381 FERTILIZABILITY AND CHROMOSOMAL INTEGRITY OF FROZEN–THAWED BRYDE's WHALE (BALAENOPTERA EDENI) SPERMATOZOA INTRACYTOPLASMICALLY INJECTED INTO MOUSE OOCYTES

Abstract

In this study, we applied intracytoplasmic sperm injection (ICSI) to mouse oocytes to evaluate the fertilizability and chromosomal integrity of the three types of frozen–thawed Bryde's whale spermatozoa. B6D2F1 female mice (7–11 weeks of age) were superovulated by injections of PMSG followed by hCG 48 h later. The oocytes recovered from oviducts between 14 and 16 h after hCG injection were denuded of their cumulus cells. Sperm samples were obtained from a Bryde's whale (Balaenoptera edeni) captured under the Japanese Whale Research Program with Special Permit in the Western North Pacific between May and August 2003 (presumptive feeding season). The whale was killed by an explosive harpoon which has been recognized as the best humane method for whales by the International Whaling Commission (IWC) and stipulated by Schedule III (Capture) of the International Convention for the Regulation of Whaling. Spermatozoa collected from vasa deferentia were cryopreserved. Frozen Bryde's whale spermatozoa were thawed at 37°C and washed with HEPES-TYH by centrifugation at 500g for 5 min. Motile and immotile spermatozoa were obtained, and some spermatozoa in HEPES-TYH were refrozen without cryoprotectant at -20°C to be completely killed. Within 24 h, they were thawed at 37°C and prepared for ICSI. Comparison of group values was performed by either Fisher's exact probability test or chi-square test where necessary. Differences at P d 0.05 were considered significant. Chromosomal normality was determined by analyses of karyotyped haploid chromosomes (n = 22) of the whale sperm. Regardless of motility and viability, whale spermatozoa efficiently led to the activation of mouse oocytes (90.3–97.4%), and sperm nuclei successfully transformed into male pronuclei within activated ooplasm (87.2–93.6%). Chromosome analysis at the first cleavage metaphase of the hybrid zygotes revealed that a majority (95.2%) of motile spermatozoa had the normal chromosome complement, whereas the percentage of chromosomal normality was significantly (P ≤ 0.001) reduced to 63.5% in immotile spermatozoa and 50.0% in dead spermatozoa, due to the increase in structural chromosome aberrations such as chromosome fragments. This is the first report showing that motile Bryde's whale spermatozoa are competent to support embryonic development. Furthermore, we have shown that chromosomal analysis of whale spermatozoa is a useful technique for measuring the influences of marine pollution on reproduction in cetacean species that occupy the top niche in the marine ecosystem.