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.

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.

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. Till now, conventional two-dimensional (2D) systems have been used for in vitro maturation of bovine cumulus-oocytes-complexes (COCs). However, using such systems may cause cell flattening and does not allow cumulus expansion in all dimensions, which is less physiological. Therefore, implementing a low-cost and highly effective in vivo-like microenvironment methodology 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 to 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 to the control. In conclusion, oocytes matured in liquid marbles or 96-well plates showed no remarkable change in terms of meiotic resumption, embryo development, and quality in both systems. None of the surface geometries influenced embryo development. These findings provide important inferences in many aspects of oocyte and embryo development. Further investigation is needed to determine other aspects like toxicity testing and ultrastructural changes in oocytes.

No abstract

No abstract