Sistein ile kombine edilen alabalık seminal plazmasının koç spermasının dondurulabilirliği üzerine etkisi
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Date
2019-07-29
Authors
Gökçe, Elif
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Publisher
Bursa Uludağ Üniversitesi
Abstract
Bu çalışmada tek başına ya da 1 mM sistein ile kombine edilen alabalık seminal plazmasının (ASP) farklı dozlarının (%0, %1, %10, %15) koç spermasının dondurulması ve eritme sonrası inkübasyon direnci üzerine etkilerinin belirlemesi amaçlanmıştır. Sperma, on baş Kıvırcık ırkı koçtan elektro-ejakülatör ile gün aşırı alındı ve birleştirildi (pooling). Pooling sonrası ortalama motilite, plazma membran bütünlüğü, akrozom ve DNA hasarı oranları sırasıyla %83,00; %89,80; %8,20; %3,40 olarak değerlendirildi. Pooling sonrası sekiz eşit hacimde bölünen sperma, %0, %1, %10, %15 oranında ASP içeren sisteinli ve sisteinsiz sulandırıcılarla final hacim 1:5 (sperma/sulandırıcı) olacak şekilde sulandırıldı ve sıcaklığı bir saat içinde 5 ºC’a indirildi. Ardından spermaya %0, %1, %10, %15 ASP ilave edilen sisteinli ve sisteinsiz sulandırıcı B (%6 gliserol) eklenerek iki saat boyunca ekilibrasyona bırakıldı. Ekilibrasyon sonrası sperma 0,25 mLhacimli payetlere çekilerek programlanabilir dondurma cihazıyla donduruldu ve sıvı azot tankında saklandı. Eritme sonrası ve inkübasyonun 3 ila 5. saatlerinde motilite, HOST, akrozomal hasar ve DNA hasarı oranları saptandı. Eritme sonrası ve inkübasyonun 3. saatinde motilite ve plazma membran bütünlüğünün %10 ASP-sistein içeren sulandırıcı ile daha yüksek oranda korunduğu saptandı (P<0,05). En düşük akrozom hasarı %10 ASP-sistein içeren gruptan elde edilirken; en düşük DNA hasarı yalnızca %10 ASP içeren gruptan elde edildi. İnkübasyonun son saatinde ise %10 ASP’nin sistein ile kombine edilmesinin tüm spermatolojik parametreler ile beraber DNA hasarı oranının korumasında da daha etkili olduğu gözlendi. Sonuç olarak, koç sperma sulandırıcısına çeşitli dozlarda eklenen ASP ve sisteinin eritme sonrası incelenen spermatolojik parametreler üzerine koruyucu etkilerde bulunduğu kanaatine varıldı.
The aim of the current study was to evaluate the effect of unaccompanied or cystein (1 mM) combinated Rainbow Trout’s Seminal Plasma’s (RTSP) doses (0%, 1%, 10%, 15%) on cryopreservation and incubation resilience of ram semen. Ram semen was collected from ten Kivircik rams via electroejaculator every other day and then collected semen was pooled. After pooling procces the mean of motility, plasma membrane integrity, acrosome defect and DNA fragmentation rates were detected as 83,00%; 89,80%; 8,20% and 3,40% respectively. After pooling process semen was divided into eight equal volumes and diluted with extenders which containing different doses of RTSP (0%, 15, 10%, 15%) and cysteine (0 mM, 1 mM). Diluted samples were cooled to 5 ºC in an hour and rediluted with extender containing 0%, 1%, 10%, 15% RTSP (with or without cystein) and 6% glycerol before two-hour equilibration. After equlibration, semen were placed into 0,25 mLfrench straws and frozen with a programmable freezing device and they were stored in liquid nitrogen tanks. Motility, HOST, defected acrosome and DNA fragmentation rates were assessed after thawing, 3th hour and 5th hour of incubation. It is detected that the extender containing 10% RTSP-cysteine was more protective on motility and plasma membrane integrity after thawing and 3rd hour of incubation (P<0,05). Although the least defected acrosome rates were detected with 10% RTSP-cysteine containing group, the least DNA fragmentation rates were observed with only 10% RTSP group. At the end of incubation, it is observed that the 10% RTSP-cysteine combination is more efficient for protecting all spermatological parameters without excepting DNA integrity. It was a reached conclusion that, supplementation of RTSP and cystein have a protective effect on cryosurvival of ram semen and on assessed spermatological parameters.
The aim of the current study was to evaluate the effect of unaccompanied or cystein (1 mM) combinated Rainbow Trout’s Seminal Plasma’s (RTSP) doses (0%, 1%, 10%, 15%) on cryopreservation and incubation resilience of ram semen. Ram semen was collected from ten Kivircik rams via electroejaculator every other day and then collected semen was pooled. After pooling procces the mean of motility, plasma membrane integrity, acrosome defect and DNA fragmentation rates were detected as 83,00%; 89,80%; 8,20% and 3,40% respectively. After pooling process semen was divided into eight equal volumes and diluted with extenders which containing different doses of RTSP (0%, 15, 10%, 15%) and cysteine (0 mM, 1 mM). Diluted samples were cooled to 5 ºC in an hour and rediluted with extender containing 0%, 1%, 10%, 15% RTSP (with or without cystein) and 6% glycerol before two-hour equilibration. After equlibration, semen were placed into 0,25 mLfrench straws and frozen with a programmable freezing device and they were stored in liquid nitrogen tanks. Motility, HOST, defected acrosome and DNA fragmentation rates were assessed after thawing, 3th hour and 5th hour of incubation. It is detected that the extender containing 10% RTSP-cysteine was more protective on motility and plasma membrane integrity after thawing and 3rd hour of incubation (P<0,05). Although the least defected acrosome rates were detected with 10% RTSP-cysteine containing group, the least DNA fragmentation rates were observed with only 10% RTSP group. At the end of incubation, it is observed that the 10% RTSP-cysteine combination is more efficient for protecting all spermatological parameters without excepting DNA integrity. It was a reached conclusion that, supplementation of RTSP and cystein have a protective effect on cryosurvival of ram semen and on assessed spermatological parameters.
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Keywords
Koç sperması, Ram semen, Dondurma, Alabalık seminal plazması, Sistein, Cryopreservation, Cysteine, Rainbow trout seminal plasma
Citation
Gökçe, E. (2019). Sistein ile kombine edilen alabalık seminal plazmasının koç spermasının dondurulabilirliği üzerine etkisi. Yayınlanmamış doktora tezi. Bursa Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.