Viabilidade de oócitos bovinos transportados por diferentes períodos em meio de maturação suplementado com análogo da Vitamina E

Abstract

Oocytes with decreased development capacity have been identified as the main cause for the reduced potential of embryos in vitro produced. The transport of the oocytes from properties to the laboratories is a fundamental factor, since the maturation begins immediately after the oocyte removal from its follicle, and the interval between the recovery and the laboratory maturation directly influence its development. The transport duration can last for several hours and can hinder subsequent embryonic development. Furthermore, the in vitro conditions results in higher oxygen concentrations, leading to an increase in the Reactive Oxygen Species (ROS) levels, and the cell membranes is very susceptible to lipid peroxidation caused by these agents. The Trolox® is a vitamin E analogue and acts as a protective agent against lipid peroxidation. Thus, it was assessed the viability of oocytes transported with a controlled gaseous atmosphere for different periods of time, and the effects of adding Trolox® the medium used on the transport. Were used 1107 oocytes from slaughterhouse ovaries, divided into eight groups according to the simulated transport period and the presence of Trolox®: zero (G0), six (G6), twelve (G12) and eighteen (G18) hours of simulated transport without antioxidant, and same periods (GT0, GT6, GT12, GT18), with the addition of 0.1 mg / ml Trolox® to the transport medium. Oocytes were placed in 1.5 ml cryogenic tubes containing 500μL transport medium (TCM-199® supplemented with HEPES, glutamine, pyruvate, antibiotics, estradiol, LH, FSH and SFB), covered with 350μL of mineral oil, gassed with a mixture of 5% CO2, 5% O2, 90% N2 balance and sealed. Oocytes of groups G0 and G10 were placed unclosed in the incubator and matured for 24 hours at 38.5 ° C with 5% CO2 in air. The other groups were placed closed inside the incubator and opened as pasted their respective transport period, completing the remaining time of maturation under the same conditions of the control group. This system would mimic the transport of oocytes in a portable incubator, simulating what

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occurs from the countryside to the laboratory after OPU. The fertilization period was 18-22 hours in similar conditions of temperature and gaseous atmosphere in medium Talp-Stock plus BSA, pyruvate, gentamicin, heparin and PHE. Presumptive zygotes were cultured for seven days in SOFaaci medium + 5% fetal calf serum, in an incubator at 38.5 ° C with 5% CO2, 5% O2 and 90% N2. The rates of cleavage, embryo production, cell count and oxidative stress were evaluated. Data were analyzed with SAS® version 9.2. Parametric data were analyzed using ANOVA and Tukey's test, as the nonparametric using the Wilcoxon test. All analyzes used 5% level of significance. Cleavage rates were similar among all groups, with difference (p <0.05) only between G0 (87.05%) and G12 (68.30%). The blastocyst production, total number of embryonic cells and TBARS concentration in the transport medium were not different (p> 0.05). These results indicate that transportation of bovine oocytes up to 18 hours can happen without any losses to the subsequent embryonic development and the addition of Trolox® in the transport medium does not influence the cleavage and blastocyst rates.