Cryopreservation of feline oocytes by vitrification using commercial kits and slush nitrogen technique

Abstract

Assisted reproductive techniques are a valuable tool for conservation breeding of endangered species. Cryopreservation methods are the basis of gamete banks, supporting genetic diversity preservation. Unfortunately, cryopreservation of feline oocytes is still considered an experimental technique. The aim of this study was to compare two commercial kits, with our protocol for vitrification of cat oocytes (IZW), which comprises a three-step method with ethylene glycol, DMSO, fetal calf serum, trehalose and Ficoll PM-70. Furthermore, we applied slush nitrogen (SN₂ ) for ultra-rapid freezing to improve survival rates. Cumulus-oocyte complexes were collected from domestic cat ovaries by slicing and vitrified at immature stage using Cryotop as storage device. Vit Kit^® Freeze/Thaw (n = 89) showed the lowest maturation percentage obtained after warming (10.1%). A significant difference in maturation percentage of oocytes was found between Kitazato^® kit (38.7%, n = 137) and IZW protocol (24.5%, n = 143). The cleavage after ICSI of warmed and matured oocytes (20.7% and 28.6%, respectively) and the morula percentage (18. 2% and 22.5%, respectively), however, did not reveal any significant difference between the two methods. Application of SN₂ did not result in any improvement of oocytes' cryopreservation. Maturation percentage of the oocytes vitrified by IZW method with SN₂ (n = 144) decreased until 6.1%, without any cleavage after fertilization. For Kitazato^® (n = 62), only 17.7% were able to undergo maturation and cleavage percentage dropped to 18.2%, not reaching morula stage. These data demonstrate that feline oocytes can be vitrified either by our IZW method or by commercial Kitazato^® kit, but the use of SN₂ is improving neither maturation nor cleavage percentages when combined with these procedures.