Fare sperminin dondurulabilirliği üzerine standart raffinoz + yağsız süt tozu sulandırıcısına eklenen düşük dozda dimetil sülfoksitin etkisi
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Date
2022
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Hatay Mustafa Kemal Üniversitesi
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info:eu-repo/semantics/openAccess
Abstract
Sunulan çalışma, fare sperminin başarılı olarak dondurulabilmesi için standart raffinoz + yağsız süt tozu sulandırıcısına eklenen düşük dozda dimetil sülfoksit (DMSO) oranlarının çözüm sonrası sperm kalite parametreleri üzerine etkilerini ortaya koymak için dizayn edilmiştir. Araştırma 12-24 haftalık yaşta 40 adet CD-1 ırkı erkek fareler üzerinde yürütülmüştür. Farelerden cerrahi yöntem ile diseke edilerek elde edilen cauda epididimislerdeki spermatozoonlar kullanılmıştır. Çalışmada %18 rafinoz + %3 yağsız süt tozu içeren standart sperm sulandırıcısına 0.25M, 0.5M, 1M ve 1.5 Molar oranlarında ayrı ayrı DMSO eklenmiştir. DMSO ilavesiz %18 Rafinoz + %3 yağsız süt tozu kombinasyonu kontrol olarak tutulmuştur. Dondurma işlemi sıvı azot kullanılarak 0.25ml'lik payetlerde yapılmış ve ardından çözdürme işlemi ise 37 o C'lik su banyosunda 30 saniyede gerçekleştirilmiştir. Çözüm sonrası sperm örenklerinin motilite, ölü-canlı, akrozom, membran bütünlüğü (HOST: Hiper ozmotik şişme testi) ve HTF (Human Tubal Fluid) içerisinde 4 saat süresince motilite dayanıklılık testleri yapılmıştır. Dondurup çözdürme sonrası gruplar arası değerlendirmede en yüksek motilite değerleri %40.0±2.88 ile 0.5 M DMSO gurubunda bulunmuştur. Motilite açısından Kontrol, 0.25 M ve 0.5 M DMSO grupları arasında önemli istatistiki bir fark bulunmazken (p>0.05), bu guruplar ile 1 M ve 1.5 M DMSO grupları arasında önemli farklar bulunmuştur (p<0.05). Dondurup çözdürme sonrası en düşük ölü spermatozoon oranı ise %38.4±2.42 ile 0.5 M DMSO grubunda tespit edilmiştir. Canlı spermatozoon oranı açısından 0.25 M ve 0.5 M DMSO grupları Kontrol grubuna göre daha iyi bulunmuştur (p<0.05). Çözüm sonrası gruplar arası en yüksek membran bütünlüğü değeri %62.8±2.17 ile 0.5 M DMSO grubunda saptanmıştır. Membran bütünlüğü açısından 0.5 M DMSO grubunun Kontrol grubun'dan daha iyi olduğu tespit edilmiştir (p<0.05). Gruplar arası değerlendirmede dondurup çözdürme sonrası en düşük akrozom hasarı oranı ise %8.6±0.85 ile 0.25 M DMSO grubunda tespit edilmiştir. Akrozom hasarı açısından kontrol grubu ile deneme grupları arasında, 1.5 M DMSO grubu hariç (p<0.05), istatistiki bir fark bulunmamıştır (p>0.05). Çalışmada dondurma ve çözdürme sonrası bir'er saatlik zaman aralığında gerçekleştirilen motilite dayanıklılık (longevity) testin 'de 3. saate'e kadar motilite oranlarında en iyi sonuç 0.5 M DMSO grubu olmuş (p<0.05), ve 4. saatte tüm gruplarda motilite değeri saptanmamıştır. Sonuç olarak, Fare sperminin dondurulabilirliği üzerine standart raffinoz + yağsız süt tozu sulandırıcısına 0.5 M DMSO ilavesinin çözüm sonrası sperm kalite parametrelerini geliştirdiği ve başarılı bir şekilde kullanılabileceği belirlenmiştir.
The present study was designed to reveal the effects of low dose dimethyl sulfoxide (DMSO) ratios added to standard raffinose + skimmed milk powder extender on post-thaw sperm quality parameters for successful freezing of mouse sperm. The study was carried out on 40 male CD-1 mice aged 12-24 weeks. Sperm from cauda epididymis obtained by surgical dissection from mice were used. In the study, DMSO was added separately at 0.25M, 0.5M, 0.1M and 1.5 Molar ratios to the standard sperm extender containing 18% raffinose + 3% skimmed milk powder. The combination of 18% Raffinose + 3% skimmed milk powder without DMSO was kept as a control. The freezing process was carried out in 0.25 ml straws using liquid nitrogen, and then the thawing process was carried out in 30 seconds in a 37 oC water bath. Motility, dead-live spermatozoon rate, acrosome and membrane integrity (HOST: Hyperosmotic swelling test) of sperm samples after thawing were performed in HTF (Human Tubal Fluid) for 4 hours. In the evaluation between groups after freezing - thawing, the highest motility values were found in the 0.5 M DMSO group with 40.0±2.88%. In terms of motility, there was no statistically significant difference between the Control, 0.25 M and 0.5 M DMSO groups (p>0.05), while significant differences were found between these groups and the 1 M and 1.5 M DMSO groups (p<0.05). The lowest rate of dead sperm after freezing-thawing was found in the 0.5 M DMSO group with 38.4±2.42%. In terms of viable spermatozoa ratio, 0.25 M and 0.5 M DMSO groups were found to be better than the control group (p<0.05). The highest membrane integrity value between the groups after thawing was found in the 0.5 M DMSO group with 62.8±2.17%. It was determined that the membrane integrity of the 0.5 M DMSO group was better than the Control group (p<0.05). In the evaluation between groups, the lowest rate of acrosome damage after freezing-thawing was found in the 0.25 M DMSO group with 8.6±0.85%. There was no statistical difference between the control and experimental groups in terms of acrosome damage, except for the 1.5 M DMSO group (p<0.05). In the study, the 0.5 M DMSO group had the best result in motility rates up to the 3rd hour in the longevity test, which was performed at one hour time interval after freezing and thawing, and no motility value was detected in all groups at the 4th hour. As a result, it was determined that the addition of 0.5 M DMSO to the standard raffinose + skimmed milk powder extender on the freezability of mouse sperm improved the sperm quality parameters after thawing and could be used successfully.
The present study was designed to reveal the effects of low dose dimethyl sulfoxide (DMSO) ratios added to standard raffinose + skimmed milk powder extender on post-thaw sperm quality parameters for successful freezing of mouse sperm. The study was carried out on 40 male CD-1 mice aged 12-24 weeks. Sperm from cauda epididymis obtained by surgical dissection from mice were used. In the study, DMSO was added separately at 0.25M, 0.5M, 0.1M and 1.5 Molar ratios to the standard sperm extender containing 18% raffinose + 3% skimmed milk powder. The combination of 18% Raffinose + 3% skimmed milk powder without DMSO was kept as a control. The freezing process was carried out in 0.25 ml straws using liquid nitrogen, and then the thawing process was carried out in 30 seconds in a 37 oC water bath. Motility, dead-live spermatozoon rate, acrosome and membrane integrity (HOST: Hyperosmotic swelling test) of sperm samples after thawing were performed in HTF (Human Tubal Fluid) for 4 hours. In the evaluation between groups after freezing - thawing, the highest motility values were found in the 0.5 M DMSO group with 40.0±2.88%. In terms of motility, there was no statistically significant difference between the Control, 0.25 M and 0.5 M DMSO groups (p>0.05), while significant differences were found between these groups and the 1 M and 1.5 M DMSO groups (p<0.05). The lowest rate of dead sperm after freezing-thawing was found in the 0.5 M DMSO group with 38.4±2.42%. In terms of viable spermatozoa ratio, 0.25 M and 0.5 M DMSO groups were found to be better than the control group (p<0.05). The highest membrane integrity value between the groups after thawing was found in the 0.5 M DMSO group with 62.8±2.17%. It was determined that the membrane integrity of the 0.5 M DMSO group was better than the Control group (p<0.05). In the evaluation between groups, the lowest rate of acrosome damage after freezing-thawing was found in the 0.25 M DMSO group with 8.6±0.85%. There was no statistical difference between the control and experimental groups in terms of acrosome damage, except for the 1.5 M DMSO group (p<0.05). In the study, the 0.5 M DMSO group had the best result in motility rates up to the 3rd hour in the longevity test, which was performed at one hour time interval after freezing and thawing, and no motility value was detected in all groups at the 4th hour. As a result, it was determined that the addition of 0.5 M DMSO to the standard raffinose + skimmed milk powder extender on the freezability of mouse sperm improved the sperm quality parameters after thawing and could be used successfully.
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Keywords
Veteriner Hekimliği, Veterinary Medicine, Fare, spermatozoon, dondurma, DMSO., Mouse, spermatozoa, cryopreservation, DMSO.
