Folliküler ve luteal faz sırasında elde edilen sığır oositlerinin in-vitro değerlendirilmesi

Bu araştırmada inek ovaryumlardan elde edilen oositlerin morfolojik yapı ve kalitelerine göre sınıflandırılması yapılmıştır. Bu amaçla mezbaha materyalinden elde edilen 105 adet ovaryum kesimden sonraki 2-4 saat içinde laboratuara ulaştırıldı. Ovaryum yüzeyindeki fonksiyonel yapıların inspeksiyonu ile siklus fazı belirlenen ovaryumlar folliküler (n=66), luteal (n=33) ve nonsiklik (n=6) olarak sınıflandırıldı. Aspire edilen kumulus hücre kompleksleri (COC) stereomikrokroskopta incelendi. Her bir COC belirtilen morfolojik kriterlere göre sınıflandırıldı. COC-A: çok sıralı ( >3 ) kompakt kumulus hücreleri ile sarılı homojen ooplasma, COC-B: Koyu renkli ve daha az sıralı kumulus hücreli oosit , COC-C: Koyu renkli ve tamamen yayılmış ya da soyulmuş oosit. A sınıfı (n=341), B sınıfı (n=374) ve C sınıfı (n=260) olmak üzere toplam 975 COC görüldü. Buna göre folliküler faz içerisindeki ovaryumlardan 3,52±0,38 adet A kalite, 3,42±0,28 adet B kalite ve 2,36±0,24 adet C kalite COC; luteal faz içerisindeki ovaryumlardan ise 2,79±0,51 adet A kalite, 3,82±0,41 adet B kalite ve 2,64±0,43 adet C kalite COC elde edildi. Folliküler ve luteal faz grubuna ait ovaryumlardan elde edilen A, B ve C sınıflarındaki ortalama COC sayıları arasında istatistiksel yönden fark gözlenmedi (p>0,05). Sonuç olarak, seksüel siklus fazınının elde edilen COC kalitesi üzerinde belirgin bir etkisi olmadığı, in-vitro çalışmalar için her iki faza ait oositlerin kullanılabileceği görülmüştür.

In-vitro classification of bovine oocyte recovered at follicular and luteal phases

In this study, bovine oocytes were classified according to morphology and quality. A total of 105 ovaries recovered from slaughterhouse, 2-4 h to our laboratory. After inspection of the functional mass on the ovarian surface for determination of cyclic phases, ovaries were classified as follicular (n=66), luteal (n=33) and noncyclic (n=6). Cumulus oocyte complexes (COCs) were aspirated and examined stereomicroscopically. Each COCs was assigned to one of three quality based groups on the appearance of the cumulus cells: COC-A: the oocytes with homogenous ooplasm surrounded by multilayer (>3 layers) compact and bright cumulus cells, COC-B: less compact and surrounded by a layer dark cumulus cells, and COC-C: strongly expanded cumulus with dark spots or denuded. Totally 975 COCs, consisting of A class (n=341), B class (n=374) and C class (n=260) were determined. The mean COCs counts were 3.52±0.38 in A class, 3.42±0.28 B class and 2.36±0.24 C class in follicular phase group; 2.79±0.51 A class, 3.82±0.41 B class and 2.64±0.43 C class in luteal phase group. There was no significant difference between the mean COCs counts recovered from follicular and luteal ovarian phase (p>0,05). In conclusion, there is no effect of ovarian phase on COCs quality and counts. Therefore, recovered oocytes from follicular and/or luteal phases are suitable for in-vitro studies.

PDF

___

1. Farin PW, Crosier AE, Farin CE. Influence of in vitro systems on embryo survival and fetal development in cattle. Theriogenology 2001; 55:151-170
2. Leibfried-Rutledge ML. Factors determining competence of in vitro produced cattle embryos. Theriogenology 1999; 51: 473-485
3. Wit AAC, Wurth YA, Kruip AM. Effect of ovarian phase and follicle quality on morphology and developmental capacity of the bovine cumulus-oocyte complex. J Anim Sci 2000; 78: 1277–1283.
4. Varisanga MD, Sumantri C, Murakami M, Fahurdin M, Suzuki T. Morphological classification of the ovaries in relat ion to the subsequent oocyte quality for IVF produced bovine embryos. Theriogenology 1998; 50: 1015-1023.
5. Noakes DE. Endogeneous and exogenous control of ovarian cyclicity In: Noakes DE, Parkinson TJ, England GCW. (Editors). Arthur’s veterinary Reproduction and Obstetrics. 8th Edition Philadelphia: Saunders: 21. 2003
6. Salamone DF, Adams GP, Mapletoft RJ. Changes in the cumulus-oocyte complex of subordinate follicles relative to follicular wave status in cattle. Theriogenology 1999; 52: 549-561.
7. Hagemann LJ, Beaumont SE, Berg M, Donnison MJ, Ledgard A, Peterson AJ Development during single IVP of bovine oocytes from dissected follicles: interactive effects of estrous cycle stage follicle size and atresia. Mol Reprod Dev 1999; 53: 451-458.
8. Machatkova M, Krausova K, Jokesova E, Tomanek M. Developmental competence of bovine oocytes: effects of follicle size and the phase of follicular wave on in vştro embryo production. Theriogenology 2004; 61: 329-335.
9. Goeseels SB, Panich P. Effects of oocyte quality on development and transcriptional activity in early bovine embryos. Anim Reprod Sci 2002; 71: 143–155.
10. Vassena R, Mapletoft RJ, Allodi S, Singh J, Adams GP. Morphology and development competence of bovine oocytes relative to follicular status. Theriogenology 2003; 60: 923-932.
11. Leibfried L, First NL. Characterization of bovine follicular oocytes and their ability to mature in vitro. J Anim Sci 1979; 48: 76-86.
12. De Loos FAM, van Vliet C, van Maurik P, Kruip THAM. Morphology of immature bovine oocytes. Gamete Res 1989; 24: 197-204.
13. Yang YB, Lu KH. The influence of bovine oocyte type on invitro fertilization and subsequent in vitro development. Theriogenology 1990; 33: 355.
14. Hazeleger NL, Stubbings RB. Developmental potential of selected bovine oocyte cumulus complexes. Theriogenology 1992; 37: 219.
15. Hazeleger NL, Hill DJ, Stubbings RB, Walton JS. Relationship of morphology and follicular fluid enviroment of bovine oocytes to their developmental potential in vitro. Theriogenology 1995; 43: 509-522.
16. Smith LC, Olivera-Angel M, Grome NP, Bathia B, Price CA. Oocyte quality in small antral follicle in the presence and absence of a large dominant follicle in cattle. J Reprod Fert 1996; 106: 193-199.
17. Knuth KM, Hunter AG. Effects of a corpus luteum and source of mineral oil overlay on in-vitro maturation and fert ilizat ion of bovine oocytes. J Dairy Sci 1996; 79 (Suppl-I): 147.
18. Boedino A, Rajamagendran R, Saha S, Sumantri C and Suzuki T. Effect of presence of CL on ovary on the oocyte number, cleavage rate and blastocyst production in-vitro in cattle. Theriogenology 1995; 43: 169.
19. Behboodi E, Anderson GB, BonDurant RH. Development of in-vitro fert ilized oocytes from pregnant and non pregnant cows in oviductal epithelial and cumulus cell coculture systems. Theriogenology 1992; 38: 1077-1084.
20. Vajta G, Machaty Z, Barandi ZS, Varga ZS. Embryos derived from the in-vitro fertilization of oocytes of pregnant cows. Theriogenology 1992; 37: 811-815.
21. Sivakumaran K, Calder M, Rajamahendran R. The influence of reproductive status of bovine on oocyte number and maturat ion in-vitro and the effect of coculture systems on fert ilizat ion and subsequent development. J Anim Sci 1991; 69 (Suppl.): 438.