How to beat the bull: Lycopene as a tool to improve in vitro fertilization efficiency in bulls with high polyspermy.

High levels of reactive oxygen species (ROS) derived from in vitro conditions compromise oocyte quality and subsequent polyspermy prevention by the zona and membrane block. Antioxidant supplementation, like lycopene, during in vitro maturation (IVM) mitigates ROS effects, yet, its efficacy in blocking polyspermy remains uncertain. This study aims to evaluate the effect of lycopene supplementation during IVM on oocyte maturation, fertilization, and developmental parameters. To this end, bovine oocytes were supplemented with 0.2 µM lycopene and fertilized with semen from three bulls. The three bulls showed different fertilization potential in vitro, with bull 1 showing the highest penetration and polyspermy rates and the lowest in vitro fertilization (IVF) efficiency. Interestingly, in bull 1, the treatment with lycopene improved IVF efficiency (p = 0.043) and reduced the polyspermy rate (p = 0.028). However, none of these effects were observed in bulls 2 and 3. Bulls with higher penetration rates exhibited better blastocyst rates although those rates did not seem to be associated with polyspermy or IVF efficiency. Oocyte mitochondrial distribution and activity and cortical granule migration and distribution were not influenced by lycopene. In conclusion, we demonstrated that lycopene addition during oocyte maturation had a positive impact on IVF efficiency by reducing polyspermy rates in a bull-dependent manner. The reduction in polyspermy rates was not caused by changes in cortical granule migration or oocyte mitochondrial distribution. Lycopene must therefore induce other changes in the oocyte that lower the in vitro penetration rates of specific bulls prone to polyspermy.

Alternative Culture Systems for Bovine Oocyte In Vitro Maturation: Liquid Marbles and Differentially Shaped 96-Well Plates.

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.

Transcriptomics Reveal Molecular Differences in Equine Oocytes Vitrified before and after In Vitro Maturation.

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.

Embryo morphokinetics derived from fresh and vitrified bovine oocytes predict blastocyst development and nuclear abnormalities.

Embryo development is a dynamic process and critical stages may go unnoticed with the use of traditional morphologic assessments, especially the timing of embryonic divisions and aberrant zygotic cleavage patterns. Bovine embryo development is impaired after oocyte vitrification, but little is known about the underlying morphokinetic behavior. Here, bovine zygotes from fresh (n = 708) and vitrified oocytes (n = 182) were monitored by time-lapse imaging and the timing and nature of early blastomere divisions were modeled to find associations with blastocyst development at day 8. The predictive potential of morphokinetic parameters was analyzed by logistic regression and receiver operating characteristic curve analysis to determine optimal cut-off values. Lag-phase was highly correlated with embryo development. Remarkably, 100% of zygotes that reached the blastocyst stage showed a lag-phase. Fast first cleavage increased the chance of blastocyst development to 30% with a cut-off of 32 h and 22 min. Aberrant zygotic cleavage events, including multipolar division, unequal blastomere sizes, and membrane ruffling resulted in decreased blastocyst development. Multipolar division leads to uneven blastomeres, which was associated with anuclear and multinuclear blastomeres, indicating genome segregation errors. Moreover, we described for the first time morphokinetics of embryos derived from vitrified bovine oocytes. Vitrification severely affected blastocyst development, although lower cryoprotectant concentration in equilibration solutions seems to be less detrimental for embryo yield. Impaired development was linked to slow cleavages, lower lag-phase incidence, and increased early embryonic arrest. Typically, less than 15% of the embryos produced from vitrified oocytes reached more than eight cells. Interestingly, the rate of abnormal first cleavage events was not affected by oocyte vitrification. In conclusion, time to first cleavage, the presence of a lag-phase, and the absence of aberrant zygotic cleavage were the best predictors of bovine blastocyst development for both fresh and vitrified oocytes.

New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification.

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.