Approaches to improve ICSI mediated transgenesis (TM-ICSI) and maximize the use of sex sorted sperm in bovine.

CANEL N; BEVACQUA R; HIRIART, M.I; CHAVEZ RABELO; CAMARGO; DANIEL SALAMONE

Congreso; 41th Anual Conference of the International Embryo transfer Society (IETS).; 2015

TM-ICSI is an effective tool for transgenic animal production. However, ICSI in cattle remains inefficient. In this work, we assayed approaches to improve egfp expressing blastocysts production by ICSI: the sperm pre treatment with heparin and L-glutathione (Hep-GSH), the use of sex sorted sperm (SS), the re frozen/thawing of SS sperm, and the combination of them. Quality of ICSI blastocysts was analyzed by studying the expression of 4 genes, and the rates of DNA fragmentation. Methods: COCs from slaughtered cow ovaries were IVM for 21 h. Non sorted (NS) and sex sorted (SS) frozen straws were thawed. Some of them were incubated with 80 μM Hep-15 mM GSH for 20 h (Hep-GSH+). Hep-GSH- control group was not pre treated. Semen samples were co-incubated with 50 ng/µl of pCX-EGFP for 5 min before ICSI. Moreover, the SS sperm that are usually discard after ICSI were cryopreserved and used for ICSI after a second thawing (ICSI SS Re Frozen). ICSI NS, Sham and diploid parthenogenetic (Diplo PA) controls were included. Oocytes were activated with 5 µM ionomycin for 4 min, TCM-199 for 3 h (except for Diploid PA) and 1.9 mM DMAP for 3 h. Cleavage and blastocyst/egfp expression rates were evaluated on days 2 and 7 post ICSI, respectively. Results are shown in Table 1. Relative expression of HMGN1, GLUT5, AQP3 and OCT4 genes from ICSI NS Hep-GSH+ and IVF blastocysts were compared by qPCR. Data was analyzed by the Pair Wise Fixed Reallocation Randomisation TEST. None of the 4 genes showed expression differences between groups. DNA fragmented nucleus index/blastocyst cell number were determined by TUNEL assay, not showing differences between groups (Kruskal-Wallis test, p