Effect of Parthenogenetic Activation on Vitrified Canine Oocytes

Abstract

Biotechnological research in pet animals has been run up in recent years. Raised questions about unsuccessful assisted reproductive technologies in canids are probably related to poor information in the reproductive physiology of canids. But on the other hand, applications in pet biology are accepted as a model for human diseases. Apart from this, the development of gamete cryopreservation is an important tool for genetic banking and conservation of endangered species. In spite of the low maturation rates of canine oocytes, the results of parthenogenetic activation were tried to maturated and vitrified-warmed canine oocytes in this study. Oocytes were collected from 20 healthy bitches at Yıldırım Municipality Stray Animals Sterilization and Rehabilitation Center. After slicing of ovaries, selected (COCs) cumulus-oocyte complexes were maturated for 72 h at 39°C in four-well petri dishes containing 500 μl TCM-199 under mineral oil in a 5%CO2 incubator. After maturation, oocytes were exposed to 50 ml PBl containing 0%, 10%, 20% ethylene glycol for 10, 10 minutes, and 30 seconds, respectively. They were vitrified in cryovials containing 30 μl VS3 in liquid nitrogen. The oocytes in this group (n=257) were grouped as ‘vitrified oocyte-VO’. After warming, the oocytes were parthenogenetically activated with ionomycin for 5 minutes and followed by cycloheximide for 3 h. Oocytes were then cultured for 72 h and assessed for nuclear maturation. The oocytes (n=257), grouped as ‘non-vitrified oocytes-FO’ were used as the control group. After maturation, oocytes were directly incubated with ionomycin and cycloheximide for parthenogenetic activation and cultured for 72 h. All oocytes were stained with Hoechst33342 for 30 min and nuclear maturation rates were assessed using a microscope. Maturation rates (MI+MII) between groups had not been found statistically different (p>0,05). GV, GVBD, MI, and MII rates were also not statistically different between the two groups (p>0,05). To our knowledge, there is no information available about the influence of parthenogenetic activation on nuclear maturation after the vitrification of maturated canine oocytes. However, low maturation rates should be clarified by further molecular studies.

Keywords

• ine oocytes
• vitrification
• in vitro maturation
• parthenogenetic activation

Partenogenetik Aktivasyonun Vitrifiye Köpek Oositleri Üzerine Etkisi

Abstract

Pet hayvanlarında biyoteknolojik çalışmalar son yıllarda hız kazanmaya başlamıştır. Köpeklerde başarısız yardımcı üreme teknikleriyle ilgili oluşan sorular, muhtemelen köpek türlerinin reproduktif fizyolojisine ait yetersiz bilgiden kaynaklanmaktadır. Fakat diğer taraftan pet biyolojisindeki uygulamalar, insan hastalıkları için model oluşturmaktadır. Bunun ötesinde gamet kriyopreservasyonunun gelişmesi, nesli tükenmekte olan türlerin korunması ve genetik banka oluşturulması için önemlidir. Bu çalışmada, köpek oositlerindeki düşük maturasyon oranlarına rağmen, partenogenetik aktivasyonun etkileri vitrifiye oositlerde test edildi. Köpek oositleri, Yıldırım Belediyesi Sokak Hayvanları Bakım ve Rehabilitasyon merkezinden alınan, 20 adet sağlıklı köpekten toplandı. Ovaryumların tekrarlı parçalanmasından sonra, seçilen COCs (kumulus oosit kompleksleri), 5% CO2 inkübatörde, mineral yağla kaplanmış 500 μl TCM-199 içeren dört-gözlü petrilerde, 39°C’de, 72 saat boyunca maturasyona bırakıldı. Maturasyondan sonra oositler, 0%, 10%, 20% etilen glikol içeren 50 ml PBl içinde sırasıyla, 10, 10 dakika ve 30 saniye muamele edildi. Oositler, 30 μl VS3 içeren kriyoviallere yerleştirilerek sıvı nitrojende donduruldu. Bu grubun oositleri (n=257) ‘vitrifiye oosit-VO’ olarak gruplandı. Çözdürme sonrasında, oositler ionomisinle 5 dakika ve sikloheksimid ile 3 saat muamele ederek partenogenetik aktivasyona bırakıldı. Sonrasında oositler 72 saat kültüre edilerek nükleer maturasyon değerlendirildi. Kontrol grubu olarak kullanılan oositler (n=257), ‘non vitrifiye oosit-FO’ olarak gruplandırıldı. Maturasyondan sonra, oositler direkt olarak ionomisin ve sikloheksimid ile muamele edilerek aktivasyona bırakıldı ve 72 saat kültüre edildi. Tüm oositler Hoechst33342 ile 30 dakika boyandıktan sonra nükleer maturasyon oranları mikroskopta değerlendirildi. Maturasyon oranları (MI+MII) gruplar arasında istatistiksel olarak anlamlı bulunmadı. (p>0,05). Gruplar arasında GV, GVBD, MI, ve MII oranlarında da istatistiksel fark bulunmadı (p>0,05). Maturasyon sonrasında, vitrifiye köpek oositlerinde partenogenetik aktivasyona bağlı nükleer değerlendirmeye çalışması bulunmamaktadır. Fakat bu uygulamada elde edilen düşük maturasyon oranlarının, ileri moleküler çalışmalarla açıklanması gerektiği kanısındayız.

Keywords

• Köpek oositleri
• vitrifikasyon
• in vitro maturasyon
• partenogenetik aktivasyon

References

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