization of the protocol.

In brief: Bulls are selected for field fertility and semen quality, but traits such as polyspermy are not considered and can increase aneuploidy during in vitro embryo production. This study links bull-specific proteomic signatures to polyspermy and embryo quality, further refining bull selection criteria. Male fertility plays a pivotal role in the success rates of in vitro embryo production. While livestock breeding programs rigorously select bulls according to their predicted field fertility, specific traits such as polyspermy rates are not routinely evaluated. Despite the known negative impact of polyspermy on embryo survival, the paternal factors involved remain unclear. In this study, we aimed to address this gap by evaluating the in vitro outcomes of four bulls, focusing on sperm motility, fertilization rates, polyspermy incidence, embryo development and quality. In addition, we analyzed the proteome profiles of sperm, 2–4 cell stage embryos and blastocysts derived from those bulls to identify potential molecular factors associated with male fertility. Bulls with comparable sperm motility parameters displayed varying in vitro fertilization outcomes. Notably, the bull with the highest polyspermy rate achieved blastocyst rates similar to those of bulls with lower polyspermy rates. The number of apoptotic cells in the blastocysts was bull-dependent. Proteomic analysis revealed bull-specific signatures in sperm and blastocysts, with no differences at the 2–4 cell stage. Differences in the sperm proteome suggested that bull-dependent penetration and polyspermy rates might be associated with the ability of the sperm to undergo capacitation and acrosomal reaction. At the blastocyst level, the bull with the highest polyspermy rates produced lower quality blastocysts due to imbalances in key proteins and pathways for embryo development. In conclusion, bulls with similar blastocyst rates may differ in polyspermy rates and resulting embryo quality underscoring the importance of careful bull selection for in vitro embryo production.

In the last decade, in vitro embryo production in horses has become an established clinical practice, but blastocyst rates from vitrified equine oocytes remain low. Cryopreservation impairs the oocyte developmental potential, which may be reflected in the messenger RNA (mRNA) profile. Therefore, this study aimed to compare the transcriptome profiles of metaphase II equine oocytes vitrified before and after in vitro maturation. To do so, three groups were analyzed with RNA sequencing: (1) fresh in vitro matured oocytes as a control (FR), (2) oocytes vitrified after in vitro maturation (VMAT), and (3) oocytes vitrified immature, warmed, and in vitro matured (VIM). In comparison with fresh oocytes, VIM resulted in 46 differentially expressed (DE) genes (14 upregulated and 32 downregulated), while VMAT showed 36 DE genes (18 in each category). A comparison of VIM vs. VMAT resulted in 44 DE genes (20 upregulated and 24 downregulated). Pathway analyses highlighted cytoskeleton, spindle formation, and calcium and cation ion transport and homeostasis as the main affected pathways in vitrified oocytes. The vitrification of in vitro matured oocytes presented subtle advantages in terms of the mRNA profile over the vitrification of immature oocytes. Therefore, this study provides a new perspective for understanding the impact of vitrification on equine oocytes and can be the basis for further improvements in the efficiency of equine oocyte vitrification.

In brief: In vitro maturation is an essential tool in reproductive technologies, though its impact on oocyte quality remains a concern. This study shows that in vitro maturation alters gene expression and DNA methylation in bovine oocytes compared to in vivo matured oocytes, potentially compromising oocyte quality and developmental competence. In vitro maturation of oocytes is a routine step in assisted reproduction but is associated with lower embryo development rates compared to oocyte maturation in vivo . We analyzed the genomic profiles of oocytes from the same cow, either matured in vivo or in vitro , using single-cell methylome and transcriptome sequencing, along with transcriptome analysis of corresponding cumulus cells. Both the transcriptome and methylome of the oocytes matured in vitro were altered. Notable changes included alterations in CpG islands associated with imprinted genes, including decreased methylation levels in MEST (PEG1), NNAT (both implicated in large offspring syndrome), and MIMT1 . Transcriptomic analysis of their cumulus cells highlighted impaired mitochondrial function, hypoxia responses, and cell adhesion. Our findings highlight the extent to which the maturation environment can influence key epigenetic regulators and mRNA profiles that affect oocyte quality and subsequent developmental outcomes. The data provide a valuable resource for optimizing assisted reproductive technologies.

In vivo-matured oocytes exhibit higher developmental competence than those matured in vitro but mimicking the in vivo environment by in vitro conditions has been challenging. Until now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems present certain limitations. Therefore, alternative low-cost methodologies may help to optimize oocyte in vitro maturation. Here, we used two different systems to culture COCs and evaluate their potential influence on embryo development and quality. In the first system, we used treated fumed silica particles to create a 3D microenvironment (liquid marbles; LM) to mature COCs. In the second system, we cultured COCs in 96-well plates with different dimensions (flat, ultra-low attachment round-bottom, and v-shaped 96-well plates). In both systems, the nuclear maturation rate remained similar to the control in 2D, showing that most oocytes reached metaphase II. However, the subsequent blastocyst rate remained lower in the liquid marble system compared with the 96-well plates and control 2D systems. Interestingly, a lower total cell number was found in the resulting embryos from both systems (LM and 96-well plates) compared with the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption. None of the surface geometries influenced embryo development while oocyte maturation in liquid marbles led to reduced embryo development. These findings show that different geometry during maturation did not have a large impact on oocyte and embryo development. Lower embryo production after in vitro maturation in liquid marbles was probably detected because in vitro maturation was performed in serum-free medium, which makes oocytes more sensitive to possible toxic effects from the environment.