Boa Spermatozoonlar›nda in vitro Kapasitasyon ve Fertilizasyon*

Ozet : Araflt›rmada iki farkl› kapasitasyon medium'unda tutularak in vitro kapasite edilen boa spermatozoonlar›n›n in vitro mature edilmifl oositleri fertilize etme yetenekleri araflt›r›lm›flt›r. Mezbahadan salanan ovaryumlar 30-35oC' deki serum fizyolojik icinde laboratuvara getirilerek, 2-8 mm cap›ndaki antral follikullerden aspirasyon yontemi ile kumulus oosit kompleksleri toplanm›flt›r. Toplanan oositler deerlendirildikten sonra maturasyon medium'una aktar›larak %5CO2 ve %95 nemli atmosferde 38,5oC'de 24 saat inkubasyona al›nm›flt›r. Bu sure sonunda kumulus ekspansiyonu gosteren oositler olgunlaflm›fl kabul edilerek fertilizasyon ortam›na al›nm›fllard›r. Maturasyon medium'u olarak %20 ECS, 10 IU/ml FSH, 10 IU/ml LH iceren Ham's F-10 kullan›lm›flt›r. Olgunlaflm›fl oositleri fertilize etmek amac›yla dondurulmufl boa spermas› kullan›lm›flt›r. Motil spermatozoonlar›n kapasitasyonu amac›yla 0,5 mM hipotaurin ve %25 follikul s›v›s› iceren mDM solusyonu kullan›lm›flt›r. Hipotaurin icin 7 saat, follikul s›v›s› icin 4 saat inkubasyon uygulanm›flt›r. Elde edilen sperma suspansiyonu, final konsantrasyon 1-3x10 6 spermatozoon/ml olacak flekilde oositlerin bulunduu ortama kat›lm›flt›r. Oositler ve spermatozoa 18 saat inkubasyona b›rak›lm›flt›r. Bu sure sonunda oositler fikse edilmifl ve boyanm›fllard›r. Oosit sitoplazmas›nda difli ve erkek pronukleuslar›n›n gozlenmesi ile fertilizasyon deerlendirilmifltir. Sonuc olarak, maturasyona b›rak›lan 878 oositten 385 tanesi (%43,84) mature olarak fertilizasyona al›nm›flt›r. Fertilizasyon sonucunda, hipotaurin ve follikul s›v›s› ile kapasite edilen spermatozoonlarda fertilizasyon oranlar› s›ras›yla %34,21 ve %29,23 olarak saptanm›flt›r. Yap›lan istatistiki deerlendirmede her iki fertilizasyon oran› aras›ndaki fark p<0.01 duzeyinde onemli bulunmufltur. Abstract : In this study, the fertilizing ability of in vitro capacitated bull spermatozoa in two different capacitation media (Hypotaurine and Follicular Fluid) was investigated. Ovaries were obtained from slaughterhouse and were transported to the laboratory in saline (0.9% NaCl) at 30-35oC. Cumulus oocyte complexes were collected from antral follicles of 2 to 8 mm in diameter. After collected oocytes were classified, they were transferred to maturation medium. Oocytes were cultured for 24 hours in a 100 ml drops of maturation media overlaid with mineral oil under 5% CO2, 95% humidity in air at 38.5oC. At the end of this incubation, oocytes which displayed cumulus expansion were considered as matured. Ham's F-10 was used as the maturation medium containing 20% ECS, 10 IU/ml FSH, 10 IU/ml LH. Cryopreserved bull semen was used for fertilization of in vitro matured oocytes. Motile spermatozoa were separated by the swim- up technique. MDM solution containing 0.5 mM hypotaurine and 25% follicular fluid was used for the capacitation of separated motile spermatozoa. Hypotaurine treated spermatozoa were incubated for 7 hours and follicular fluid treated spermatozoa were incubated for 4 hours. At the end of these incubations, the obtained sperm suspension was added to fertilization medium. The final concentration of sperm in the medium was 1-3x10 6 sperm/ml. Oocytes and sperm were cocultured for 18 h. At the end of this culture period, oocytes were fixed and stained for evaluation of fertilization. Oocytes were considered to be fertilized when female and male pronuclei were visible in the cytoplasm. Finally, from 878 collected oocyes, 385 oocytes were matured (43.84%). Fertilization rates of capacitated spermatozoa with hypotaurine and follicular fluid were 34.21% and 29.23% respectively. The difference between fertilization rates were found to be significant (p<0.01).