Effect of freezing rate on acrosome and chromatin integrity in ram semen

Abstract

The objective of the present study was to investigate the effect of different freezing rates on post-thaw sperm motility, acrosome defect, and sperm chromatin structure and apoptotic activity in ram semen. Collected semen was diluted at 1:5 (semen/extender) with Bioxel® (IMV technologies France) at 30ºC and then cooled to 5ºC within 1h. Cooled semen was subjected to the equilibration for 2 hours. Equilibrated semen was frozen in 0.25 ml straw at two different cooling rates (slow: 0.5°C/min from 5 to -20°C and fast: 5°C/min from 5 to -20°C). Both groups were frozen from -20 to -120°C at 25°C/min and stored in liquid nitrogen until use. Post-thaw (37°C/30 min) sperm motility, defected acrosome (Pisum sativum agglutinin fluorescein conjugate, FITC PSA), sperm chromatin structure determined by Acridin Orange (AO) and apoptotic activity using terminaldeoxynucleotidyl transferasemediated dUTP nick-end labeling (TUNEL) were evaluated. Post-thaw sperm motility, acrosome defect, AO and TUNEL for slow frozen semen were 42.8±8.8%, 31.6±12.9%, 2.9±2.4% and 2.8±1.6%, and for fast frozen semen were 36.5±9.9%, 24.7±11.1%, 3.3±2.2% and 6.3±3.4%, respectively. Post-thaw semen analyses showed that there was no significant difference between two freezing curves in terms of acrosome defect, sperm chromatin damage (AO). However, a significant difference was found for postthaw semen motility between two groups (P<0.05). In conclusion, while the slow freezing procedure improved post-thaw sperm motility, acrosome and chromatin integrities and apoptotic index in ram spermatozoa did not show any significant difference between freezing rates

Keywords

• Apoptosis
• freezing rate
• ram semen

Koç spermasında dondurma hızının akrozom ve kromatin bütünlüğü üzerine etkisi

Abstract

Bu çalışma, farklı dondurma hızlarının, eritme sonrası motilite, akrozom bütünlüğü, kromatin yapısı ve apoptozis üzerine olan etkilerini araştırmak amacıyla gerçekleştirildi. Toplanan sperma örnekleri 30ºC’de 1/5 oranında (sperma/sulandırıcı) Bioxel® (IMV, Fransa) ile sulandırıldı ve 1 saatte +5ºC’ye düşürülerek 2 saat süre ile ekilibrasyona bırakıldı. Ekilibre edilen sperma 0.25 ml’lik payetlere çekilerek iki farklı soğutma hızında (yavaş: +5°C’tan -20°C’a 0.5°C/dak ve hızlı: +5°C’tan -20°C’a 5°C/dak) donduruldu. Her iki grup -20°C’tan -120°C’a 25°C/dak hızla donduruldu. Eritme sonrası (37°C/30 dak) aşamada; akrozom bozukluğu Pisum sativum agglutinin fluorescein conjugate ( FITC PSA) ile kromatin yapısı Acridin Orange (AO) ile ve apoptozis ise terminaldeoxynucleotidyl transferase-mediated dUTP nick-end labeling tekniği (TUNEL) kullanarak belirlendi. Motilite muayeneleri ısıtma tablalı faz kontrast mikroskop ile gerçekleştirildi. Eritme sonrası motilite, akrozom bozukluğu, AO ve TUNEL değerleri sırasıyla yavaş dondurma için %42.8±8.8, %31.6±12.9, %2.9±2.4 ve 2.8±1.6 hızlı dondurma için ise %36.5±9.9, %24.7±11.1, %3.3±2.2 ve %6.3±3.4 olarak saptandı. Spermanın eritme sonrası bulgularının değerlendirilmesinde akrozom bozukluğu ve sperma kromatin yapısı (AO) yönünden iki soğutma hızı arasında fark bulunmazken, eritme sonrası motilitede gruplar arasında istatistiksel fark bulundu (P<0.05). Sonuç olarak, koç spermasında yavaş soğutma prosedürü ile dondurma, eritme sonrası motiliteyi iyileştirirken, akrozom ve kromatin bütünlüğünü ve apoptotik indeksi soğutma oranları arasında önemli bir fark göstermemiştir

Keywords

• Apoptozis
• dondurma hızı
• koç sperması

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