Title: Genetically modified animals, in particular mice, are unique mutants with a major scientific potential. Cryopreservation of pre-implantation embryos or of spermatozoa is a common approach to protect those lines against loss or to archive them. Prerequisite to take a mutant line out of a breeding nucleus is the cryopreservation of a sufficient number of samples and a powerful quality assessment.
Background: Several common protocols to cryopreserve spermatozoa and subsequently to forward these to an in vitro-fertilization under standardized conditions are published using donors of a strictly limited age-span. In practice, several limitations happen frequently for those approaches, e.g. the availability of genetically modified males, in particular if a line is in an experimental use or the mutation leads to restrictions. Female donors are often received from external sources and are subsequently not available before weaning. They are subject of several delays to induce an early superovulation. The aim of this report is to study the suitable age span of sperm and oocyte donors.
Methods and Findings: A total of 441 GM sperm donors and the production of wild type oocytes with the genetic backgrounds BALB/c, C57BL/6N, and DBA/2 were investigated. The quality was assessed with simple, but purposive scores. Polynomial statistical analyses investigating increasing and decreasing yields show that the age of sperm-donors can be extended to ten months; the suitable age of oocyte donors is strain specific up to twelve weeks.
Conclusions: The extended age spans demonstrated allow a more flexible use of donors and contribute subsequently to the 3Rs.
Michael Ramin, Tim Holland-Letz, Anna Schwab, Johannes Schenkel