137 Enhancing blastocyst formation rate using bovine freeze-dried sperm: investigation of oocyte activation and sperm pretreatment

Currently, bovine sperm are cryopreserved in liquid nitrogen (LN), but alternative technologies are desired due to high maintenance costs, the need for specialised storage facilities and a continuous supply of LN, energy dependency, safety concerns, and loss due to disaster. We have succeeded in producing cattle using freeze-dried (FD) sperm, but the development rate of blastocysts is very low, and the technology has not been yet practical. In this study, with the aim of improving the blastocyst rate, we investigated activation treatment of oocytes and pretreatment of sperm before freeze drying, and evaluated in vitro development by intracytoplasmic sperm injection (ICSI). In Experiment 1, assuming a procedure with ICSI, the conditions for activation were studied. Oocytes were cultured for 22, 23, 24, and 25 h for IVM. Cumulus cells were stripped from COCs, treated with Ionophore A23187 (IA) 2 h later, and cultured in 6DMAP for 4 h (PA group) or 2 h later in 6DMAP for 2 h (ICSI group). The rate of pronuclear formation after in vitro culture was investigated. In Experiment 2, the effects of cycloheximide (CHX) on second polar body extrusion and pronuclear formation were examined. Oocytes matured in vitro for 23 h and were stripped and treated with IA 2 h later. The rates of second polar body extrusion and pronuclear formation in the PA group, ICSI group, and ICSI+CHX group (cultured in CHX-added medium for 2 h and then in 6DMAP for another 2 h) were investigated. In Experiment 3, the effect of sperm pretreatment on DNA damage after freeze drying was investigated. Freshly ejaculated bovine sperm were divided into no treatment, inducing capacitation (Cap group), dithiothreitol treatment (DTT group), and glutathione treatment (GSH group), and the DNA damage rates were compared by modified comet assay after freeze drying. In Experiment 4, the development of embryos produced from FD sperm by ICSI was examined. Intracytoplasmic sperm injection was performed using FD sperm from untreated, Cap, DTT, and GSH groups. After IA treatment, the embryos were cultured in CHX-added medium for 2 h and then in 6DMAP for another 2 h. In vitro culture was performed to examine the rates of pronuclear formation, cleavage, and blastocyst formation. The results for Experiment 1 are as follows: the pronuclear formation rate in the PA and ICSI group reached a plateau at 24 h of IVM, with the PA group having a significantly higher pronuclear formation rate than the ICSI group (P < 0.05). In Experiment 2, the ICSI+CHX group showed significantly higher rates of second polar body extrusion and pronuclear formation than the ICSI group. In Experiment 3, the DNA damage rate of FD spermatozoa was less than 6% in the nontreated, Cap, DTT, and GSH groups. In Experiment 4, the rate of male and female pronuclear formation was significantly higher in the DTT group (55%) than in the nontreated group (17%) (P < 0.05). Furthermore, the blastocyst rate was significantly higher in the GSH group (29%) than in the nontreated (5%; P < 0.05). In conclusion, these results suggest that the addition of CHX to the activation medium improves the rate of second polar body extrusion and pronuclear formation, and that pretreatment of sperm with GSH is effective to produce normal fertilized embryos and improve blastocyst rate using FD sperm.