Redução do volume de percoll para seleção espermática de sêmen sexado na produção in vitro de embriões bovinos

Abstract

For in vitro production (IVP) of bovine embryos, different sperm selection techniques are used to recover the highest number of sperm with good quality and to remove components of the semen and diluent. Among these techniques, the selection by discontinuous Percoll gradients is currently the most used in bovine IVP laboratories. However, this technique has been modified mainly with the increase of the use of sex-sorted semen. The sex-sorted semen has distinct characteristics of conventional semen, such as low number of cells per dose and low post-thaw motility. Thus, the development of an efficient method for sperm selection, which increases the rate of recovery without impairing the quality of sperm is essential. The aim of the present study was to evaluate the effects of Percoll volume on recovery rate, sperm quality, oxidative stress and embryo development kinetics in bovine embryos IVP. Conventional or sex-sorted semen straws were thawed at 35°C for 20 s and distributed in equal aliquots into three different Percoll volume: 300 μL of each Percoll gradient (90, 60, and 30%), Control; 100 μL of each Percoll gradient, P100; and 200 μL of each Percoll gradient, P200. Sperm quality, fertilization rate, and embryonic development kinetics up to 48 h post-insemination were evaluated. For conventional semen, motility, vigor, and recovery x

rate improved in the P100 and P200 treatments compared to the Control (P < 0.05), whereas reactive oxygen species (ROS) levels, lipid peroxidation, and superoxide dismutase enzyme (SOD) activity were not influenced by treatments. For sex-sorted semen, P100 increased sperm curvilinear velocity, average path velocity, and amplitude of lateral head displacement (P < 0.05). Recovery rate was higher in P100 than in the Control and P200 (P < 0.05), formation of ROS was lower in P100 than in the Control and P200, and SOD activity was lower in P100 than in the Control. Fertilization and cleavage rates, moment of first cleavage, and cell number were similar between P100 and the Control (P > 0.05). Therefore, a Percoll volume of 100 μL increased sperm recovery rate without damage to sperm quality or early embryonic development, allowing to increase the number of inseminated drops / dose and to reduce costs.