Activation of bovine oocytes following ICSI and effect of activation on embryo according to developmental stages

Bu çalışmada amaç ICSI uygulanan oositlerin Ca iyonofor ile aktivasyonu ve bu aktivasyon yönteminin embriyonik gelişim aşamalarına göre etkilerinin araştırılmasıdır. Çalışma materyalini mezbahada kesilen inek ve düve ovaryumlarından aspirasyon metodu ile toplanan kumulus oosit kompleksleri oluşturdu. A kalite kumulus oosit komplekslerinin (n=500) in vitro maturasyonu TCM-199 vasatında, 39°C’de %5 CO2 içeren maksimum neme sahip ortamda 22 saat süreyle inkübe edilerek sağlandı. Matur oositler seçilerek rastgele iki gruba ayrıldı ve ICSI işlemi gerçekleştirildi. Grup I’de (n=250) bulunan oositler 5 µM Ca iyonofor ile 38.5°C’de, 5 dak. süreyle aktive edildi. Grup II’de (n=250) bulunan oositler ise kontrol grubu olarak alındı ve herhangi bir aktivasyon işlemi uygulanmadan ICSI sonrası kültür vasatına kondu. Grup I’de bölünme, morula ve blastosist oranları sırasıyla %44.8, %17.2 ve %11.6 olarak bulunurken, Grup II’de bu oranlar sırasıyla %26.8, %8.2 ve %3.2 olarak belirlendi. Bölünme oranları açısından gruplar arasındaki fark önemli bulunurken (P0.05). Sonuç olarak, sığır oositlerinde ICSI sonrası aktivasyonun zorunlu olduğu, bölünmenin başlatılmasında Ca iyonofor ile aktivasyonun pratik ve hızlı bir yöntem olduğu fakat embriyoların morula ve blastosist aşamalarına ulaşmasında yetersiz kaldığı belirlendi.

Anahtar Kelimeler:

oosit olgunlaşması, Bovidae, blastosist, embriyonik gelişim, oosit, geviş getiren hayvanlar

Sığır oositlerinin ICSI sonrası aktivasyonu ve embriyonun gelişim aşamalarına göre aktivasyonun etkileri

In the present study the aim was investigate to Ca ionophore activation in fertilized oocytes by using Intracytoplasmic sperm injection (ICSI) method and the effects of activation considering the stages of embryo development. Cumulus-oocyte complexes (COCs) were collected by aspiration method from the ovaries of cattle and heifers slaughtered at an abattoir. In vitro maturation of A quality COCs (n=500) was performed in TCM 199 medium at 39°C in an atmosphere of 5% CO2 in air with maximum humidity for 22 h. Subsequently, ICSI was carried out to oocytes and randomly divided into two groups. In Group I (n=250; study group) consisted of oocytes activated with 5 μM Ca ionophore at 38.5°C for 5 min, and in Group II (n=250; control group) contained oocytes without any activation. In Group I cleavage, morula and blastocyst rates were determined as 44.8%, 17.2% and 11.6%, respectively. In Group II the rates were established as 26.8%, 9.2% and 3.2%, respectively. Statistically the difference between groups considering the cleavage rates were obtained significantly (P<0.001); however, difference between groups considering morula and blastocyst rates was not determined significantly (P>0.05). In conclusion, the activation of bovine oocytes after ICSI is obligated to apply, and Ca ionophore was a practical and rapid activator to incite cleavage, but inadequate for embryos to reach the morula and blastocyst stages.

Keywords:

oocyte maturation, Bovidae, blastocyst, embryonic development, oocytes, ruminants,

PDF

___

1. Chung JT, Keefer CL, Downey BR: Activation of bovine oocytes following intracytoplasmic sperm injection (ICSI). Theriogenology, 53, 1273-1284, 2000.
2. Galli C, Vassiliev I, Lagutina I, Galli A, Lazzari G: Bovine embryo development following ICSI: Effect of activation, sperm capacitation and pre-treatment with dithiothreitol. Theriogenology, 60, 1467-1480, 2003.
3. Van Steirteghem AC, Nagy Z, Liu J, Joris H, Staessen C, Smitz J, Wisanto A, Devroey P: High fertilization and implantation rates after intracytoplasmic sperm injection. Hum Reprod, 8, 1061-1066, 1993.
4. Keffer CL: Fertilization by sperm injection in the rabbit. Gamete Res, 22, 59-69, 1989.
5. Kimura Y, Yanagimachi R: Intracytoplasmic sperm injection in the mouse. Biol Reprod, 52, 709-720, 1995.
6. Galli C, Crotti G, Turini P, Duchi R, Mari G, Zavaglia G, Barbacini S, Galli C: Equine embryos at the compacted morula and blastocyst stage can be ontained by intracytoplasmic sperm injection (ICSI) of in vitro matured oocytes with frozen-thawed spermatozoa from semen of different fertilities. Theriogenology, 58, 709-712, 2002.
7. Horiuchi T, Emuta C, Yamauchi Y, Oikawa T, Numabe T, Yanagimachi R: Birth of normal calves after intracytoplasmic sperm injection of bovine oocytes: A methodical approach. Theriogenology, 57, 1013-1024, 2002.
8. Keefer CL, Younis AI, Brackett BG: Cleavage development of bovine oocytes fertilized by sperm injection. Mol Reprod Dev, 25, 281-285, 1990.
9. Ushijima H, Nakane T: Present status and prospects for bovine Intracytoplasmic sperm injection with in vitro-matured oocytes and frozen semen. J Mamm Ova Res, 23, 107-113, 2006.
10. Goto K, Kinoshita, Takuma Y, Ogawa K: Fertilization of bovine oocytes by the injection of immobilized, killed spermatozoa. Vet Rec 24, 517-520, 1990.
11. Ware CB, Barnes FL, Maki-Laurila M, First NL: Age dependence of bovine oocyte activation. Gamete Res, 22, 265-275, 1989.
12. Nagai T: Parthenogenetic activation of cattle follicular oocytes in vitro with ethanol. Gamete Res, 16, 243-249, 1987.
13. Keskintepe L, Pacholczyk G, Machnicka A, Norris K, Curuk MA, Khan I, Barckett BG: Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa. Biol Reprod, 67, 409-415, 2002.
14. Sirard MA, Florrnan HM, Leibfried-Rutledge ML, Barnes FL, Sirns ML, First NL: Timing of nuclear progression and protein synthesis necessary for meiotic maturation of bovine oocytes. Biol Reprod, 40, 1257- 1262, 1989.
15. Murase Y, Araki Y, Mizuno S, Kawaguchi C, Naito M, Yoshizawa M, Araki Y: Pregnancy following chemical activation of oocytes in a couple with repeated failure of fertilization using ICSI: Case report. Hum Reprod, 19, 1604-1607, 2004.
16. Wang WH, Abeydeera LR, Day BN: Functional analysis of activation of porcine oocytes by spermatozoa, calcium ionophore and electrical pulse. Theriogenology, 49, 158, 1998.
17. Brackett BG, Zuelke KA: Analysis of factors involved in the in vitro production of bovine embryos. Theriogenology, 39, 43-64, 1993.
18. Hansen PJ: Preparation of media. http://www.animal.ufl.edu/hansen/ ivf/mediaprep.htm, Accessed: 15.10.2005.
19. Tosti E, Boni R, Cuomo A: Fertilization and activation currents in bovine oocytes. Reproduction, 124, 835-846, 2002.
20. Rho GJ, Kawarsky S, Johnson WH, Kochhar K, Betteridge KJ: Sperm and oocyte treatments to improve the formation of male and female pronuclei and subsequent development following intra- cytoplasmic sperm injection into bovine oocytes. Biol Reprod, 59, 918- 924, 1998.
21. Oikawa T, Takada N, Kikuchi T, Numabe T, Takenaka M, Horiuchi T: Evaluation of activation treatments for blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection. Anim Reprod Sci, 86, 187-194, 2005.
22. Li GP, Seidel Jr GE, Squires EL: Improved cleavage of bovine ICSI ova cultured in heparin-containing medium. Theriogenology, 61, 1077-1084, 2004.
23. Goto K: Bovine microfertilization and embryo transfer. Mol Reprod Dev, 36, 288-290, 1993.