94 USE OF β-MERCAPTOETHANOL AND CYSTEINE FOR IN VITRO MATURATION AND CULTURE OF SHEEP EMBRYOS

Abstract

During in vitro production (IVP), embryos are sensitive to suffering negative effects from catabolites, such as reactive oxygen species (ROS). Under physiological conditions, the action of the ROS is blocked by antioxidants such as glutathione, but glutathione's concentration is reduced during the main steps of the IVP process. The objective of the present study is to evaluate the effect of the supplementation of the media for in vitro maturation (IVM) and in vitro culture (IVC) with β-mercaptoethanol and cysteine on the rates of embryo development and viability after vitrification in open pulled straws (OPS). Ten IVP routines were conducted for IVP, using ovaries form pubertal sheep collected in a slaughterhouse. The ovaries were kept in a saline/antibiotic solution at 30°C during transport to the laboratory. The cumulus oophurus–oocytes complexes (COC) selected for IVM were allocated to 2 treatments: T1 (control), including no antioxidants in the IVM and IVC media (n = 676); and T2, including 50 μM β-mercaptoethanol and 600 μM cysteine, in the IVM and IVC media (n = 729). The IVM was conducted using the TCM 199 medium including oestradiol, FSH, LH, pyruvate, heat inactivated sheep serum and antibiotics, for 22 to 24 h. Sperm selection was conducted by swim-up in medium with tris-glucose-citric acid with fresh semen. For IVF, conducted for 18 to 22 h, 1 × 106 spermatozoa per mL were used in SOF medium including 2% heat-inactivated sheep serum. Both IVM and IVF were conducted with incubation with 5% CO2 at 39°C with saturated humidity. After IVF, the probable zygotes were denuded and cultured for 8 days in SOF medium with 0.4% BSA, at 39°C, in bags with 3 gases (5% CO2, 90% N2 and 5% O2). The criteria considered for embryo viability were: cleavage rate at Day 2 (cleaved/inseminated), embryo development at Day 7 (blastocysts/cleaved) and the reexpansion rate 24 h post-vitrification. Such frequencies were compared between treatments by the chi-squared test. The cleavage rate did not differ (P > 0.05) for T1 (60.3%) and T2 (64.3%). The rate of embryro development at Day 7 was also similar (P > 0.05) for T1 (33.6%) and T2 (36.6%). The reexpansion rate for T1 (76.9%) and T2 (54.1%) were also similar (P > 0.05). Thus, supplementation of IVM and IVC media with β-mercaptoethanol and cysteine presented no effect in the development and viability of vitrified sheep embryos. CAPES, MARFRIG Group.