In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.
Keywords: Cryopreservation; Cryoprotectant; Hoechst 33342; Mare; Meiotic competence.
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