216 EFFECT OF SPERM PRETREATMENT WITH LYSOLECITHIN AND Triton X-100 ON PRONUCLEAR FORMATION AND QUALITY OF BOVINE EMBRYOS PRODUCED BY INTRACYTOPLASMIC SPERM INJECTION

Abstract

In cattle, intracytoplasmic sperm injection (ICSI) has low efficiency and has application for sexed semen, semen of poor quality of high genetic value, or semen of endangered breeds. The acrosome content has a harmful effect within the oocyte because of the presence of hydrolytic enzymes. With the aim of removing the acrosome and destabilising the membranes, cryopreserved bovine spermatozoa were treated with lysolecithin (LL) and Triton X-100 (TX). Frozen straws of bull semen were thawed and the spermatozoa were selected by Percoll gradient. The selected spermatozoa were incubated at concentrations of 0.05% LL (ICSI-LL), 0.05% TX (ICSI-TX), and a control group (without treatment) for 1 min with vortex, and were then immediately washed by centrifugation at 400 × g in Sp-TALP medium. Following ICSI, oocytes were activated with 5 mM ionomycin and 5 µg mL^–1 cycloheximide. Embryos were cultured in KSOM medium at 38.5°C, 5% CO₂, 5% O₂, and 90% N₂, to Day 8 to record blastocyst rate. Pronuclear formation was evaluated at 19 h after activation, and the presumptive zygotes were fixed in methanol-glacial acetic acid (3 : 1), stained with Hoechst, and analysed with a fluorescence microscope. The results were evaluated by a chi-squared test with Bonferroni’s correction. Significance was set at P < 0.05. The quality of embryos was evaluated by counting the total cell numbers (T), inner cell mass cells (ICM), and trophectoderm cells (TE) after staining with Hoechst and propidium iodide. We injected 345 oocytes in the control group, 335 in ICSI-LL, 304 in ICSI-TX, and 351 were parthenogenetically activated (12 replicates for each group). A higher cleavage rate was observed in ICSI-TX (66%; 231/351) and ICSI-LL (62%; 207/335) compared the control group (53%; 183/345). In addition, a significantly higher blastocyst rate was observed in both treatments: 27% (82/304) and 27.5% (92 of 335) for ICSI-TX and ICSI-LL groups, respectively, compared with the control group (22.6%; 78/345). In the parthenogenetic activation group without ICSI, the cleavage rate was 41.9% (147/351) and blastocyst rate was 15.4% (54/351). Differences among treatments were analysed using one-way ANOVA after arcsine transformation, and significance was set at P < 0.05. No differences were observed in pronuclear formation (with male and female pronuclei and 2 polar bodies) for ICSI-LL (77%; 48/53), ICSI-TX (76.4%; 39/51), and the control group (71.6%; 38/53) with 3 replicates for each group. No significant differences were observed in the quality of the embryos, ICM cell number (control: 35.7 ± 2.8, ICSI-LL: 38 ± 4.5, and ICSI-TX: 40.8 ± 8.2), TE (control: 139 ± 31.5, ICSI-LL: 124 ± 10.4, and ICSI-TX: 129 ± 18.5), T (control: 170 ± 25.6, ICSI-LL: 156 ± 14.3, and ICSI-TX: 176 30.3), with 8 replicates for each group. In conclusion, this study proposed treatments able to improve the rate of development without affecting the quality of embryos produced by ICSI.