322 DIFFERENTIAL DEVELOPMENT OF RABBIT EMBRYOS FOLLOWING MICROINSEMINATION USING SPERM AND SPERMATIDS

Abstract

Microinsemination is a technique that delivers male germ cells directly into the ooplasm. The efficiency of fertilization and subsequent embryo development after microinsemination varies with species and the male germ cells used. This study examined the developmental ability of rabbit embryos in vitro and in vivo following microinsemination using haploid male germ cells at different stages. First, we injected rabbit spermatozoa, elongated spermatids, and round spermatids into mouse oocytes to assess their oocyte-activating capacity. Mouse oocytes are a good experimental model for assessing the oocyte-activating capacity of male germ cells from different species. The majority of mouse oocytes were activated irrespective of the stage of rabbit male germ cells injected (77, 61, and 73% for spermatozoa, elongated spermatids, and round spermatids, respectively). By contrast, these male germ cells activated homologous rabbit oocytes at rates of 100, 59, and 29%, respectively. After 120 h in culture, 69, 55, and 13% of these activated rabbit oocytes (pronuclear eggs) developed into blastocysts, respectively. The rate of embryo development into blastocysts following round spermatid injection was significantly improved when oocytes were activated by an electric pulse shortly before microinsemination. The total number of cells was counted in embryos that reached the morula/blastocyst stages in culture using nuclear-staining with propidium iodide. The average cell number of embryos derived from elongated (89 ± 41; mean ± SD) or round spermatid (98 ± 34) injection was significantly lower than that of control embryos (in vivo fertilization) (211 ± 44) (P < 0.01). After 24 h in culture, some four- to eight-cell-stage embryos were transferred into the oviducts of pseudopregnant females. Normal pups were born from embryos involving sperm (4 offspring/16 transfers; 25%) and elongated spermatid (3/26; 12%) injection, but none from those involving round spermatid injection (0/68). These findings indicate that rabbit male germ cells acquire the ability to activate oocytes and to support subsequent embryo development as they undergo spermiogenesis. Immaturity of the nuclear genome or difficulty in coordinating the behavior of the male and female chromosomes might compromise embryo development.