İn Vitro Elde Edilen Sığır Embriyolarının Dondurulmasında Vitrifikasyon Medyumuna Maruz Kalma Sürelerinin Çözünme Sonrası Gelişim Üzerine Etkisi

Year 2012, Volume: 38 Issue: 1, 29 - 36, 01.11.2011

Özen Özdaş , Ümüt Cirit Kamber Demir Süleyman Bacınoğlu Alper Baran Serhat Pabuccuoğlu , Tülat İrez Serhat Alkan Kemal Ak

Abstract

ÖZET

Günümüzde dondurulmuş embriyoların transferi ile üstün verim özelliklerine sahip sürülerin oluşturulması, hastalıkların kontrolü ve genetik materyallerin uzun süre saklanması mümkün olabilmektedir. Ancak donmuş embriyolarda eritme sonrası tranfer edilebilir embriyo eldesi ve gebelik oranları istenilen düzeyde değildir. Özellikle embriyoların dondurulması sırasında dejenerasyonlar oluşmaktadır. Dejenerasyonların, embriyoların kriyoprotektif maddelere maruz kalma süreleri ile toksik etkilerinden meydana geldiği düşünülmektedir. Çalışmada mezbahada kesilen sığırların ovaryumları kullanıldı. Aspirasyon yöntemi ile elde edilen oositler (1290 adet) TCM-199 da 22-24 saat süreyle %5 CO₂, %5 O₂, %90N₂ gaz atmosferinde 38,8 ºC de in vitro olarak olgunlaştırıldılar. Olgun oositler IVF-TALP medyumunda 18-24 saat süreyle fertilize edildiler. Fertilizasyon sonrası 48. saatte cleavage %67,05 (865/1290) saptandı. Embriyolar %10 FCS'li SOF medyumunda 7 gün süreyle %5 CO₂, %5 O₂, %90N₂ gaz karışımında blastosist (%34,91; 302/865) aşamasına kadar inkübe edildiler. Erken blastosist-blastosist aşamasına ulaşan 302 adet embriyodan 254 tanesi vitrifikasyon solüsyonunda farklı sürelere (15, 30, 60, 90 sn) maruz bırakılarak donduruldular. Bu amaçla sırasıyla 4 grup oluşturuldu. Her grup sırasıyla 67, 64, 63 ve 60 embriyo içerdi (Grup 1, 2, 3, 4). Embriyolar önce % 10 Gliserol + % 10 FCS içeren PBS solüsyonunda (Vs1) 5 dk, sonra %10 Gliserol + %10 FCS+ %20 Etilen Glikollü PBS'de (Vs2) 5 dk. bekletildiler. Daha sonra embriyolar payet içindeki %25 Gliserol + %20 Etilen Glikol + %10 FCS + 0,1 M Sükroz vitrifikasyon solüsyonuna (Vs3) aktarıldılar ve değişik sürelere (15, 30, 60, 90 sn) maruz bırakıldıktan sonra sıvı azota daldırılarak donduruldular. Eritme sonrası (37ºC) embriyolar birkaç kez yıkama medyumunda ve SOF medyumunda yıkandıktan sonra, her bir gruba ait embriyolar 48 saat süreyle tekrar inkübe edildiler. Çalışmada istatistiki analizde ki-kare testi kullanıldı. Eritme sonrası, genişlemiş blastosist-zonadan çıkma safhasında en iyi gelişim %52,2 (35/67) ile Grup 1 de saptanırken, bunu %45,3 (29/64) ile Grup 2, %22,2 (14/63) ile Grup 3 ve %5 (3/60) ile Grup 4 takip etti. Grup I ve II arasında istatiksel bir fark bulunmadı. Grup 1 ile Grup 3 arasındaki istatistiksel fark P<0,01, Grup 1 ile Grup 4 arasında ise P<0,001 düzeyinde anlamlı bulundu.

Anahtar Kelimeler: Sığır, embriyo, in vitro, vitrifikasyon, kriyoprotektan

 

ABSTRACT

 

EFFECT OF EXPOSURE DURATION TO THE VITRIFICATION MEDIUM ON THE

POST THAW DEVELOPMENT OF IN VITRO DERIVED BOVINE EMBRYOS

 

Transfer of frozen embryos enables the establishment of elite herds, control of diseases and storage of genetic materials for longer periods. However, although intercontinental transfer of frozen embryos is possible, post-thaw degenerations are encountered and pregnancy rates are not at the expected level. Embryos especially degenerate during freezing and thawing procedures. These degenerations are thought to be due to the exposure time and toxic effects of used cryoprotectants. In this study slaughtered cattle ovaries were used. Oocytes were collected from ovaries using the aspiration method and matured in their own group in 700 microliter TCM-199 for 22-24 h at a gas atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 38.8 ºC. Matured oocytes were fertilized for 18-24 h in IVF-TALP medium. After fertilization cleavage was 67.05% (865/1290) at 48^th h. Embryos were cultured up to early blastocyst-blastocyst stage (34.91%; 302/865) in SOF medium supplemented with 10 % FCS for 7 days at a gas atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 38.8 ºC. 302 embryos at the early blastocyst stage were frozen after an exposure to vitrification solution for various time periods (15, 30, 60, and 90 sec). Four groups have been established for this purpose (Groups 1, 2, 3, 4). Each group has included 67, 64, 63 and 60 embryos, respectively. All embryos were first kept in PBS solution containing 10% Glycerol + 10% FCS (Vs1) for 5 minutes and then in PBS containing 10% Glycerol+10% FCS+20% Ethylene Glycol (Vs2) solution for 5 minutes. Later, embryos were taken to straw containing vitrification solution (Vs3), 25% Glycerol + 10% FCS + 25% Ethylene Glycol + 0.1 M sucrose, and exposed for various time periods (15, 30, 60 and 90 sec), then frozen by dipping into liquid nitrogen. After thawing (37 ºC) embryos were washed several times in washing medium supplemented with 0.5 M sucrose and SOF medium, then embryos of each group were incubated again for another 48 hours. Chi-square test was used in this study. Post thaw development to expanded blastocyst stage was highest in Group 1 with 52.2% (35/67) followed by Group 2 with 45.3% (29/64), Group 3 with 22.2% (14/63) and Group 4 with 5% (3/60). No statistical significance was observed between Groups 1 and 2. The statistical difference between Group 1 and 3 and between Group 1 and 4 were at P<0.01 and P<0.001 levels, respectively.

KeyWords: Cattle, embryo, in vitro, vitrification, cryoprotectant

Keywords

Sığır, embriyo, in vitro, vitrifikasyon, kriyoprotektan

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