The use of domestic cat (Felis silvestris catus) freeze-dried sperm to produce in vitro embryos via intracytoplasmic sperm injection (ICSI)

Abstract

More than thirty-six wild feline species are threatened or even close to extinction (Cites, 1973). Hence, the in vitro production (IVP) of embryos in the domestic cat has been an alternative in the conservation of wild species since the developed technology may be transferred to other species. As the freeze-drying process has been successfully used in the murine, bovine and equine, yielding viable offspring following intracytoplasmic sperm injection (ICSI), an improvement in the conservation of endangered species has arisen. It has already been shown the satisfactory freeze-drying process for cat sperm (MAGALHÃES, 2009), but it is yet necessary to prove their fertility by the production of embryos. This study aims at verifying the viability of freeze-dried cat sperm by producing in vitro embryos through the ICSI. Sperm will be collected from male donors and they will undergo conventional assessments - motility, sperm concentration, sperm morphology, and membrane integrity. Semen samples will be frozen in SOFaa using cryotubes (Magalhães et al. 2009) and freeze-dried in a freeze-drier (Edwards do Brasil) where they will be kept for 48 hours at -40ºC (time enough to provide a stable sample with 1% moisture. Oocytes will be collected from ovaries of queens that will undergo elective ovariohisterectomy (OSH). Soon after OSH ovaries will be sliced and oocytes will be analyzed and selected. Complex-cumulus-oocytes (CCO) with a uniform and dark cytoplasm, and presenting at least 2 layers of compact cumulus cells will undergo in vitro maturation. Then, matured oocytes and rehydrated sperm will undergo ICSI. The resulting structures will be stained and assessed for the presence of pro-nuclei and / or cleavage rate. (AU)