Vitrification of in vitro produced porcine blastocysts: influence of cryoprotectants toxicity and embryo age

ABSTRACT

Background: Porcine embryos are sensible to all assisted reproduction manipulations, especially the ones that involvecryopreservation. 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 embryoproduction, the speed of development is often unevenly distributed. It is possible that their development speed, affectsembryo tolerance to cryoprotectants. This study aimed to evaluate the toxicity of porcine embryos of days 5 or 6 of cultureto cryoprotectant agents; as well as to assess embryo survival to vitrification.Materials, Methods & Results: Parthenogenetic porcine blastocysts and expanded blastocysts of days 5 and 6 of culturewere exposed to toxicity tests (experiments 1 and 2) and vitrification (experiment 3) using different protocols. In the firstexperiment, 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 0.5 M SUC; G2 16% EG + 16%DMSO with 0.4 M SUC; G3 18% EG + 18% DMSO with 0.5 M SUC). In the fresh Control, embryos of day 6 are moresensible 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 6compared 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 day6, 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 hatchedembryos, whereas in G1 the TCN was not affected. The second experiment compared EG combined to one of these threeextracellular...