218 IMPROVEMENT OF INTRACYTOPLASMIC SPERM INJECTION MEDIATED TRANSGENESIS (TM-INTRACYTOPLASMIC SPERM INJECTION) USING BULL SPERM PRETREATED WITH HEPARIN AND GLUTATHIONE

Abstract

TM-intracytoplasmic sperm injection (ICSI) was demonstrated to be an effective technique for the production of transgenic animals. However, this method has not been widely applied for transgenesis in cattle, because of the low embryo developmental rates. This problem may be related to the incomplete sperm decondensation and subsequent pronuclei formation that occurs in cattle after ICSI (Malcuit et al. 2006 Reprod. Fertil. Dev. 18, 39–51). Delgado et al. showed that pretreatment with heparin-sodium salt combined with reduced glutathione (Hep-GSH) could improve bull sperm decondensation (2001 Archives of Andrology 47, 47–58). The objective of this work was to test the use of pretreated sperm with Hep-GSH for TM-ICSI, because an improvement of male pronucleus formation could cause an increase on the frequency of exogenous DNA integration. To this aim, cumulus-oocyte complexes were collected from slaughtered cow ovaries and in vitro matured for 21 h. Frozen sperm from a bull that was previously determined to produce low developmental rates post ICSI and IVF was used. It was thawed and washed twice by centrifuging at 390 × g for 10 min. After that, sperm were incubated with Tris medium supplemented with 80 μM Hep and 15 mM GSH for 20 h. After washing, semen was co-incubated with 50 ng μL^–1 of pCX-EGFP plasmid for 5 min on ice and used for ICSI (Hep-GSH ICSI group). An ICSI control group was injected with semen not treated with Hep-GSH. Sham controls were injected with 50 ng μL^–1 of pCX-EGFP. Haploid and diploid parthenogenetic controls were also included (Haplo PA and Diplo PA groups). Oocytes were activated by a 4 min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h, and treated with 1.9 mM DMAP for 3 h; Diplo PA were immediately exposed to DMAP after ionomycin treatment. Embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post ICSI, respectively. Expression of egfp was assayed at Day 4 and at the blastocyst stage. Results: Hep-GSH ICSI group showed higher cleavage rates than ICSI control (68.5%, n = 89 v. 35%, n = 60), and lower than Sham, Diplo PA, and Haplo Pa groups (94% n = 50, 95.1% n = 61, and 85.1% n = 47, respectively; Fisher's exact test, P ≤ 0.05). Although blastocyst rates from ICSI groups did not differ from Haplo PA (21.2%) and Sham groups (8%), Hep-GSH ICSI produced higher rates than ICSI control (19.1 v. 5%). The higher blastocyst rates were observed for Diplo PA (47.5%; P ≤ 0.05). Transgene expression levels at Day 4 were higher for both Hep-GSH ICSI and ICSI control than for Sham control (24.7 and 11.7% v. 0%, respectively; P ≤ 0.05). Rates of egfp expressing blastocysts/injected oocytes were significantly higher for Hep-GSH ICSI than for ICSI and Sham control groups (13.5 v. 1.7 and 0%, respectively; P ≤ 0.05). Conclusions: Pretreatment of bull sperm with Hep-GSH can increase blastocyst rates after ICSI, even when low quality semen is used. Additionally, the employment of Hep-GSH treatment increased rates of transgene expressing blastocysts. It could be a useful strategy for massively implementing TM-ICSI in bovine, for the production of transgenic animals.