Gamete and embryo cryopreservation are core assisted reproductive tools that facilitate transport, use and long-term storage of valuable genetics. It has been demonstrated that the cryoprotectants contained in the cryopreservation extenders as well as the process itself alter the chemical and physical proprieties of the plasma membrane of gametes/embryos. Specifically cryopreservation induces structural changes in the plasma membrane and cytoskeleton, increases mitochondrial depolarization, enhances ROS production and in turn triggers apoptosis. Furthermore, tolerance for cryopreservation notably varies depending on the type of genetic material. Specifically, epididymal sperm cryopreservation is less successful than ejaculated sperm because it has had no contact with seminal plasma (the main source of spermatozoal antioxidant enzymes and cholesterol). On the other hand, oocytes and early embryonic stages (zygotes to 8 cells) exhibit lower tolerance for vitrification due to the low permeability of their zona pellucida; in addition, the quality of the embryos produced in vitro is lower than the quality of those produced in vivo being more susceptible to irreversible damage during vitrification. Therefore, in the experiments carried out in the present Doctoral Thesis we focused our efforts on the improvement of gamete and embryo cryopreservation at the more susceptible stages to freezing-induced damage. To achieve this goal we analyzed the effect of N-acetylcysteine addition to vitrified murine oocytes and embryos (2-cell stage) at different time points as well as to cryopreserved Lidia bull epididymal spermatozoa in order to determine the usefulness of this antioxidant. In addition, an attempt was made to determine whether the use of alternative cryoprotectants such as dimethylformamide (DMF) improved post-thaw semen quality of Lidia bull epididymal sperm after prolonged storage at 4°C (24-96 hours).Our results showed that prolonged storage of Lidia bull epididymis up to 96 hours did not seem to negatively affect sperm motility, as significant differences in their main advanced motility parameters (VCL, VSL, VAP, ALH and LIN) were not found when sperm were cryopreserved using an extender added with glycerol (7%, v/v) or glycerol and DMF (at 3.5%, v/v each). When 1 or 2.5 mM of NAC were added to any of the sperm freezing extenders total and progressive motility, viability, mitochondrial membrane potential, ROS production, and DNA fragmentation or acrosomal intactness remained unaffected despite prolonged cooled storage.On the other hand, when murine 2-cells embryos produced in vivo or in vitro were vitrified and warmed, vitrification or addition of 1 mM of NAC did not negatively affect their development to the expanded blastocyst stage compared to the fresh controls. Interestingly, the quality of the expanded blastocysts derived from vitrified 2-cell embryos produced in vivo significantly increased when the antioxidant was added at days 1.5 and 2.5 after freezing and at day 1.5 in IVF-derived embryos, as the total blastomere count at day 4 was significantly enhanced compared to the vitrified control devoid of NAC.In our last batch of experiments, murine oocytes were vitrified in presence or absence of NAC (1 mM) which was added prior to or after vitrification. In oocytes added with NAC prior or after vitrification higher mitochondrial polarization was observed compared to vitrified controls. Interestingly, ROS production and ATP content was similar in fresh and vitrified oocytes. However, ATP content significantly decreased when NAC was added prior vitrification, compared to NAC supplementation after warming. ROS production was consistently enhanced in NAC-supplemented oocytes compared to the vitrified controls, disregarding the time at which the antioxidant was added. On the other hand, a significant increase in the number of blastomeres was observed when the addition of NAC was carried out after vitrification compared to its addition prior to vitrification.In conclusion, the addition of N-acetylcysteine does not seem to exert any beneficial effect on the post-thaw quality of Lidia bull epididymal sperm; however, its addition to murine oocytes and early embryos improves the quality of the derived expanded blastocysts depending upon the moment of addition.
