Koyun oositlerinin seçim kriterleri ve kültür ortamına göre in vitro gelişim düzeylerinin araştırılması
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Tarih
2024
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Yayıncı
Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Sunulan çalışmada boyama yöntemi ve morfolojik yapılarına göre seçilen koyun oositlerinin maturasyon, fertilizasyon ve blastosist gelişim oranlarına drop hacmi ve oosit yoğunluğunun etkisinin belirlenmesi amaçlanmıştır. Sunulan çalışmanın laboratuvar uygulamaları UHAEM Müdürlüğü bünyesinde bulunan Embriyo Transferi laboratuvarında yürütüldü. Lokal mezbahadan alınan ovaryumlar antibiyotik içeren %0,9 fizyolojik tuzlu su içerisinde ve 25-30°C'lik sabit sıcaklık sağlayan termosta 2-3 saat içerisinde laboratuvara getirildi. Oositlerin maturasyonunda TCM-199, fertilizasyonunda BSOF, kültüründe ise SOF kullanıldı. Oositler; 2-8 mm çaplı antral foliküllerden 18-G kanül takılan 5 ml'lik enjektör kullanılarak aspire edildi. Alınan oositler rastgele iki gruba ayrıldı. Birinci gruptaki oositler Brilliant Cresyl Blue (BCB) ile boyandı. İkinci gruptaki oositler ise morfolojik yapılarına göre değerlendirildi. Boya alan oositler (BCB+) ve morfolojik değerlendirme sonunda A ve B kalitede olan oositler çalışmaya dahil edildi. Seçilen oositler, oluşturulan mikro damlacıklarda oosit/solüsyon hacim oranları; 1 adet /5 µl, 1 adet /10 µl, well of the well (WOW) kültür sisteminde bireysel ve 16 adet /100 µl grubunda toplu halde ve olacak şekilde in vitro maturasyon, fertilizasyon ve kültür aşamalarına tabi tutuldu. Mezbahadan getirilen 352 adet ovaryumdan 2-8 mm çapındaki foliküllerden toplam 1179 oosit aspire edildi. Elde edilen oositlerin 627'si BCB boyasına maruz bırakıldı ve bunların 367'si boya alırken (BCB+) 260'ı boya almadı (BCB-). Kalan 554 oosit ise morfolojik yapılarına göre sınıflandırıldı ve 393 adet oosit A ve B kaliteli olarak değerlendirildi. Çalışmada, boya alan 367 oosit (BCB+) ve A-B kalitedeki 393 oosit olmak üzere toplam 760 oosit kullanıldı. Hem morfolojik yapısına göre hem de BCB ile boyama sonrası değerlendirilen oositlere bütün aşamalarda aynı şartlarda IVP uygulamaları gerçekleştirildi. Yapılan analizde BCB ile seçilen oositlerde; IVM, IVF ve IVC sonuçları sırası %86,82; %64,57; %26,15 olarak gerçekleşirken morfolojik seçime göre yapılan değerlendirmede ise bu oranlar %83,20; %57,25; %21,37 olarak gerçekleşti ve gruplar arasında istatistiki farkın önemli olduğu tespit edildi. A ve B kalite oositler ile farklı büyüklükteki drop hacimlerine göre yapılan çalışmada ise maturasyon oranları sırası ile %84,86; %76,56; %84,37 ve %87,50; fertilizasyon oranları %73,46; %64,21; %69,73 ve %69,25 blastosist oranları ise %30,49; %16,21; %27,72 ve %28,54 olarak tespit edildi. Boyama yöntemi ile seçilen BCB+ oositlerde ise maturasyon oranları sırası ile %88,20; %79,91; %88,54 ve %90,62; fertilizasyon oranları %78,44; %67,80; %75,28 ve %74,84; blastosist oranları ise %34,33; %19,52; %30,85 ve %33,33 olarak tespit edildi. Her iki çalışmada da gruplar arasındaki fark istatistiki bakımdan anlamlı bulundu (P<0,05). Sonuç olarak, koyunlarda BCB boyası kullanılarak seçilen oositlerin, morfolojik yapısına göre seçilen oositlere göre in vitro embriyo üretiminin tüm aşamalarında daha iyi gelişim gösterdiği, oositler boyama işleminden zarar görmediği için subjektif değerlendirmeden daha yararlı bir araç olarak IVF çalışmalarında kullanılabileceği sonucuna ulaşıldı. Aynı zamanda sunulan çalışma ile koyunlarda in vitro embriyo üretiminde; hücrelerin maturasyon, fertilizasyon ve kültür ortamında 5 μl büyüklüğündeki droplarda bireysel olarak da gelişebileceği, grup kültürü ve WOW sistemi ile benzer oranlarda blastosist geliştirilebildiği belirlenmiştir.
The present study aimed to determine the effect of drop volume and oocyte density on the maturation, fertilization, and blastocyst development rates of sheep oocytes selected according to staining method and morphological structures. The laboratory applications of the present study were carried out in the Embryo Transfer Laboratory of the UHAEM Directorate. Ovaries were obtained from slaughterhouses close to the laboratory. TCM-199 was used for maturation, BSOF for fertilization, and SOF for oocyte culture. Oocytes were aspirated from antral follicles measuring 2-8 mm in diameter. The oocytes were randomly divided into two groups. The oocytes in the first group were stained with Brilliant Cresyl Blue (BCB). The oocytes in the second group were scored according to their morphological structures. Stained oocytes (BCB+) and oocytes with A and B quality at the end of morphological evaluation were included in the study. The selected oocytes were subjected to in vitro maturation, fertilization, and culture stages individually in micro drops of 5 µL, 10 µL volume, and well of the well (WOW) culture system and groups of 16 oocytes in 100 µL drops. A total of 1179 oocytes were collected from follicles 2-8 mm in diameter from 352 ovaries. Of the collected oocytes, 627 were exposed to BCB staining, of which 367 were stained (BCB+) and 260 were not (BCB-). The collected 554 oocytes were classified according to their morphological structures and 393 oocytes were classified as A and B quality. A total of 760 oocytes were used in the study, including 367 oocytes that received dye (BCB+) and 393 oocytes of A-B quality. Both A-B quality oocytes and BCB+ oocytes were subjected to IVP with the same volume and cell density at all stages. The results of IVM, IVF, and IVC were 86.82%, 64.57%, and 26.15% respectively in oocytes selected by BCB, while these rates were 83.20%, 57.25%, and 21.37% in the evaluation according to morphological selection, and it was found that the statistical difference between the groups was significant. In the study carried out with A and B quality oocytes according to different drop volumes, the maturation rates were 84.86%, 76.56%, 84.37%, and 87.50%; the fertilization rates were 73.46%, 64.21%, 69.73%, and 69.25%; the blastocyst rates were 30.49%, 16.21%, 27.72%, and 28.54%. In BCB+ oocytes selected by staining, maturation rates were 88.20%, 79.91%, 88.54%, and 90.62%; fertilization rates were 78.44%, 67.80%, 75.28%, and 74.84%; and blastocyst rates were 34.33%, 19.52%, 30.85%, and 33.33%, respectively. In both studies, the difference between the groups was statistically significant (P<0.05). As a result, it was concluded that oocytes selected by BCB staining in sheep showed better development at all stages of in vitro embryo production than oocytes selected by morphological structure, and since the oocytes were not damaged by the staining process, it could be used as a more useful tool in IVF studies than subjective evaluation. At the same time, it was found that in vitro embryo production in sheep, cells can develop individually in 5 μL drops in the maturation, fertilization, and culture environment, and blastocysts can be developed at similar rates using group culture and the WOW system.
The present study aimed to determine the effect of drop volume and oocyte density on the maturation, fertilization, and blastocyst development rates of sheep oocytes selected according to staining method and morphological structures. The laboratory applications of the present study were carried out in the Embryo Transfer Laboratory of the UHAEM Directorate. Ovaries were obtained from slaughterhouses close to the laboratory. TCM-199 was used for maturation, BSOF for fertilization, and SOF for oocyte culture. Oocytes were aspirated from antral follicles measuring 2-8 mm in diameter. The oocytes were randomly divided into two groups. The oocytes in the first group were stained with Brilliant Cresyl Blue (BCB). The oocytes in the second group were scored according to their morphological structures. Stained oocytes (BCB+) and oocytes with A and B quality at the end of morphological evaluation were included in the study. The selected oocytes were subjected to in vitro maturation, fertilization, and culture stages individually in micro drops of 5 µL, 10 µL volume, and well of the well (WOW) culture system and groups of 16 oocytes in 100 µL drops. A total of 1179 oocytes were collected from follicles 2-8 mm in diameter from 352 ovaries. Of the collected oocytes, 627 were exposed to BCB staining, of which 367 were stained (BCB+) and 260 were not (BCB-). The collected 554 oocytes were classified according to their morphological structures and 393 oocytes were classified as A and B quality. A total of 760 oocytes were used in the study, including 367 oocytes that received dye (BCB+) and 393 oocytes of A-B quality. Both A-B quality oocytes and BCB+ oocytes were subjected to IVP with the same volume and cell density at all stages. The results of IVM, IVF, and IVC were 86.82%, 64.57%, and 26.15% respectively in oocytes selected by BCB, while these rates were 83.20%, 57.25%, and 21.37% in the evaluation according to morphological selection, and it was found that the statistical difference between the groups was significant. In the study carried out with A and B quality oocytes according to different drop volumes, the maturation rates were 84.86%, 76.56%, 84.37%, and 87.50%; the fertilization rates were 73.46%, 64.21%, 69.73%, and 69.25%; the blastocyst rates were 30.49%, 16.21%, 27.72%, and 28.54%. In BCB+ oocytes selected by staining, maturation rates were 88.20%, 79.91%, 88.54%, and 90.62%; fertilization rates were 78.44%, 67.80%, 75.28%, and 74.84%; and blastocyst rates were 34.33%, 19.52%, 30.85%, and 33.33%, respectively. In both studies, the difference between the groups was statistically significant (P<0.05). As a result, it was concluded that oocytes selected by BCB staining in sheep showed better development at all stages of in vitro embryo production than oocytes selected by morphological structure, and since the oocytes were not damaged by the staining process, it could be used as a more useful tool in IVF studies than subjective evaluation. At the same time, it was found that in vitro embryo production in sheep, cells can develop individually in 5 μL drops in the maturation, fertilization, and culture environment, and blastocysts can be developed at similar rates using group culture and the WOW system.
Açıklama
Anahtar Kelimeler
Brilliant Cresyl Blue, Embriyo, İn Vitro Fertilizasyon, Koyun, Well Of The Well, Embryo, In Vitro Fertilization, Sheep
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Sevgi, S. Ü. (2024). Koyun oositlerinin seçim kriterleri ve kültür ortamına göre in vitro gelişim düzeylerinin araştırılması. (Doktora Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
