MİTHAT EVECEN, ÜMÜT CİRİT, KAMBER DEMİR, Elif KARAMAN, GÜL BAKIRER ÖZTÜRK, Asiye İzem HAMZAOĞLU, SEMA BİRLER, SERHAT PABUCCUOĞLU

Comparison of two different media for In vitro production of dog embryos

Köpeklerde in vitro fertilizasyon yöntemi ile embriyo üretilebilmiş ve nükleer transfer yöntemiyle klonlanmış embriyolardan canlı bir doğum elde edilebilmiş olmasına karşın, bu alandaki teknolojilerin başarısı oldukça sınırlı düzeydedir. Bu güne kadar yapılan in vitro çalışmalarda, sadece iki adet morula ve bir adet blastosist elde edilebilmiştir. Sunulan çalışmanın amacı, iki farklı medyumun (mSOF ve TCM 199) köpek oositlerinin in vitro maturasyon (İVM), in vitro fertilizasyon (İVF) ve in vitro kültürü (İVK) üzerine etkilerini araştırmaktır. Çalışma iki aşamada gerçekleştirildi. Birinci aşamada, iki farklı medyumun köpek oositlerinin İVM’u üzerine etkisi araştırıldı. İVM sürecinin sonunda fikze edilen oositlerin nükleer olgunlaşma değerlendirmesi, aseto-orsein ile boyanarak yapıldı. Çalışmanın ikinci aşamasında ise, 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. Sonuç olarak, İVM oranları açısından mSOF medyum grubunda bulunan oositlerin TCM 199 grubundakilere nazaran daha başarılı olduğu belirlendi (P

Anahtar Kelimeler:

oosit, morula, blastosist, hücre kültürü, yarılma, kültür ortamı, embriyo kültürü, embriyo nakli, embriyonik gelişim, embriyo, in vitro kültür, köpek, in vitro döllenme

İn vitro köpek embriyosu üretimi üzerine iki farklı medyumun etkisi

Embryo production via in vitro fertilization and nuclear transfer has been accomplished in the dog, and the transfer of the cloned embryos has recently resulted in the birth of puppies. However, the efficiency of these technologies is still very limited. Until now, only two morulas and single blactocyst have been achieved in vitro. Therefore, the aim of the present study was to examine the effects of two different media (mSOF and TCM 199) 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, the effects of two different media, on IVM of dog oocytes were investigated. At the end of the IVM period, the nuclear maturation rates were evaluated by aceto-orcein staining method. At the second step, after the IVM period the oocytes inseminated with fresh spermatozoa for 24 h and left for IVC for 7 d. At the end of the IVC period, embryonic development was assessed by microscopic observation at 24 h intervals and then fixed and stained by the same method. Consequently, maturation rates of oocytes in mSOF medium were significantly higher than those of the TCM 199 (P<0.001). After 7 d of the IVC period, only one oocyte was cleaved in the TCM 199, while eight oocytes cleaved and one of them developed to morula stage in mSOF medium group (P=0.037).

Keywords:

oocytes, morula, blastocyst, cell culture, cleavage, culture media, embryo culture, embryo transfer, embryonic development, embryos, in vitro culture, dogs, in vitro fertilization,

PDF

___

1. Hewitt DA, England GCW: Synthetic oviductal fluid and oviductal cell coculture for canine oocyte maturation in vitro. Anim Reprod Sci, 55, 63-65, 1999.
2. 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.
3. Mahi CA, Yanagimachi R: Maturation and sperm penetration of canine ovarian oocytes in vitro. J Exp Zool, 196, 189-196, 1976.
4. Luvoni GC, Chigioni S, Beccaglia M: Embryo production in dogs: From in vitro fertilization to cloning. Reprod Dom Anim, 41, 286-290, 2006.
5. Songsasen N, Wildt DE: Oocyte biology and challenges in developing in vitro maturation systems in the domestic dog. Anim Reprod Sci, 98, 2-22, 2007.
6. Otoi T, Shin T, Kraemer D, Westhusin ME: Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization. Reprod Nutr Dev, 44, 631-637, 2004.
7. Hatoya S, Sugiyama Y, Torii R, Wijewardana V, Kumagai D, Sugiura K, Kida K, Kawate N, Tamada H, Swada T, Inaba T: Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes. Theriogenology, 55, 1083-1090, 2006.
8. Otoi T, Murakami M, Fujii M, Tanaka M.Ooka A, Une S, Suzuki T: Development of canine oocytes matured and fertilized in vitro. Vet Rec, 146, 52-53, 2000.
9. Lee BC, Kim M.K., Jang G, Oh HJ, Yuda F, Kim HJ, Shamin MH, Kim JJ, Kang SK, Schatten G, Hwang WS: Dogs cloned from adult somatic cells. Nature, 436, 641, 2005.
10. England GWW, Verstegen JP, Hewitt DA: Pregnancy following in vitro fertilisation of canine oocytes. Vet Rec, 148, 20-22, 2000.
11. Farstad W: Current state in biotechnology in canine and feline reproduction. Anim Reprod Sci, 60-61, 375-387, 2000.
12. Evecen M, Cirit Ü, Demir K, Karaman E, Hamzaoğlu Aİ, Bakırer G: Developmental competence of domestic cat oocytes from ovaries stored at various durations at 4oC temperature. Anim Reprod Sci, 116, 169-172, 2009.
13. Tervitt HR, Whittingham DG, Rowson LEA: Successful culture in vitro of sheep and cattle ova. J Reprod Fertil, 30, 493-497, 1972.
14. 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.
15. Hewitt DA, England GCW: The effect of oocyte size and bitch age upon oocyte nuclear maturation in vitro. Theriogenology, 49, 957-966, 1998.
16. 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.
17. 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.
18. Taş M, Evecen M, Özdaş ÖB, Cirit Ü, Demir K, Birler S, Pabuccuoğlu S: Effect of transport and storage temperature of ovaries on in vitro maturation of bitch oocytes. Anim Reprod Sci, 96, 30-34, 2006.
19. Rodrigues BA, Rodrigues JL: Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome. Theriogenology, 66, 1667-1672, 2006.
20. Rodrigues BA, Santos, DLC, Rodrigues JL: Effect of maturation medium on in vitro cleavage of canine oocytes fertilized with fresh and cooled homologous semen. Zygote, 15, 43-53, 2007.
21. Rota A, Cabianca G: In vitro maturation rates of canine oocytes from anoestrous bitches in simple media. Reprod Nutr Dev, 44, 105-109, 2004.
22. Massip A, Mermillod P, Van Langendonct A, Touze JL, Dessy F: Survival and viability of fresh and frozen-thawed in vitro bovine blastocyst. Reprod Nurt Dev, 35, 3-10, 2005.
23. Cui XS, Jin YX, Shen XH, Lee JY, Lee HS, Yin XJ, Kong IK, Kim NH: Epidermal growth factor enhances meiotic resumption of canine oocytes in the presence of BSA. Theriogenology, 66, 267-274, 2006.
24. Keskintepe L, Brackett BG: In vitro developmental competence of in vitro-matured bovine oocytes fertilized and cultured in completely defined media. Biol Reprod, 55, 333- 339, 1996.
25. Evecen M, Şahin BE, Baran A, Ak K, İleri İK: In vitro fertilization of cat oocytes. J Fac Vet Univ Istanbul, 29, 253- 258, 2003.
26. 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.
27. Farstad W, Hyttel P, Grondahl C, Monval MM, Smith AJ: Fertilization and early embryonic development in the blue fox (Alopex lagopus). J Reprod Fertil, 47, 219-226. 1993.