Animal production via SCNT provides a unique tool for protection of valuable individuals, conservation of vulnerable and endangered species and production of transgenic animals. A total of 167 MI and 219 MII stage oocytes were used as the material of the study. The oocytes were enucleated at 44 h after in vitro maturation by aspiration of the polar body and the MI or MII plates. Cycling granulosa cells were used for nuclear transfer. Cell fusion was induced with DC pulses of 2.0 kV/cm 60µs, 0.1s apart (2x) delivered by a BTX Electrocell Manipulator 200 (BTX, San Diego, CA, USA). After fusion, the embryos were activated by 1.0 kV/cm 20µs DC pulses 0.1s apart (2x) followed by 2 mM 6-DMAP (6-dimethylaminopurine) incubation in culture medium for 4 h in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38°C. The somatic cell transferred embryos were cultured for 8 days in mSOF medium supplemented with 0.4% BSA in a humidified 5% CO2, 5% O2, and 90% N2 atmosphere at 38°C. After in vitro culture period, all embryos transferred to HSOF containing Hoechst 33342 (5 μg/mL) and the cell numbers were counted under ultraviolet light using a fluorescent microscope. The fusion (66.66 vs 21.55%) and cleavage rates (15.75 vs 11.11%) were significantly higher in MII stage oocytes than MI stage oocytes (P<0.02). While SCNT embryos were developed to morula stage in MII group (14; 9.58%), all the cleaved embryos were arrested at the 2-4 cell stage in MI group. None of the embryos was developed to blastocyst stage in both groups. Keywords: Cat, SCNT, In vitro, MI -MII oocytes Kedilerde MI ve MII Oositleri Kullanilarak Somatik Klonlama ÖzetSomatik klonlama yoluyla hayvan üretimi, üstün değerdeki bireylerin korunması, savunmasız ve tehlike altında bulunan türlerin korunması ile transgenik hayvanların çoğaltılmasına hizmet eder. Çalışmanın materyalini 167 adet MI ve 219 adet MII dönemdeki oosit oluşturdu. Polar cisimciklerin (MII) ve kromatin setlerin (MI ve MII) enükleasyonu, 44 saatlik in vitro olgunlaştırma periyodunun ardından gerçekleştirildi. Nükleer transfer amacıyla siklik dönemlerdeki granüloza hücreleri kullanıldı. Oositsomatik hücre komplekslerinde füzyon işlemi, DC akımın sağlandığı 2.0 kV/cm 60 µs, 0.1s ara (2x), BTX Electrocell Manipulator 200 (BTX, San Diego, CA, USA) ile gerçekleştirildi. Aktivaston işlemi için ise, 1.0 kV/cm 20µs DC akım 0.1s ara (2x) kullanıldı ve ardından 2 mM 6-DMAP (6-dimethylaminopurine) içerisine alınarak, 38°C'lik sıcaklık ve %5 CO2, %5 O2 ve %90 N2 gaz karışımının sağlandığı inkübatörde 4 saat süresince kültüre edildi. Somatik hücrelerin nakledildiği klon embriyolar daha sonra aynı inkübatör koşullarında %0.4 BSA katkılı mSOF medyumu içerisinde 8 gün boyunca in vitro kültüre bırakıldılar. Ardından klon embriyolar, embriyonik hücre sayılarının tespiti amacıyla Hoechst 33342 (5 μg/mL) içeren HSOF medyumu içerisine alındı ve ultraviyole küplü floresan mikroskobunda değerlendirildi. Sonuçta, füzyon (%66.66-21.55) ve yarıklanma oranlarının (%15.75-11.11) MII dönem oositleri lehine önemli...

SummaryThis study was conducted to determine the most successful techniques on inmature and in vitro-matured cat oocytes that were parhtenogenically activated using 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX), in combination with electrical stimulation and calcium ionophore. After 44 h of in vitro maturation, the oocytes with a polar body were separated as mature (M II) and those without a polar body were considered as immature. Four different activation treatments and two control groups were used for parthenogenetic activation with both mature and immature cat oocytes. After 48 h of activation, the oocytes were examined and the non-cleaved oocytes removed. The cleaved oocytes/embryos were cultured in vitro in mSOF medium for an additional four days. After six days of in vitro culture (IVC), embryo quality was evaluated. The results in the present study suggested that (I) both in vitro matured and immature cat oocytes have a potential to develop to morula and blastocyst stages after parthenogenetic activation, (II) electrical stimulation + 6-DMAP is a more useful technique for both matured and immature cat oocytes and (III) to our knowledge, this is the first report that describes morula and blastocyst formation from parthenogenetically activated immature cat oocytes.