Freeze Preservation of Spermatozoa Recovered from Male Mice that had been Refrigerated after Death

Abstract

To develop a model for utilizing germ cells collected from dead animals, male mice of ICR and BDF1 were euthanized and refrigerated at 4-6 C for various periods, then the epididymal spermatozoa collected from the carcasses were frozen in liquid nitrogen, and the viability of the spermatozoa after thawing was examined by in-vitro fertilization, embryo culture and embryo transfer. When males had not been refrigerated, 41-52% of freshly collected oocytes were fertilized. The fertilization rate after 1 day of refrigeration was 29-49%, and the rate dropped as the refrigeration period increased, reaching 1-3% after 3 days. Partial zona dissection improved the fertilization rate when males were refrigerated for 2 days (5-9% vs 12-24%). High proportions of the resulting embryos developed in vitro, regardless of the strain of male mice, the refrigeration period and the partial zona dissection of oocytes. Embryos derived from 2-day refrigerated male sperm developed to young after transfer to recipients. The present system, using carcass refrigeration and sperm freezing, should be applicable to rescuing valuable genetic variants in laboratory animals or livestock animals as well as wild species in the future.