KAMBER DEMİR, Gül ÖZTÜRK BAKIRER, Ümit CİRİT, H. Hakan BOZKURT, ABİT AKTAŞ, Sema BİRLER, KEMAL AK, SERHAT PABUCCUOĞLU

Effects of cooling rate on membrane integrity and motility parameters of cryopreserved ram spermatozoa

Bu çalışmada koç spermasının 26°Cden +5°Cye indirilmesinde farklı soğutma hızlarının (0.3°C/dk., 0.6°C/dk. ve 0.9°C/dk.) eritme sonrası spermatolojik özellikler ve spermatozoonların ultrastrüktürel yapısı üzerindeki etkilerinin incelenmesi amaçlanmıştır. Altı adet koçtan elektro ejakülatörle alınan spermalar 26°Cdaki su banyosunda pooling işlemine tabii tutuldu. Tris bazlı sulandırıcıyla sulandırılan birleştirilmiş sperma üç eşit hacme bölünerek 3 farklı hızda (0.3, 0.6 ve 0.9°C/dk.) +5°C ye soğutuldu. Sperma iki basamakta sulandırıldı, gliserol sperma ısısının +5°Cye indiği ikinci basamakta katıldı. Sulandırma sonrası sperma 1 saat ekilibre edildi daha sonrasında 0.25 ml payetlere çekilerek sıvı azot buharında donduruldu. Sperma pooling, sulandırma, soğutma, ekilibrasyon ve eritme sonrası gibi tüm aşamalarında motilite değerleri Bilgisayar Destekli Analiz Sistemleri (CASA) ile değerlendirildi. Pooling ve soğutma sonrasında elektron mikroskop incelemeleri gerçekleştirildi. 0.3°C/dk. soğutma grubunun, spermanın +5°Cye soğutma, ekilibrasyon ve eritme sonrasındaki hem total motilite hem de progressive motilite değerleri 0.9°C/dk. soğutma grubuna göre önemli derecede yüksek bulundu (P0.05). Soğutma ve eritme sonrasında ise progressive motilite değerleri daha yüksek bulunurken(P0.05). Yapılan TEM incelemesinde, tüm soğutma hızı gruplarında eritme sonrasında tespit edilen toplam hasarlı spermatozoit oranı, pooling sonrasına göre önemli derecede yüksek bulunmuştur (P

Koç spermasının dondurulmasında kullanılan soğutma oranlarının membran bütünlüğü ve motilite özelliklerine etkisi

In this study we aimed to determinate the efects of three diferent cooling rates from +26°C to +5°C at (0.3°C/min 0.6°C/min and 0.9°C/min) on spermatologic and ultrastructure properties of ram semen. For this purpose semen from 6 rams was collected by electroejaculator and was pooled in a +26°C waterbath. Pooled semen was diluated with tris based extender and divided into three equal parts according cooling rates (0.3°C/min., 0.6°C/min. and 0.9°C/min). Cooled semen was reextended with extender B +5°C in the second step. Diluated samples were equilibrated for 1 h and then were loaded in 0.25 mL straws and freezed in liquid nitrogen vapor. After each freezing stage semen was evaluated motility with computer-assisted semen analysis (CASA). Electron microscobic evaluation was done for pooled and chilled samples. It has been observed that 0.3°C/min. cooled group had meaningfully higher values of motility and progressive motility at +5°C after equilibration and post-thaw stages when compared with the 0.9°C/min. group (P<0.05). When compared to the 0.6°C/min., the 0.3°C/min. cooled group had higher total motility values at after cooling to +5°C (P<0.05), equilibration (P<0.05) and post thaw stages (P>0.05) and had higher progressive motility at after cooling to +5°C (P<0.05), equilibration (P>0.05) and post-thaw stage (P<0.05). The TEM evaluation showed that at cooling to the +5°C increases the total damaged spermatozoa in all groups (P<0.05). In conclusion, cooling the ram semen to +5°C with a rate above 0.3°C/min. afected negatively the spermatological characteristics. Reaching the cooling rates of 0.6 and 0.9°C/min. increasingly deteriorated the post-thaw motility and progressive motility values. Also, low temperature related to ultrastructural damage was observed at the first dilution step and localized at diferent regions of the sperm head depends upon the processes and cooling rates.

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