Estudo in vitro da fertilidade de espermatozoides criopreservados obtidos na cauda do epidídimo de touros

Abstract

This work aimed to study fertility of sperm obtained from epididymis tail of Nelore bulls and subjected to cryopreservation in extenders with three different concentrations of glycerol, and stabilization times at 5 °C and antioxidants addition to semen extender. Epididymides were obtained from slaughterhouse up to one hour after the death of the animal, placed in individual plastic bags and transported in box at room temperature (28 °C). At the laboratory, spermatozoa were recovered from epididymis tail using flotation technique and then diluted in Tris-yolk egg. In experiment 1 three concentrations of glycerol (3%, 5% and 7%) was evaluated at different stabilization times (0h, 2h and 4h). In the second experiment was evaluated the effect of adding enzymatic antioxidants in epididymal spermatozoa cryopreservation, resulting in following experimental groups: (Control; CAT 50 and 100 U/mL, and SOD 50 and 100 U/mL) at a concentration of 60 x 106 sperm/mL, packaged in straws (0.25 mL) and frozen in automated system. In both experiments, semen samples were thawed at 37 °C/30 sec was evaluated for plasma membrane integrity (PMi), acrosomal integrity (Aci), mitochondrial membrane potential (MMP) and sperm kinematics. In Experiment 1, PMi and Aci analyzes demonstrated values lower (P<0.05) for glycerol 3%, at stabilization time at 4h compared to other times. In kinematic evaluation, sperm values were lower (P<0.05) for BCF parameter in glycerol 3% and 7% groups at 2h of stabilization time compared to glycerol 5%; for ALH, glycerol 3% group in stabilization time at 4h demonstrated lowest (P<0.05) compared to other groups. Glycerol 5% group, settling time 0h, showed lower values (P <0.05) for the hPMM and Aci parameters, when compared to other groups. For IVF experiment, only glycerol 5% at stabilization time at 4h was used. Forty percents of embryonic viability were obtained from oocytes used. In experiment 2, parameters of MT, MP, VCL, VSL, VAP, STR, ALH and BFC did not differ (P> 0.05) between control and treated groups. However, significant differences (P <0.05) were observed between groups for Linearity (LIN) and oscillation index (WOB), being less than 100 SOD treatment (P<0.05) than other groups. Addition of SOD 100 determined lower (P<0.001) percentages of blastocysts post-cleavage. Based on results of kinematic and in vitro fertilization, it is possible to conclude that spermatozoa cryopreservation obtained from epididymis tail of bulls should be performed using extender with glycerol at 5% of concentration and stabilization time at 5 °C during 4h. Addition of CAT concentrations of 50 and 100 U/mL and SOD at concentration of 50 U/mL did not affect the sperm quality. Addition of SOD at a concentration of 100 U/mL reduces fertility of epididymal sperm after cryopreservation. However, it is noteworthy that sperm obtained from epididymis tail of bulls can be successfully used in IVF programs.