New Alternative Mixtures of Cryoprotectants for Equine Immature Oocyte Vitrification

SIMPLE SUMMARY: Oocyte cryopreservation allows female gametes to be conserved for long periods, which would be of benefit for mares of high genetic merit, but its efficiency is not satisfactory yet. Therefore, the aim of this study was to optimize a vitrification protocol for equine oocytes using a systematic approach. We performed a side-by-side comparison of different cryoprotective agents (CPAs) during the vitrification and warming of equine oocytes. In the first experiment, a fixed mixture of CPAs that enter the oocyte was used, and three sugars were compared, which cannot penetrate the oocyte but provide protection through an osmotic effect. In the second experiment, one sugar from the first experiment was selected to compare three mixtures of CPAs that enter the oocyte. Overall, the embryo development was reduced after oocyte cryopreservation when compared to fresh oocytes. Yet, we were able to produce embryos with all six cryoprotective agent mixtures, and we identified one promising combination of cryoprotectants, consisting of propylene glycol, ethylene glycol, and galactose, that resulted in blastocyst rates in the same range as the fresh control group. ABSTRACT: 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.