217 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION EMBRYO DEVELOPMENT IN BOVINE USING HIGH CYSTEAMINE CONCENTRATION DURING IN VITRO MATURATION AND SPERM CO-CULTURE WITH CUMULUS‐OOCYTE COMPLEXES

Abstract

In bovine, the intracytoplasmic sperm injection (ICSI) technique remains inefficient probably because of low levels of male sperm decondensation. In species with frequent fertilization failure, high cysteamine (Cys) concentration during in vitro maturation (IVM) has been used to improve IVF. Cysteamine, a precursor of glutathione, plays a critical role on sperm decondensation. The aim of this work was to improve ICSI efficiency in bovine by (1) increasing endogenous glutathione levels from oocytes using high Cys during IVM; and (2) incubating sperm with cumulus-oocyte complexes (COC) before ICSI, to mimic the physiological capacitation process. In experiment 1, we tested the effect of high Cys concentrations during IVM over the development of IVF embryos. In experiment 2, we performed ICSI after IVM with 1 mM Cys, based on IVF results. The COC were collected from slaughtered cow ovaries and IVM for 21 h with 10, 1, and 0.5 mM Cys v. 0.1 mM Cys (standard condition). Then, IVF was performed using 16 × 10⁶ sperm mL^–1 for 5 h on BO medium. For ICSI, COC were IVM with 1 mM Cys (ICSI 1 mM groups), and sperm used for injection was previously incubated with COC for 3 h (Inc. groups), as was done for IVF. Sham and diploid parthenogenetic (PA Diplo) controls were also included. Metaphase II oocytes were selected for ICSI, and injected oocytes were activated by a 4-min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h (except for PA Diplo) and treated with 2 mM DMAP for 3 h. For ICSI control groups, COC were matured using 0.1 mM Cys. All embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post-IVF/ICSI, respectively. The total cell numbers of blastocysts were counted at Day 7, after Hoechst 33342 staining. Results are shown in Table 1. In conclusion, an increase of 5- to 10-fold of Cys concentration during IVM was not detrimental for development to blastocyst after IVF. The use of 1 mM Cys during IVM combined with the use of sperm co-cultured wit IVM COC before sperm injection is a good strategy to improve in vitro development of bovine ICSI embryos.

Table 1.  Effect of 1 mM cysteamine (Cys) during IVM over the development of IVF bovine embryos (top part) and effect of 1 mM Cys during IVM over embryo development of ICSI embryos, using sperm previously incubated (Inc.) with COC (bottom part)