Cryopreservation of canine oocytes by vitrification method and parthenogenetic activity

Biotechnologic researches in pet animals have been run up in recent years. Raised questions about unsuccesful assisted reproductive technologies in canid species are probably related with poor information in reproductive physiology of canid species. The studies about this subject has been thought to be motivated by emotional reasons. But on the other hand the applications in pet biology is accepted as model for human diseases. Apart from this, development of gamete cryopreservation is an important tool for genetic bank and conservation of endangered species. In spite of low maturation rates of canine oocytes, the results of parthenogenetic activation has been tried to maturated and vitrified-warmed canine oocytes in this thesis. Oocytes were collected from 10 healthy bitches at Yıldırım Municipality Stray Animals Sterilization and Rehabilitation Center. After slicing of ovaries, selected cumulus oocyte complexes were maturated for 72 h at 39°C in four-well petri dishes containing 500 µl TCM-199 under mineral oil in a 5%CO2 incubator. After maturation, oocytes were exposed to 50 ml PBl containing 0%, 10%, 20% ethylene glycol for 10, 10 minutes and 30 seconds respectively. They were vitrified in cryovials containing 30 µl VS3 in liquid nitrogen. The oocytes in this group (n=257) were grouped as vitrified oocyted 'VO'. After warming, the oocytes were parthenogenetically activated with ionomycin for 5 minutes and followed by cycloheximide for 3 hr. Oocytes were then cultured for 72 hr and assessed for nuclear maturation. The oocytes (n=257), grouped as fresh oocytes 'FO' were used as control group. After maturation, oocytes were directly incubated with ionomycin and cycloheximide for parthenogenetic activation and cultured for 72 h. All oocytes were stained with Hoechst33342 for 30 min and nuclear maturation rates were assessed using a phase contrast microscope. Maturation rates (MI+MII) between groups had not been found statistically different (p>0,05). To our knowledge there is no information available about the influence of parthenogenetic activation on nuclear maturation after vitrification of maturated canine oocytes. However, low maturation rates should be clarified by further molecular studies. Keywords: Canine oocytes, vitrification, in vitro maturation, parthenogenetic activation