132 VITRIFICATION SYSTEM (OPEN AND CLOSED) IN NEW INCUBATOR WITH REDUCED OXYGEN

Abstract

Most IVF laboratories uses high oxygen tension (20%) during embryonic culture. However, it is known that under physiological conditions, oxygen tension in the female reproductive tract ranges between 2 and 8%. Therefore, the aim of this study was to evaluate survival and hatching rate after in vitro culture of vitrified/thawed bovine in vitro-produced blastocysts cultured under different oxygen concentrations. The experiment consisted of comparing 2 culture systems using different concentrations of oxygen: conventional incubator (20% O₂, Thermo Scientific, model 3130) and a new incubator (5% O₂, WTA Watanabe Tecnologia Aplicada, model Eve). Only Day 7 expanded blastocysts grade 1 were used. Embryos were produced according to conventional IVF protocols. Briefly, cumulus-oocyte complexes were aspirated from postmortem ovarian follicles, matured in TCM199 + 10% FCS + 0.5 mg FSH mL^–1 + 50 mg LH mL^–1 + 1 mg oestradiol mL^–1, for 24 h at 38.5°C, 5% CO₂ in air. Live spermatozoa from Nellore bull were obtained by centrifugation in Percoll gradients (45 and 90%) and cultured with cumulus-oocyte complexes at 1 million of sperm mL^–1 in TALP medium + 10 mg of heparin mL^–1. After 20 h incubation, zygotes were transferred to CR2 + 2.5% FCS + 4 mg of BSA mL^–1 and granulosa monolayer for 7 days. Expanded blastocysts were randomly allocated to 2 treatments for vitrification (open system – cryotop and closed system – HSV Kit, IMV-Technologies) using the same vitrification media and protocol (VS1: 10% ethylene glycol + 10% dimethyl sulfoxide and VS2: 20% ethylene glycol + 20% dimethyl sulfoxide for 8 min and 50 s, respectively). After exposure to the vitrification solutions, 2 embryos were loaded/straw, and the straws were plunged into LN. The warming procedure consisted of, immediately after removal from LN2, transferring the embryos in 2 successive warming solutions with decreased concentrations of sucrose (1 M and 0.50 M for 5 min each). The vitrified/rewarmed embryos were transferred to in vitro culture. There were no differences in survival rates (P < 0.05) between the open and closed vitrification system for blastocysts produced in reduced oxygen in the Eve incubator – 5% O₂ (96% – 109/114 and 98% – 158/161, respectively) compared with embryos produced in the high oxygen environment in the Thermo incubator – 20% O₂ (93% – 214/230 and 92% – 94/102, respectively). Hatching rates were increased for blastocysts cultured in the lower oxygen environment (EVE treatment: 95 and 98%, respectively, for open and closed vitrification protocols) when compared with the high oxygen environment (Thermo treatment: 86 and 87%, respectively, for open and closed systems); P < 0.05. In vitro culture in a reduced-oxygen environment improves blastocysts competence after vitrification.

Financial support was received from CNPq-RHAE.