Three replicates were performed. Embryos from each group were transferred individually to a cryotube, rapidly frozen in liquid N2 and learn more stored at −80 °C for further RNA extraction and PCR analysis. Total RNA was extracted from three pools of five blastocysts of both groups and quantification of Aqp3 and ATPase1 transcripts relative to β-actin gene was performed in duplicate by real time PCR for further comparison between groups. Expanded blastocysts co-cultured in CR2aa plus 10% (FCS) were vitrified by the Open Pulled Straw (OPS) method [35] in a solution with 20% dimethyl sulphoxide (DMSO) and 20% ethylene glycol (EG).

After warming, embryos were co-cultured in CR2aa medium with granulosa cell monolayer for 72 h. The control group consisted of fresh embryos (non-vitrified). Post warming survival was assessed by their re-expansion and hatching at 72 h. Total of eight replicates were performed. Vitrified-warmed and fresh embryos were transferred individually to a cryotube, rapidly frozen in liquid N2 and stored at −80 °C for further

RNA extraction and PCR analysis. Total RNA was extracted from two pools of five re-expanded embryos at 72 h and relative quantification of Aqp3 and ATPase1 transcripts was performed in duplicate by real time PCR. Ovaries were obtained at a local slaughterhouse and shipped to laboratory in saline solution (0.9% NaCl with 0.1 g/L streptomycin) at 36.0 °C. Follicles were aspirated and cumulus–oocyte complexes (COCs) with more than three compact layers of cumulus cells and oocyte with homogeneous cytoplasm were matured in tissue culture medium (TCM-199, Obeticholic Acid solubility dmso Gibco Life Technologies, Inc., Grand Island, NY, USA) supplemented with 20 μg/mL follicle stimulating hormone (FSH; Pluset, Serono, Italy), 0.36 mM sodium pyruvate, 10 mM sodium bicarbonate and 50 mg/mL streptomycin/penicillin in a humidified atmosphere of 5% CO2 at 38.5 °C for

24 h. For in vitro fertilization, frozen/thawed semen was centrifuged at 9000g for 5 min in a Percoll discontinuous density gradient (45–90%) to obtain motile spermatozoa. The pellet was centrifuged again at 9000g for 3 min in Fert-TALP medium [12]. In vitro fertilization was performed in 100-μL drops of Fert-TALP supplemented with 2 × 106 spermatozoa/mL, 20 μg/mL of heparin and Sitaxentan 6 mg/mL of fatty acid free BSA fraction V, covered with mineral oil, for 21 h in a humidified atmosphere of 5% CO2 and 38.8 °C in air. Presumptive zygotes were partially denuded and co-cultured in CR2aa or SOFaac media with 10% FCS (Nutricell, Campinas, SP, Brazil) with their own cumulus cells under 5% CO2 and 39 °C in high humidity for 192 h post-insemination (hpi). Cleavage was assessed at 72 hpi and blastocyst at 168 (day 7) and 192 (day 8) hpi. Grade I (according to the IETS Manual [29] blastocysts and expanded blastocysts underwent osmotic challenge.