Effect of oocyte diameter on in vitro embryo production in dogs
Köpeklerde, in vitro embriyo üretimi amacıyla henüz başarılı bir yöntem bulunmamaktadır. Günümüze kadar yapılan in vitro çalışmalarda, sadece bir adet blastosist elde edildiği bildirilmiştir. Sunulan çalışmada, köpeklerde farklı oosit çaplarının (≤100 μm ve >100 μm) in vitro maturasyon (İVM), in vitro fertilizasyon (İVF) ve in vitro kültür (İVK) sonrası embriyonik gelişim üzerine etkisinin araştırılmasını amaçlandı. Çalışma iki aşamada gerçekleşti. İlk aşamada (Deney I), farklı oosit çaplarının İVM üzerine etkisi araştırıldı. Bu aşamanın sonunda oositlerin olgunlaşma durumları, aseto-orsein boyama metoduyla belirlendi. Çalışmanın ikinci aşamasında ise (Deney II), İVM sonrasında oositler taze sperma ile 24 saat İVF’a tabii tutuldular ve ardından yedi gün boyunca in vitro kültüre edildiler. Embriyoların gelişimsel kontrolleri her 24 saatte bir mikroskop bakısıyla kontrol edildikten sonra, yedinci günün sonunda aynı metotla fikze edilip boyanarak değerlendirildiler. İVM-İVF sonuçları karşılaştırmasında, büyük çapa sahip olan oositlerin, küçük olanlara göre daha yüksek oranda olgunlaşabildiği ve daha fazla oranda bölünmeler gösterebildiği saptandı (P
Köpeklerde oosit çapının in vitro embriyo üretimi üzerine etkisi
In vitro embryo production has not been confidently applied to the dog successfully. Up to date, only one blactocyst have been achieved by in vitro culture. Therefore, the aim of the present study was to examine the effects of different oocyte diameters (≤100 μm and >100 μm) on in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of immature dog oocytes. The study was performed in two steps. At the first step (experiment I), effects of two different oocyte diameters on IVM of dog oocytes were investigated. The nuclear maturation rates were evaluated by aceto-orcein staining method at the end of the IVM. At the second step (experiment II), in vitro matured oocytes were fertilized with fresh spermatozoa for 24 h and in vitro cultured for 7 d. At the end of the IVC period, embryonic development was assessed by microscopic observation at 24 h intervals and fixed for staining by aceto-orcein staining method after 7 days. In comparison relating in IVM and IVF rates, larger oocytes have higher maturation (P<0.05) and cleavage (P<0.01) rates than the smaller ones. Unfortunately, none of the oocyte was reached to morula or blactocyst stage in both groups. In conclusion, it is demonstrated that the oocyte diameter may be a helpful selection criteria for dog in vitro embryo production.
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- 1. Concannon PW, McCann JP, Temple M: Biology and endocrinology of ovulation, pregnancy, and parturition in the dog. J Reprod Fertil, 39 (Suppl.): 3-25, 1989.
- 2. Yamada S, Shimazu Y, Kawaji H, Nakazawa M, Naito K, Toyoda Y: Maturation, fertilization, and development of dog oocytes in vitro. Biol Reprod, 46, 853-858, 1992.
- 3. Otoi T, Fujii M, Tanaka M, Ooka A, Suzuki T: Canine oocyte diameter in relation to meiotic competence and sperm penetration. Theriogenolog, 54, 535-542, 2000.
- 4. Farstad W: Current state in biotechnology in canine and feline reproduction. Anim Reprod Sci, 60-61, 375-387, 2000.
- 5. Otoi T, Yamamoto K, Koyama N, Tachikawa S, Suzuki T: Bovine oocyte diameter in relation to developmental competence. Theriogenology, 48, 769-774, 1997.
- 6. Hewitt DA, England GCW: The effect of oocyte size and bitch age upon oocyte nuclear maturation in vitro. Theriogenology, 49, 957-966, 1998.
- 7. Evecen M, Cirit Ü, Demir K, Özdaş ÖB, Taş M, Birler S, Pabuccuoğlu S: Effects of estrous cycle stage and transport temperature of ovaries on in vitro maturation of canine oocytes. Anim Reprod Sci, 117, 160-165, 2010.
- 8. Tervitt HR, Whittingham DG, Rowson LEA: Successful culture in vitro of sheep and cattle ova. J Reprod Fertil, 30, 493-497, 1972.
- 9. Rodrigues BA, Santos DLC, Rodrigues JL: Embryonic development of in vitro matured and in vitro fertilized dog oocytes. Mol Reprod Dev, 67, 215-223, 2004.
- 10. Reynaud K, Fontbonne A, Marseloo N, Thoumire S, Chebrout M, de Lesegno CV, Chastant-Maillard S: In vivo meiotic resumption, fertilization and early embryonic development in the bitch. Reproduction, 130, 193-201, 2005.
- 11. Songsasen N, Gomez M, Leibo SP: Effects of meiosisinhibiting agents and equine chorionic gonadotropin on nuclear maturation of canine oocytes. Mol Reprod Dev, 65, 435-445, 2003.
- 12. Songsasen N, Wildt DE: Size of the donor follicle, but not stage of reproductive cycle or seasonality, influences meiotic competency of selected domestic dog oocyte. Mol Reprod Dev, 72, 113-119, 2005.
- 13. Songsasen N, Fickes A, Pukazhenthi BS, Wildt DE: Follicular morphology, oocyte diameter and, localisation of fibroblast growth factors in the domestic dog ovary. Reprod Dom Anim, 44 (Suppl. 2): 65-70 (2009); doi: 10.1111/j.1439- 0531.2009.01424.x
- 14. Favetta LA, Robert C, St John ET, Betts DH, King WA: P66shc but not p53 is involved in early arrest of in vitro produced bovine embryos. Mol Human Reprod, 10, 383-392, 2004.