274 LYSOLECITHIN TREATMENT OF ELAND, BONGO, AND BOVINE SPERMATOZOA AND CLEAVAGE OF BOVINE OOCYTES AFTER INTERSPECIES INTRACYTOPLASMIC SPERM INJECTION

Abstract

Compared to success rates in human, intracytoplasmic sperm injection (ICSI) is inefficient in ungulate species. Although factors such as injection of membrane-intact sperm and toxic effects of acrosome contents are suspected causes, the reasons for the inefficiency are unclear. A recent report in mice demonstrated that ICSI using spermatozoa treated with a physiological detergent, lysolecithin, improved oocyte activation, cleavage, and offspring production after embryo transfer (Morozumi K et al. 2006 PNAS 109, 17 661–17 666). The objectives of the present study were to evaluate the effects of detergent treatment on motility and membrane integrity of frozen thawed eland, bongo and bovine spermatozoa and to examine sperm decondensation/embryonic cleavage following ICSI of in vitro-matured bovine oocytes. In experiment 1, sperm motility was observed on a warm microscope stage during exposure to 3 lecithin concentrations, 0.04, 0.02, and 0.01%, and the time at which 100% of the spermatozoa lost motility was recorded. In experiment 2, spermatozoa were exposed to 0.02% lecithin for 22 s, and the membrane integrity and acrosome status of spermatozoa were determined using a combined trypan blue-Giemsa staining (Nagy et al. 1999 Theriogenology 52, 1153–1159). In experiment 3, bovine oocytes were injected, using the piezo drill, with lecithin-treated (0.02%, immobilized) or untreated (piezo pulse immobilized) eland, bongo, or bovine spermatozoa and subsequently cultured for 2 days in CR1aa containing 3 mg mL^–1 BSA. Each experiment was replicated at least 3 times. Lecithin induced time- and concentration-dependent loss of sperm motility. The average time to loss of motility in 100% of the spermatozoa at 0.04, 0.02, and 0.01% lecithin was 107, 222, and 344 s in bovine; 82, 135, and 179 s in eland; and 65, 115, and 158 in bongo, respectively. Data on membrane integrity (intact or nonintact) and acrosome status (reacted or nonreacted) of detergent-treated or control spermatozoa are shown in Table 1. Sperm head decondensation and embryonic cleavage were observed following homologous and interspecies (antelope into bovine) ICSI of lecithin-treated or control spermatozoa. In conclusion, lecithin treatment induced concentration and time-dependent loss of motility and was effective in damaging the sperm membrane and acrosome in eland, bongo, and domestic bulls. Eland and bongo spermatozoa underwent decondensation and activated bovine oocytes after interspecies ICSI.

Table 1.