Improvement of ICSI embryo development in bovine using high cysteamine concentration during IVM and sperm co culture with COCs.

CANEL N; SUVÁ M; BEVACQUA R; SALAMONE D

Congreso; 42nd Annual Conference of the IETS; 2016

In bovine, ICSI technique remains inefficient mainly due to low levels of male pronucleus formation. High concentration of the glutathione precursor cysteamine (Cys) during IVM has improved the IVF outcome in species with frequent fertilization failure. The aim of this work was to improve ICSI efficiency in bovine by: a) increasing endogenous glutathione levels of oocytes using high Cys during IVM; and b) incubating sperm with COCs shortly before ICSI, for mimicking 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. COCs were collected from slaughtered cow ovaries and IVM for 21 h with 10, 1 and 0.5 mM Cys vs. 0.1 mM Cys (standard condition). IVF was performed following Brackett and Oliphant protocol, using 16x106 sperm/ml. For ICSI, COCs were IVM with 1 mM Cys (ICSI 1 mM groups), and sperm used for injection was previously incubated with COCs for 3 h (Inc groups), as previously done for IVF. Sham and diploid parthenogenetic (PA Diplo) controls were also included. MII 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 COCs 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. Total blastocysts cell number was counted at day 7, after hoechst 33342 staining. Differences among treatments were determined by Fisher′s exact test for cleavage and blastocyst rates (p≤0.05), or by Kruskal-Wallis with Dunn?s correction for mean cell number of blastocysts ± SD (p≤0.01). Results from experiment 1 showed a decrease in cleavage (n=116, 12.9%), blastocyst rates (3.5%), and mean number of cells from IVF blastocysts (85±20) when 10 mM Cys was used on IVM. No differences were found for 1 (n=153, 77.8%, 41.2% and 120±42), 0.5 (n=135, 79.3%, 32.6% and 123±53) and 0.1 mM groups (n=115, 82.7%, 37.4% and 131±41). On experiment 2, ICSI 1 mM-Inc rates did not differ from Sham group (n=106, 88.7 and 20.8%) and were lower than PA Diplo control (n=144, 95.8 and 61.1%). However, ICSI 1 mM-Inc group showed higher cleavage (n=116, 79.3%) and blastocyst rates (23.3%) than ICSI 1 mM (n=108, 51.9 and 17.6%), ICSI 0.1 mM-Inc (n=117, 91.5 and 11.1%) and ICSI 0.1 mM (n=132, 59.9 and 13.7%). Although Inc increased cleavage rates of ICSI embryos, both using 1 and 0.1 mM Cys during IVM, the development to blastocyst was only improved for ICSI 1 mM-Inc. In conclusion, an increase of 5 to 10 folds of Cys concentration during IVM is not detrimental for development to blastocyst after IVF. The use of 1 mM Cys during IVM combined with the use of sperm co cultured with in vitro matured COCs prior to sperm injection, might be a good strategy to improve in vitro development of bovine ICSI embryos.