Arı Sperması (Apis mellifera anatoliaca) ve İnsülin Etkileşiminin Soya Lesitini Temelli Kriyoprezervasyon Sulandırıcılarında Flow Sitometri Olarak incelenmesi

Abstract

Gen havuzlarının istenen yönde gelişimine katkıda bulunan üreme kontrolü, spermanın dondurması ile alakalı biyoteknolojik yöntemleri önemli bir noktaya koymaktadır. Sperma dondurmaya yöntelik çalışmalar özellikle memelilerde yoğun olarak çalışılmış olmasına rağmen, arı sperması açısından başlangıç aşamasındadır. İnsülinin seminal plazmada bulunduğu ve çeşitli sperma parametreleri üzerine etkilerinin olduğu güncel çalışmalarla desteklenmiştir. Ayrıca, soya lesitini uzun süredir, özellikle yumurta sarısına alternatif, güvenilir bir kaynak olarak sperma dondurma sulandırıcılarında tercih edilen maddelerden biridir. Bu çalışma, soya lesitini (%2) temelli arı sperma dondurma sulandırıcılarına farklı dozlarda insülin (5, 10 ve 15 IU dozda) eklenmesi sonucunda elde edilecek sonuçların, flow sitometri veya mikroskopik incelemeler aracılığıyla önemli sperma parametreleri üzerindeki etkileri değerlendirmek amacıyla gerçekleştirişmiştir. Cinsel olgunluğa ulaşmış arılardan elde edilen örnekler bireysel farklılığı engellemek adına birleştirildi (pooling). Dondurma işlemi, üç doz insülin destekli ve bir insülin içermeyen kontrol grubu olacak şekilde düzenlendi ve yöntem planlandığı şekilde uygulandı. Dondurulup çözülen örnekler; motilite, plazma membran (hipo-osmotik şişme testi) ve DNA bütünlüğü (TUNEL testi) değerlendirmelerine, floresan ataçmanı olan bir faz kontrast mikroskobu kullanılarak gerçekleştirildi. Ayrıca, akrozomal bütünlük (PNA-FITC), nitrik oksit seviyeleri (DAF-2/DA) ve mitokondriyal membran potansiyeli (JC-1), bir flow sitometri cihazı aracılığıyla araştırıldı. Beklendiği gibi, tüm sperma parametreleri dondurma işleminin doğası gereği etkilendi, ancak insülin eklenmiş çalışma grupları, kontrol grubuna kıyasla pozitif yönde bir istatistiki farkı ortaya koyamadı. Bu sonuçlar ışığında, soya lesitini temelli arı sperması dondurma sulandırıcılarında kullanılan insülin takviyesinin tercih edilen dozlarda kullanılmasının, yardımcı biyoteknolojik yöntemler aracılığıyla daha yüksek verimli arı kolonileri elde edilmesi ve arı ürünleri konusunda ülke veriminin istenilen düzeye çıkartılması konusunda olumlu bir etkiye sahip olmadığı tespit edildi.

Keywords

• Apis mellifera anatoliaca
• Arı sperması
• Flow-sitometri
• İnsülin
• Soya lesitini
• Sperma dondurma

Flow Cytometric Examination of The Interaction Between Drone Sperm (Apis Mellifera Anatoliaca) and Insulin in Soybean Lecithin-Based Cryopreservation Extenders

Abstract

This study was conducted to evaluate the effects of different doses of insulin (5, 10, and 15 IU) added to soybean lecithin-based drone sperm freezing extenders on sperm parameters through flow cytometry or microscopic examinations. Samples obtained from sexually mature bees were pooled to eliminate individual variation. The freezing process was organized with three insulin supplemented doses and one insulin-free control group, and the method was implemented as planned. Thawed samples were evaluated for motility, plasma membrane (hypo-osmotic swelling test), and DNA integrity (TUNEL assay) using a phase-contrast microscope with fluorescent attachment. Additionally, acrosomal integrity (PNA-FITC), nitric oxide levels (DAF-2/DA), and mitochondrial membrane potential (JC-1) were investigated using a flow cytometer. As expected, all sperm parameters were affected by the freezing process inherently, however, the insulin-supplemented experimental groups did not demonstrate a statistically significant positive difference compared to the control group. In light of these results, it was determined that the use of insulin supplementation, in preferred doses, in soybean lecithin-based drone sperm cryopreservation extenders did not have a positive effect on achieving higher productive honeybee colonies and elevating the country's yield in bee products through auxiliary biotechnological methods as desired.

Keywords

• Apis mellifera anatoliaca
• Drone sperm
• Flow-cytometer
• Insulin
• Soybean lecithin
• Sperma cryopreservation

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