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Oocyte and embryo cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig oocytes, because of their greater susceptibility to cryoinjuries, have shown a reduced ability to be fertilized in vitro and a lower developmental competence. The aim of this study was to evaluate the apoptotic status of porcine oocytes vitrified by Cryotop method. We assessed three parameters linked to the activation of the apoptotic cascade: the exteriorization of phosphatidylserine using Annexin V, the caspase activation using FITC-VAD-FMK and the alteration of plasma membrane permeability employing YO-PRO-1. These assays were performed on control oocytes, oocytes exposed to vitrification solutions (toxicity control) and vitrified oocytes either immediately after warming or after incubation for 2 h into maturation medium. The exposition to vitrification solutions triggered an increase of the percentage of oocytes both faintly (VAD+ PI−) and strongly (VAD++ PI−) labeled by FITC-VAD-FMK but not a significant modification of the number of oocytes Annexin V (A+ PI−, early apoptotic) and YO-PRO-1(YP+ PI−, apoptotic) positive in comparison with control oocytes. Oocytes submitted to the whole vitrification procedure showed a rise of the percentage of early apoptotic oocytes (A+ PI−) and FITC-VAD-FMK positive oocytes (VAD+/VAD++ PI−) and a contemporaneous increase of the number of dead oocytes (PI+). On the contrary, vitrified oocytes analyzed immediately after warming did not show a significant increase in the percentage of apoptotic oocytes (YO-PRO-1+/PI−) as compared with the control. Post warming incubation for 2 h into maturation medium, in comparison with oocytes analyzed immediately after warming, did not induce any increase in the percentage of early apoptotic (A+ P−) oocytes while a decrease of the percentage of VAD+/PI− oocytes and a contemporaneous increase of VAD−/PI− oocytes were observed. Moreover, the post-warming incubation induced a rise of the percentage of apoptotic oocytes (YO-PRO-1+/PI−). All these data confirm the involvement of apoptotic mechanisms on the injuries induced by vitrification procedure in pig oocytes; explanation of this phenomenon could be useful to improve oocytes’ cryopreservation protocols.

Three experiments were designed to evaluate the effects of vitrification using Cryotop method on MII porcine oocyte viability, chromosomes configuration, meiotic spindle morphology and in vitro fertilization; to do this, in vitro matured oocytes were subjected to the cryoprotectant treatment excluding the plunging into liquid nitrogen, the whole vitrification/warming/rehydration procedure or no treatment (control). In experiment 1 viable oocytes were not reduced by either cryoprotectants or vitrification when they were evaluated immediately after warming and cryoprotectant dilution. However, after a 2 h incubation, the survival rate significantly decreased (P < 0.05). In experiment 2 cryoprotectant exposure significantly (P < 0.05) influenced spindle morphology even if chromosome organization did not vary, while vitrification significantly (P < 0.05) increased oocytes with damaged spindles and chromosomes displaced from the metaphase plate. No significant improvements in these parameters were observed after 2 h of incubation but, on the contrary, the rate of oocytes with normal chromosome configuration was reduced. In experiment 3 significant differences among the three groups in the fertilization rate but not in the percentages of monospermy fertilization were recorded; in addition, exposure to cryoprotectants and vitrification significantly (P < 0.05) increased degenerated oocyte rate. Overall, these findings confirm that porcine oocytes at MII stage are very sensitive to vitrification, which reduces the rate of viable oocytes and alters microtubule organization, thus impairing fertilization; in addition, incubation of oocytes for 2 h after devitrification seems to be detrimental rather than ameliorative. Further improvements of the current protocol will be necessary in order to optimize the Cryotop method for vitrifying pig matured oocytes.