Ethylene glycol is a CPA commonly used in vitrification solutions for bovine embryos [35] and [7] and Aqp3 channel may participate in the diffusion rate of this CPA during vitrification. Considering that dehydration and rehydration are important events during cryopreservation, this study aimed to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos. In addition, the relative expression of Aqp3 and Na/K ATPase isoform alpha 1 (ATPase1) gene was also evaluated in blastocysts with

different ability to undergo rehydration and after vitrification. http://www.selleckchem.com/products/blz945.html All chemicals were from Sigma Chemical (St. Louis, MO, USA) unless stated otherwise. Three experiments were carried out in order to evaluate: (1) the effect of culture media and stage of development in the capacity of in vitro-fertilized bovine embryos to undergo shrinkage and swelling; (2) the expression of Aqp3 and ATPase1 genes in embryos with different ability to undergo rehydration and; (3) the expression of Aqp3 and ATPase1 genes in embryos after vitrification/warming. Two trials were performed. In the first one, in vitro fertilized presumptive zygotes were co-culture with their own cumulus cells in SOFaac [14] or modified CR2aa (modified from Rosenkrans Jr. and First Selleckchem JNK inhibitor [27] – sodium chloride 108.0 mM,

potassium chloride 3.0 mM, sodium bicarbonate 26.0 mM, hemicalcium lactate 5.0 mM, sodium pyruvate 0.36 mM, glycine 10.0 mM, alanine 1.0 mM, glutamine 1.0 mM, minimal essential

medium amino acids [MEM] 10 μL/mL, basal medium Eagle [BME] amino acids 20 μL/mL and BSA 3 mg/mL), both supplemented with 10% fetal calf serum (FCS). Data of cleavage was collected at 72 h post insemination and blastocyst production at day 7 and 8 post-insemination. Six replicates were performed. The second trial evaluated the ability of blastocysts and expanded blastocysts, co-cultured in CR2aa or SOFaa as in the first trial, to undergo shrinkage and swelling. Embryos at day 7 post-insemination were exposed to a buffered hypertonic Tacrolimus (FK506) medium with 900 mOsm (TALP-HEPES supplemented with NaCl) for 5 min and then transferred to an isotonic medium where they remained for 10 min. Afterwards the embryos were cultured in CR2aa medium under 5% CO2 and 39 °C for 120 min. Pictures of embryos from each culture media were taken at 0, 5, 10 and 120 min (T0, T5, T10 and T120, respectively), for further area measurement and dehydration and rehydration calculations. Ability in dehydrate and rehydrate of embryos co-cultured in CR2aa or SOFaac and of embryos at different stages of development (blastocyst and expanded blastocyst) were compared. Six replicates were performed. In this experiment embryos cultured in CR2aa plus 10% (FCS) for 7 days post-insemination were exposed to a hypertonic medium in the same conditions of experiment 1.