PRESERVATION OF EJACULATED MOUSE SPERMATOZOA FROM FERTILE C57BL/6 AND INFERTILE Hook1/Hook1 MICE COLLECTED FROM THE UTERI OF MATED FEMALES

Abstract

Methods routinely used to preserve mouse spermatozoa require that the male be euthanized to recover spermatozoa from the epididymides. Here we obtained multiple samples of ejaculated spermatozoa from normal fertile C57BL/6 and infertile Hook1/Hook1 (formerly known as azh/azh) mutant males from uteri after mating, thus avoiding terminating the males. Ejaculated sperm were preserved by conventional cryopreservation or by rapid freezing without cryoprotection, and injected into the oocytes by intracytoplasmic sperm injection (ICSI). The proportions of oocytes that survived, became activated, and developed into two-cell embryos were similar when comparing two preservation methods, wild type vs. Hook1/Hook1 mice, and tested mice vs. controls (fresh and rapid frozen epididymal and fresh ejaculated sperm). Two-cell embryos were transferred into the oviducts of pseudo-pregnant females and fetal development was examined at day 15 of gestation. Thirty nine to 54% of transferred embryos produced with preserved ejaculated sperm implanted. Live, normal fetuses (11–17%) were obtained in all examined groups and from all males included in the study. More implants (71–82%) and fetuses (28–31%) were noted in controls. Lower developmental potentials of embryos produced with preserved ejaculated sperm might be due to their capacitation status; the majority of sperm retrieved from the uterus were capacitated. This study bears significance for the maintenance and distribution of novel mouse strains. The method is applicable for all types of mice, including those with male infertility syndromes. The sole requirement is that the male of interest is able to copulate and its ejaculate contains spermatozoa. [This material is based on work supported by NIH HD048446 grant to M.A.W] (platform)