Methionine, cysteine ve BHT ilave edilmiş TRIS-yumurta sarısı ile dondurulan koç spermasının IN-vitro olarak değerlendirilmesi
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Date
2018-12-21
Authors
Toker, Mehmed Berk
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Publisher
Bursa Uludağ Üniversitesi
Abstract
Bu çalışmada TRIS-sitrik asit yumurta sarısı (%20) temel sulandırıcısına 2,5mM, 5mM metiyonin, 1mM, 2mM sistein, 1mM, 2mM bütil hidroksitolüen (BHT) eklenerek sulandırılan ve dondurulan koç spermasında çözüm sonrası bazı spermatolojik parametreler üzerine antioksidanların etkileri ve karşılaştırılması amaçlandı. Sperma, beş baş Kıvırcık ırkı koçtan üreme sezonunda, elektro-ejakülatör ile haftada iki kez alındı ve birleştirildi. Pooling sonrası ortalama hacim (ml), motilite (%), yoğunluk (x109/ml), plazma membran bütünlüğü (HOST, %), akrozomal (%), diğer morfolojik (%) ve toplam morfolojik (%) bozukluk oranları sırasıyla 3,92, 84,0, 1,96, 93,8, 5,60, 0,4 ve 6,0 bulundu. Sulandırılan sperma örnekleri bir saat içinde 5ºC'a indirildi ve %6 gliserol eklendikten sonra iki saat boyunca ekilibrasyona tabi tutuldu. Sperma, payette (0,25ml) 200x106 spermatozoon olacak şekilde çekildi ve dondurma cihazıyla dondurularak (-120 oC) analize kadar sıvı azotun içinde saklandı. Çözdürme sonrası motilite, HOST, akrozomal bozukluk, diğer ve toplam morfolojik bozukluk muayeneleri yapıldı. Motilite ve HOST oranları, kontrol ile tüm antioksidan grupları arasında fark (P<0,05) ve pozitif korelasyon tespit edildi (r=0,880). Akrozomal bozukluk oranlarında kontrol ile 2,5mM, 5mM metiyonin ve 1mM, 2mM sistein grupları arasında fark saptandı (P<0,05). Ayrıca metiyonin ve sistein grupları kendi dozları arasında da farklıydı (P<0,05). Diğer morfolojik bozukluk oranlarında ise fark yalnızca 1mM sistein ile 2mM BHT grupları arasında tespit edildi (P<0,05). Toplam morfolojik bozukluk oranlarında ise 1mM, 2mM BHT ve 1mM sistein grupları hariç, kontrol ve diğer deneme grupları arasında fark görüldü (P<0,05). Sonuç olarak, koç sperma sulandırıcısına çeşitli dozlarda eklenen antioksidanların çözüm sonrası incelenen spermatolojik parametreler üzerine koruyucu etkilerde bulunduğu kanaatine varıldı.
The aim of this research is to study the effects and comparison of the antioxidants [2,5mM, 5mM methionine, 1mM, 2mM cysteine, 1mM, 2mM butylhyroxytoluene (BHT)] for semen parameters on freeze-thawed ram sperm that diluted in TRIS-citric acid and egg yolk (%20) sperm extender. Sperm was collected from five Kivircik rams twice a week with electro-ejaculator in breeding season and pooled. After pooling the mean of the volume (ml), motility (%), sperm number (x109/ml), plasma membrane integrity (HOST, %), acrosomal, other morphologic and total morphologic defects (%) was found 3,92, 84,0, 1,96, 93,8, 5,60, 0,4 ve 6,0 respectively. The diluted sperm samples were cooled to 5ºC within an hour and equlibrated for another two hours after adding %6 of glycerol. 200x106 sperm cells were placed into 0,25ml french straws and freezed with a programmable freezing device (-120 oC) and plunged into liquid nitrogen tanks until spermatological analyses. After thawing of the sperm straws, the motility, HOST also acrosomal, other morphologic and total morphologic defects parameters were investigated. All antioxidant added study groups were statistically different (P<0,05) from antioxidant-free control group in terms of motility and HOST parameters and also were in a positive correlation between these parameters (r=0,880). In terms of acrosomal defects, there was a statistical difference among control group with the 2,5mM, 5mM methionine and 1mM, 2mM cysteine groups (P<0,05). Also there was a difference between the doses of methionine and cysteine groups (P<0,05). The difference between 1mM cysteine and 2mM BHT groups was statistically significant (P<0,05). The statistical difference for the total morphologic defects was found among control group and 2mM cysteine, 2,5mM and 5mM methionine groups (P<0,05). The study had reached the conclusion that the effects of antioxidants that added to ram sperm diluents with different doses had protective effects on spermatological parameters.
The aim of this research is to study the effects and comparison of the antioxidants [2,5mM, 5mM methionine, 1mM, 2mM cysteine, 1mM, 2mM butylhyroxytoluene (BHT)] for semen parameters on freeze-thawed ram sperm that diluted in TRIS-citric acid and egg yolk (%20) sperm extender. Sperm was collected from five Kivircik rams twice a week with electro-ejaculator in breeding season and pooled. After pooling the mean of the volume (ml), motility (%), sperm number (x109/ml), plasma membrane integrity (HOST, %), acrosomal, other morphologic and total morphologic defects (%) was found 3,92, 84,0, 1,96, 93,8, 5,60, 0,4 ve 6,0 respectively. The diluted sperm samples were cooled to 5ºC within an hour and equlibrated for another two hours after adding %6 of glycerol. 200x106 sperm cells were placed into 0,25ml french straws and freezed with a programmable freezing device (-120 oC) and plunged into liquid nitrogen tanks until spermatological analyses. After thawing of the sperm straws, the motility, HOST also acrosomal, other morphologic and total morphologic defects parameters were investigated. All antioxidant added study groups were statistically different (P<0,05) from antioxidant-free control group in terms of motility and HOST parameters and also were in a positive correlation between these parameters (r=0,880). In terms of acrosomal defects, there was a statistical difference among control group with the 2,5mM, 5mM methionine and 1mM, 2mM cysteine groups (P<0,05). Also there was a difference between the doses of methionine and cysteine groups (P<0,05). The difference between 1mM cysteine and 2mM BHT groups was statistically significant (P<0,05). The statistical difference for the total morphologic defects was found among control group and 2mM cysteine, 2,5mM and 5mM methionine groups (P<0,05). The study had reached the conclusion that the effects of antioxidants that added to ram sperm diluents with different doses had protective effects on spermatological parameters.
Description
Keywords
Koç sperması, Dondurma, Metiyonin, Sistein, BHT, Ram sperm, Cryopreservation, Methionine, Cysteine
Citation
Toker, M. B. (2018). Methionine, cysteine ve BHT ilave edilmiş TRIS-yumurta sarısı ile dondurulan koç spermasının IN-vitro olarak değerlendirilmesi. Yayınlanmamış doktora tezi. Bursa Uludağ Üniversitesi Sağlık Bilimleri Enstitüsü.