Vitrification of In Vitro Produced Porcine Blastocysts: Influence of Cryoprotectants Toxicity and Embryo Age

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involve cryopreservation. Despite the high cryoprotectant concentrations routinely applied, vitrification is the most effective technique to date. These substances toxicity can also play a negative role in embryo viability. During in vitro porcine embryo production, the speed of development is often unevenly distributed. It is possible that their development speed, affects embryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of culture to cryoprotectant agents; as well as to assess embryo survival to vitrification.Material, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culture were exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the first experiment, three different cryoprotectants were used (Dimethyl sulfoxide - DMSO, Ethylene glycol – EG, and Sucrose - SUC), combined in three different associations (G1: 15% EG + 15% DMSO with 0.5M SUC; G2: 16% EG + 16% DMSO with 0.4M SUC; G3: 18% EG + 18% DMSO with 0.5M SUC). In the fresh Control, embryos of day 6 are more sensible than the ones of day 5, whom showed a lower hatching rate (39.7 vs. 60.8%). After the toxicity (Experiment 1) test, the G1 showed better expansion rates in day 6 (50.0 vs 31.0 and 3.6% for G2 and G3) and higher hatching of day 6 compared to G2 and G3 (23.2, vs. 8.6 and 0.0% for G2 and G3). The fresh non hatched embryos at day 8, derived at day 6, had a lower percentage of cells with cleaved caspase-3 (20.2%) compared with the G1 (30.5%), G2 (31.4%) and G3 (30.5%). The hatched embryos of day 5 from G2 had lower total cell number (TCN) compared with the day 6 hatched embryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these three extracellular cryoprotectants: Polyvinylpyrrolidone/sucrose/trehalose (respectively groups: PVP, SUC, TRE). The group SUC has raised the best results for day 5 embryos, whereas for day 6 embryos SUC and TRE were both best. The third experiment tested four vitrification protocols, being P1: EG+DMSO+TRE/warming with SUC; P2: EG+DMSO+TRE/warming TRE; P3: EG+TRE/ warming SUC; P4: EG+TRE/warming TRE. The expansion of vitrified day 5 embryos was higher in the P1 (20.0%) in comparison with the other three groups (4.3, 4.3 and 4.4% for P2, P3 and P4, respectively), with no difference for their hatching rates, been it lower comparing to the Control. Day 6 embryos showed no difference in expansion and hatching for the vitrified groups, been them lower than the Control.Discussion: Embryos obtained on day 6 are more sensible than the ones of day 5, fact observed when the embryos were exposed to cryoprotectant solution, as well by the behavior of the no treated Control embryos. The toxicity increases as it does the concentration of intracellular cryoprotectant, where over 16% of the intracellular cryoprotectors already affected the day 6 embryos development. For the day 5 embryos however, 15 or 16% of the intracellular cryoptrotectors, had similar behavior to the embryos. For the extracellular solutions, however, it is variable according the embryos development speed. Indeed, it is necessary to adjust the cryoprotectors to be used to cryopreserve porcine in vitro produced embryos obtained at days 5 and 6 of culture.