No abstract

In vitro-produced bovine embryos matured in modified Tissue Culture Medium 199 were vitrified at the 7th and 8th days of culture, and development at 48 h after warming was recorded. Ovaries were obtained from a local abattoir, and the oocytes were recovered by slicing method and divided into two main groups. The first group included cysteamine in the TCM-199 (Group A) and the second did not (Group B). They were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO 2 in humidified air. Matured oocytes were fertilized with 1-2 × 10 6 /mL of frozen-thawed bull semen. At the end of the period the embryos were divided into two subgroups in order to be developed. Therefore, four groups were established: Group AI-SOF (synthetic oviduct fluid), Group AII-CR1aa (Charles Rosencrans), Group BI-SOF, and Group BII-CR1aa. The rates of blastocyst development and expanded blastocysts were detected as 20%, 18.1%, 24.6%, and 22.5% and survival rate as 5.5%, 23.6%, 0%, and 5.2%, respectively, showing statistically significant superiority in Group AII-CR1aa at the level of P < 0.05. In conclusion, supplementing cysteamine into maturation media with subsequent culture in AII-CR1aa had a positive effect on blastocyst survival after vitrification.

No abstract

Antioxidant substances used at any stage of in vitro embryo production increase intracellular glutathione synthesis (GSH), improve nuclear maturation rates and protect embryos against endogenous or exogenous oxidative stresses, making embryos resistant to freeze, as well as increasing cell quality and number of embryos reaching blastocysts. Production of GSH depends on the availability and uptake of cysteine in the medium. However, cysteine is very unstable outside the cell and is auto-oxidized to cystine. Cysteamine reduces cystine to cysteine and promotes the uptake of cysteine by cells thereby enhancing the GSH synthesis. Consequently, cysteamine plays an important role in the synthesis of GSH and is a key factor in the defense mechanism against ROS.

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...

No abstract

Sheep is a very important source of wool, meat and milk all over the world. Oxidative stress during in vitro culture leads to defects in development of gametes and embryos. Several antioxidants such as cysteamine, L-ascorbic acid, beta mercaptoethanol, cysteine, glutathione, proteins, vitamins are used to supplement culture media to counter the oxidative stress. This study was aimed to detect the effect of cysteamine supplementation to the maturation medium and oviductal cell supplementation to culture medium on the subsequent development rates of sheep embryos with the control group. Oocytes were obtained from slaughtered sheep ovaries. Selected oocytes were incubated with or without 100 µM cysteamine in TCM-199 medium under 38.5-38.8ºC 5% CO 2 for 23 h. During IVF fresh semen was collected from ram by electroejaculation, they were washed in H-SOF medium and were fertilized in B-SOF medium with oocytes incubated for 18 hours under 38.5-38.8ºC 5% CO 2 , 5% O 2 and 90% N 2 . The oocytes were obtained from maturation medium with/without cysteamine (C+,-) and were cultured in SOF or CR1aa media with/without oviductal cells (Ov+,-) and were grouped as; Group Ia: SOF+(C+ Ov-), Group Ib: SOF+(C+Ov+), Group Ic: SOF+(C-Ov-) Group Id: SOF+(C-Ov+); Group IIa: CR1aa+(C+ Ov-), Group IIb: CR1aa+(C+ Ov+), Group IIc: CR1aa+(C-Ov-), Group IId: CR1aa+(C-Ov+). Embryos were incubated under 38.5-38.8ºC 5% CO 2 , 5% O 2 , 90% N 2 in culture medium for 7 days. Embryo developments were observed and recorded daily. GLM procedure found in SPSS packet program was used for statistical analysis in this study. In conclusion, the addition of cysteamine or oviductal cells in vitro culture media found to have any effect in terms of the capacity of reaching to blastocyst stage in SOF or CR1aa media and no statistical difference is detected between groups. Keywords: Cysteamine, Embryo, Oviductal cell, Medium, Sheep Kültür Medyumlarında Sisteamin ve Ovidukt Hücrelerinin Koyun Embriyo Gelişimi Üzerine Etkisi ÖzetKoyun yün, et ve süt kaynağı olarak dünya çapında önemli bir çiftlik hayvanıdır. İn vitro kültür esnasında oksidatif stres embriyo ve gametlerin gelişiminde defektlere neden olur. Sisteamin, L-askorbik asit, beta merkaptoetanol, sistein, glutatyon, proteinler, vitaminler gibi birçok antioksidanlar oksidatif stresi engellemek için kültür medyumunda kullanılır. Bu çalışmada maturasyon medyumuna sisteamin ve kültür medyumuna ovidukt hücreleri eklenen oositlerin kontrol grubu oluşturularak kültürleri sonucundaki gelişim aşamalarının değerlendirilmesi hedeflendi. Oositler mezbahada kesilen koyunların ovaryumlarından elde edildi. Seçilen oositler 100 µM sisteamin ilaveli ve ilavesiz TCM-199 medyumunda 38.5-38.8ºC'de %5 CO 2 altında 23 saat boyunca inkübe edildi. IVF aşamasında koçlardan elektroejakülasyon yöntemiyle elde edilen taze sperma H-SOF medyumunda yıkandıktan sonra fertilizasyon amacıyla B-SOF medyumunda bulunan oositler ile 18 saat 38.5-38.8ºC'de %5 CO 2 , %5 O 2 ve %90 N 2 'de inkübe edildi. Sisteaminli/ sisteaminsiz (S +, -) ...

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