İn Vitro koşullarda elde edilen sığır embriyolarının antifreeze protein III ile dondurulması ve çözüm sonrası değerlendirilmesi
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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 tez çalışmasında in vitro üretilen sığır embriyolarının vitrifikasyon yöntemi ile dondurulmasında vitrifikasyon solüsyonuna ilave edilen farklı dozlardaki tip III Antifreeze Protein (AFP-III)'in çözüm sonu 24. saatte reekspanse blastosist (hatched olmayan), hatched blastosist ve toplam canlı blastosist oranları ile blastosistteki hücre sayıları (iç hücre kitlesi, trofektoderm ve toplam hücre sayıları) oranları üzerindeki etkilerinin araştırılması amaçlandı. Mezbahanede kesim sonrası hayvanların ovaryumları toplanarak uygun ortamlarda laboratuvara getirildi ve ovaryumlar üzerinde bulunan 2-8 mm çapa sahip olan foliküller aspire edilerek oositler toplandı. Toplanan oositler kalitelerine göre sınıflandırılıp çalışmaya yalnızca A ve B kalite mature olmayan oositler dahil edildi. Oositlerin maturasyonu, fertilizasyonu, kültür işlemi ve blastosist oluşum süreci için IVF Bioscience firmasına ait ticari solüsyonlar kullanıldı. Oositlerin maturasyonu ve fertilizasyonu sırasıyla 22-24 saat ve 12-18 saat süreyle %5 CO2, 38,5˚C'de ve yüksek oranda nemli ortam sağlayan inkübatörde gerçekleştirildi. Fertilizasyon sonrası kültür işlemi %6 CO2, %6 O2, 38,5˚C ısı ve yüksek oranda nem içeren inkübatörde 6-9 gün süresince yapıldı. Fertilizasyon 0. gün kabul edilerek 6. günden itibaren in vitro kültür ortamında blastosist varlığı kontrol edildi. Vitrifikasyon işlemi için 3 aşamalı vitrifikasyon prosedürü (IVF Bioscience firmasının önerdiği şekilde) uygulandı. Çalışmanın vitrifikasyon deney grupları Kontrol (AFP-III 0), AFP-III 200 ng/ml, AFP-III 300 ng/ml ve AFP-III 400 ng/ml olacak şekilde oluşturuldu ve blastosistler bu deney gruplarıyla vitrifikasyon yöntemiyle donduruldu. Çözüm sonrası toplamda 166 adet embriyonun gelişimleri gözlenerek değerlendirmeye alındı. Çözüm sonrası 24. saatteki gelişmeleri izlenen embriyoların: Reekspanse (hatched olmayan), hatched blastosist ve toplam canlı blastosist oranları bakımından vitrifikasyon deney grupları arasında istatistiksel açıdan önemli bir fark görülmedi. Aynı şekilde, diferansiyel boyama sonrası elde edilen blastosistteki hücre sayıları ve oranları arasında yapılan istatistik analizlerde kontrol grubu ve farklı AFP-III dozları arasında herhangi bir önemli fark bulunmadı (p>0,05). Sonuç olarak, çözüm sonrası 24. saatteki blastosist gelişim oranları ve blastosistteki hücre sayıları üzerinde AFP-III'ün istatistiksel açıdan önemli bir fark oluşturmadığı gözlendi (p>0,05).
The purpose of this dissertation study was to freeze in vitro produced bovine embryos by the vitrification method and to investigate the effect of different doses type III antifreeze protein by adding to the vitrification solution used in the freezing process and evaluate its effect on reexpanding blastocyst (non-hatched), hatched blastocyst, total live blastocyst rates and cell numbers (inner cell mass, trophoectoderm and total cell number of blastocyst) 24th hour after thawing. After slaughter in the slaughterhouse, the ovaries of the animals were collected and brought to the laboratory in appropriate environments, and the follicles with a diameter of 2-8 mm on the ovaries were aspirated and the oocytes were collected. The collected oocytes were classified according to their quality, and only immature oocytes of quality A and B were included in the study. Commercial medias from IVF Bioscience were used for the maturation, fertilization, culture and blastocyst formation process of oocytes. Maturation and fertilization of oocytes were carried out in an incubator providing an environment of 5% CO2, 38.5˚C and high humidity for respectively 22-24 hours and 12-18 hours. Post-fertilization culture was carried out for 6-9 days in an incubator providing an environment of 6% CO2, 6% O2, 38.5˚C temperature and high humidity. Fertilization was considered to be day 0, and the presence of blastocysts in the in vitro culture medium was checked starting from day 6. For the vitrification process, the 3-stage vitrification procedure was applied as recommended by the IVF Bioscience company. The vitrification experimental groups of our study were created as Control (AFP-III 0), AFP-III 200 ng/ml, AFP-III 300 ng/ml and AFP-III 400 ng/ml, and blastocysts were frozen with these experimental groups by the vitrification method. After the warming procedure (according to IVF Bioscience procedure), the development of a total of 166 embryos was observed and evaluated. There was no statistically significant difference between the vitrification experimental groups in terms of reexpansion (non-hatched), hatched blastocyst and total live blastocyst rates of the embryos whose developments were monitored at the 24th hour after warming. Likewise, in the statistical analysis made between the cell numbers and ratios in the warmed blastocyst obtained after differential staining, no significant difference was found between the control group and different doses of AFP-III groups (p>0,05). As a result, it was observed that AFP-III did not make a statistically significant difference on blastocyst development rates and cell numbers in the vitrified blastocyst at the 24th hour after warming (p>0,05).
The purpose of this dissertation study was to freeze in vitro produced bovine embryos by the vitrification method and to investigate the effect of different doses type III antifreeze protein by adding to the vitrification solution used in the freezing process and evaluate its effect on reexpanding blastocyst (non-hatched), hatched blastocyst, total live blastocyst rates and cell numbers (inner cell mass, trophoectoderm and total cell number of blastocyst) 24th hour after thawing. After slaughter in the slaughterhouse, the ovaries of the animals were collected and brought to the laboratory in appropriate environments, and the follicles with a diameter of 2-8 mm on the ovaries were aspirated and the oocytes were collected. The collected oocytes were classified according to their quality, and only immature oocytes of quality A and B were included in the study. Commercial medias from IVF Bioscience were used for the maturation, fertilization, culture and blastocyst formation process of oocytes. Maturation and fertilization of oocytes were carried out in an incubator providing an environment of 5% CO2, 38.5˚C and high humidity for respectively 22-24 hours and 12-18 hours. Post-fertilization culture was carried out for 6-9 days in an incubator providing an environment of 6% CO2, 6% O2, 38.5˚C temperature and high humidity. Fertilization was considered to be day 0, and the presence of blastocysts in the in vitro culture medium was checked starting from day 6. For the vitrification process, the 3-stage vitrification procedure was applied as recommended by the IVF Bioscience company. The vitrification experimental groups of our study were created as Control (AFP-III 0), AFP-III 200 ng/ml, AFP-III 300 ng/ml and AFP-III 400 ng/ml, and blastocysts were frozen with these experimental groups by the vitrification method. After the warming procedure (according to IVF Bioscience procedure), the development of a total of 166 embryos was observed and evaluated. There was no statistically significant difference between the vitrification experimental groups in terms of reexpansion (non-hatched), hatched blastocyst and total live blastocyst rates of the embryos whose developments were monitored at the 24th hour after warming. Likewise, in the statistical analysis made between the cell numbers and ratios in the warmed blastocyst obtained after differential staining, no significant difference was found between the control group and different doses of AFP-III groups (p>0,05). As a result, it was observed that AFP-III did not make a statistically significant difference on blastocyst development rates and cell numbers in the vitrified blastocyst at the 24th hour after warming (p>0,05).
Açıklama
Anahtar Kelimeler
Antifreeze Protein III, İn Vitro Fertilizasyon, Sığır Blastosist, Bovine Blastocyst, İn Vitro Fertilization, Vitrification, Virtifikasyon
Kaynak
WoS Q Değeri
Scopus Q Değeri
Cilt
Sayı
Künye
Sarı, A. (2024). İn Vitro koşullarda elde edilen sığır embriyolarının antifreeze protein III ile dondurulması ve çözüm sonrası değerlendirilmesi. (Doktora Tezi). Selçuk Üniversitesi, Sağlık Bilimleri Enstitüsü, Konya.
