Koç Spermasının Dondurulmasında, İnkubasyon Direncinin Çeşitli Antioksidanlar ile Artırılması

Year 2017, Volume: 14 Issue: 3, 183 - 190, 15.12.2017

Selim Alçay , Mehmed Berk Toker Elif Gökçe Zülfiye Gül Nail Tekin Önder Burcu Üstüner , Zekariya Nur , Hakan Sağırkaya , Mustafa Kemal Soylu

Abstract

Çalışmamızda farklı antioksidanların dondurma çözdürme sonrası sperma parametreleri ve spermanın inkubasy-on direncinin artırılması üzerine etkilerinin belirlenmesi amaçlanmıştır. Birleştirilien sperma örnekleri dört eşit hacme bölünerek, antioksidan ilave edilmiş (5mM methiyonin, 5mM sisteamin, 1mM sistein) ve edilmemiş (kontrol) sulandırıcılarla iki aşamalı sulandırma yöntemi ile sulandırılmıştır. Sperma örneklerinin değerlendirilmesi amacıyla mo-tilite, plazma membran bütünlüğünün değerlendirilmesi amacıyla hipoozmotik şişme testi (HOST) ve Hoechst 33258 testi, akrozom ve DNA bozukluklarının belirlenmesi amacıyla ise sırasıyla FITC-Pisum sativum agglutinin (PSA-FITC) ve terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) boyama değerlerine bakılmıştır. Ayrıca membran lipid peroksidasyonun değerlendirilmesi için malondialdehyde (MDA) konsantrasyon ölçümü yapılmıştır. Değerlendirmeler doğrultusunda koç spermasının dondrulmasında antioksidan ilave edilen grupların mo-tilite, plazma mebran bütünlüğü ve akrozom bütünlü değerlerinin kontrol grubundan daha yüksek olduğu sonucuna varılmıştır (P<0.05). Sistein ilave edilen grubun motilite (%60.40) ve akrozomal bütünlük (%70) oranları diğer gruplar-dan daha yüksek buunmuştur.Ancak inkubasyonun 0. Saatinde grupların DNA bütünlük oranları arasında bir fark bulunmamaktadır (P>0.05). Dondurma çözdürme işlemleri ve 6 saat inkubasyon sonrası en yüksek motilite (%35), HOST (%50), Hoechst (%56.50) ve en düşük akrozomal bozukluk (%35) ve DNA bozukluk (%8.80) oranları 1mM sistein içeren sulandırıcı ile dondurulan grupta elde edilmiştir.

Keywords

Antioksidan, inkubasyon direnci, koç sperması, sperma dondurma

Improvement of Incubation Resilience with Various Antioxidants in Cryopreserved Ram Semen*

Abstract

The aim of the current study was to evaluate different antioxidant-supplemented extenders for post-thaw semen quality and incubation resilience of ram spermatozoa. Pooled semen samples were divided into four equal vol-umes and each volume were diluted with two-step dilution method in control and antoxidant supplemented groups (5mM methionine, 5mM cysteamine and 1mM cysteine). Semen samples were assessed for the sperm motility, plasma membrane integrity using hypoosmotic swelling test (HOST) and Hoechst 33258 test, damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Membrane lipid peroxidation was also analyzed using the malondialdehyde (MDA) concen-tration assay. This study showed that antioxidant supplemented extenders had beneficial effect on motility, plasma membrane integrity and acrosome integrity of ram semen compared to control group (P<0.05). The motility (60.40%) and acrosome integrity (30%) values of cysteine group was significantly higher than those of the groups (P<0.05). However, there were no differences about DNA fragmentation rates at 0 h of incubation. In addition, we achieved a higher motility (35%), HOST (50%), Hoechst (56.50%) and lower defected acrosome (35%) and DNA fragmentation (8.80%) rates in post thawed ram semen even after 6 h incubation when the extender was supplemented with 1 mM cysteine.

Keywords

Antioxidants, cryopreservation, incubation resilience, ram semen

References

• 1. Alcay S, Toker B, Ustuner B, Nur Z, Sa-girkaya H, Soylu MK. Investigation of rela-tionships between DNA integrity and fresh semen parameters in rams. Kafkas Univ Vet Fak Derg 2014; 20(5): 793-8. 2. Alcay S, Toker M.B, Gokce E, Ustuner B, Onder NT, Sagırkaya H, Nur Z, Soylu MK. Successful ram semen cryopreservation with lyophilized egg yolk-based extender. Cryobiology 2015; 71(2): 329-33. 3. Alcay S, Ustuner B, Nur Z. Effects of low molecular weight cryoprotectants on the post-thaw ram sperm quality and fertilizing ability. Small Rum Res 2016; 136: 59-64. 4. Benson JD, Woods EJ, Walters EM, Critser TK. The cryobiology of spermatozoa. Theri-ogenology 2012; 78(8): 1682-99. 5. Bergeron A, Manjunath P. New insights to-wards understanding the mechanisms of sperm protection by egg yolk and milk. Mol Reprod Dev 2006; 73(10): 1338-44. 6. Bilodeau JF, Chatterjee S, Sirard MA, Gag-non C. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol Reprod Dev 2000; 55(3): 282-8. 7. Bucak, MN, Ateşşahin, A, Varışlı, O, Yüce, A, Tekin, N, Akçay, A. The influence of tre-halose, taurine, cysteamine and hyaluronan on ram semen. Microscopic and oxidative stress parameters after freeze-thawing pro-cess. Theriogenology 2007; 67(5): 1060-7. 8. Bucak MN, Keskin N, Taspinar M, Coyan K, Baspinar N, Cenariu MC, Bilgili A, Ozturk C, Kursunlu AN. Raffinose and hypotaurine improve the post-thawed Merino ram sperm parameters. Cryobiology 2013; 67(1): 34-9. 9. Bucak MN, Sariozkan S, Tuncer PB, Sakin F, Atessahin A, Kulaksiz R, Cevik M. The effect of antioxidants on post-thawed Ango-ra goat (Capra hircusancryrensis) sperm parameters, lipid peroxidation and antioxi-dant activities. Small Rum Res 2010; 89: 24-30. 10. Bucak MN, Tuncer PB, Sarıözkan S, Akalın PP, Çoyan K, Başpınar N, Özkalp B. Effects of hypotaurine, cysteamine and aminoacids solution on post-thaw microscopic and oxi-dative stress parameters of Angora goat semen. Res Vet Sci 2009; 87(3): 468-72. 11. Bucak MN, Uysal O. The role of antioxidants in freezing of Saanen goat semen. Indian Vet J 2008; 85(2): 148-50. 12. Buckett WM, Luckas MJ, Aird IA. Farquhar-son RG, Kingsland CR, Lewis-Jones DI. The hypo-osmotic swelling test in recurrent mis-carriage. Fertil Steril 1997; 68(3): 506-9. 13. Chelucci S, Pasciu V, Succu S, Addis D, Leoni GG, Manca ME, Naitane S, Berlinguer F. Soybean lecithin–based extender pre-serves spermatozoa membrane integrity and fertilizing potential during goat semen cryopreservation. Theriogenology 2015; 83(6): 1064-74. 14. Coyan K, Baspınar N, Bucak MN, Akalin PP. Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and bio-chemical parameters. Cryobiology 2011; 63(1): 1-6. 15. Emamverdi M, Zhandi M, ZareShahneh A, Sharafi M, Akbari-Sharif A. Optimization of ram semen cryopreservation using a chemi-cally defined soybean lecithin-based extend-er. Reprod Dom Anim 2013; 48(6): 899-904. 16. Forouzanfar M, Fekri Ershad S, Hosseini SM, Hajian M, Ostad-Hosseini S, Abid A, Tavalaee M, Shahverdi A, Vosough Dizaji A, Nasr Esfahani MH. Can permeable super oxide dismutase mimetic agents improve the quality of frozen–thawed ram semen? Cryo-biology 2013; 66(2): 126-30. 17. Forouzanfar M, Sharafi M, Hosseini SM, Ostadhosseini S, Hajian M, Hosseini L, Abedi P, Nili N, Rahmani HR, Nasr-Esfahani MH. In vitro comparison of egg yolk-based and soybean lecithinbased extenders for cryopreservation of ram semen. Theri-ogenology 2010; 73(4): 480-7. 18. Hammadeh ME, Dehn CH, Hippach M, Ze-giniadou T, Stieber M, Georg T, Rosenbaum P, Schmidt W. Comparison between com-puterized slow-stage and static liquid nitro-gen vapour freezing methods with respect to the deleterious effect on chromatin and mor-phology of spermatozoa from fertile and subfertile men. Int J Androl 2001; 24(2): 66-72. 19. López-Fernández C, Fernández JL, Gosálbez A, Arroyoa F, Vázquez JM, Holtd WV, Gosálvez J. Dynamics of sperm DNA fragmentation in domestic animals: III. ram. Theriogenology 2008; 70(6): 898-908. 20. Makarevich AV, Kubovicova E, Sirotkin AV, Pivko J. Demonstration of the effect of epi-dermal growth factor on ram sperm parame-ters using two fluorescent assays. Veterinar-ni Med 2010; 55(12): 581-9. 21. Martí JI, Martí E, Cebrián-Perez JE, Muiño-Blanco T. Survival rate of antioxidant en-zyme activity of ram spermatozoa after dilu-tion with different extenders or selection by a dextran swim-up procedure. Theriogenolo-gy 2003; 60(6): 1013-20. 22. Mata-Campuzano M, Álvarez-Rodríguez M, Álvarez M, Tamayo-Canul J, Anel L, de Paz P, Martínez-Pastor F. Post-thaw quality and incubation resilience of cryopreserved ram spermatozoa are affected by antioxidant supplementation and choice of extender. Theriogenology 2015; 83(4): 520-8. 23. Mazor D, Golan E, Philip V. Red blood cell permeability to thiol compounds following oxidative stress. Eur J Haematol 1996; 57(3): 241-6. 24. Merton JS, Knijn HM, Flapper H, Dotinga F, Roelen BA, Vos PL, Mullaart E. Cysteamine supplementation during in vitro maturation of slaughterhouse- and opu-derived bovine oocytes improves embryonic development without affecting cryotolerance, pregnancy rate, and calf characteristics. Theriogenolo-gy 2013; 80(4): 365-71 25. Motamedi-Mojdehi R, Roostaei-Ali Mehr M, Rajabi-Toustani R. Effect of different levels of glycerol and cholesterol-loaded cyclodex-trin on cryosurvival of ram spermatozoa. Reprod Dom Anim 2014; 49(1): 65-70. 26. Motlagh MK, Sharafi M, Zhandi M, Moham-madi-Sangcheshmeh A, Shakeri M, So-leimaniet M, Zeinoaldini S. Antioxidant effect of rosemary (Rosmarinus officinalis L.) ex-tract in soybean lecithin-based semen ex-tender following freeze–thawing process of ram sperm. Cryobiology 2014; 69(2): 217-22. 27. Najafi A, Kia HD, Mohammadi H, Najafi MH, Zanganeh Z, Sharafi M, Martinez-Pastor F, Adeldust H. Different concentrations of cys-teamine and ergothioneine improve micro-scopic and oxidative parameters in ram se-men frozen with a soybean lecithin extend-er. Cryobiology 2014; 69(1): 68-73. 28. Nur Z, Zik B, Ustuner B, Sagirkaya H, Ozguden CG. Effects of different cryoprotec-tive agents on ram sperm morphology and DNA integrity. Theriogenology 2010; 73(9): 1267-75. 29. O’Connell M, Mcclure M, Lewis SEM. The effects of cryopreservation on sperm mor-phology, motility and mitochondrial function. Hum Reprod 2002; 17(3): 704-79. 30. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979; 95(2): 351-8. 31. Perez LJ, Valcarcel A, De Las Heras MA, Moses D, Baldassarre H. Evidence that fro-zen/thawed ram spermatozoa show acceler-ated capacitation in vitro as assessed by chlortetracycline assay. Theriogenology 1996; 46(1): 131-40. 32. Reed DJ. Glutathione: toxicological implica-tions. Annu Rev Pharmacol Toxicol 1990; 30: 603-31. 33. Reed DJ, Orrenius S. The role of methionine in glutathione biosynthesis by isolated hepatocytes. Biochem Biophys Res Com-mun 1977; 77(4): 1257-64. 34. Riha L, Apolen D, Pıvko J, Grafenau P, Ku-bovıcova E. Influence of implementers on sheep fertility out of season. Slovak J Anim Sci 2006; 4: 180-2. 35. Sagara J, Miura K, Bannai S. Cysteine up-take and glutathione level in fetal brain cells in primary culture and in suspension. J Neu-rochem 1993; 61(5): 1667-71. 36. Silva ECB, Cajueiro JFP, Silva SV, Vidal AH, Soares PC, Guerra MMP. In vitro evalu-ation of ram sperm frozen with glycerol, eth-ylene glycol or acetamide. Anim Reprod Sci 2012; 132(3-4): 155-8. 37. Szczesniak-Fabianczyk B, Bochenek M, Smorag Z, Ryszka F. Effect of antioxidants added to boar semen extender on the se-men survival time and sperm chromatin structure. Reprod Biol 2003; 3(1): 81-7. 38. Tuncer PB, Bucak MN, Buyukleblebici S, Sarıozkan S, Yeni D, Eken A, Akalın PP, Kinet H, Avdatek F, Fidan AF. The effect of cysteine and glutathione on sperm and oxi-dative stress parameters of post-thawed bull semen. Cryobiology 2010; 61(3): 303-7. 39. Ustuner B, Alcay S, Nur Z, Sagirkaya H, Soylu MK. Effect of egg yolk and soybean lecithin on tris-based extender in post-thaw ram semen quality and in vitro fertility. Kaf- kas Univ Vet Fak Derg 2014; 20(3): 393-8. 40. Ustuner B, Alcay S, Toker MB, Nur Z, Gokce E, Ak Sonat F, Gul Z, Duman M, Ceniz C, Uslu A, Sagirkaya H, Soylu MK. Effect of rainbow trout (Oncorhynchus mykiss) seminalplasma on the post-thaw quality of ram semen cryopreservedin a soy-bean lecithin-based or egg yolk-based ex-tender. Anim Reprod Sci 2016; 164: 97-104. 41. Wang W, Luo J, Sun S, Xi L, Gao Q, Haile AB, Shi H, Zhang W, Shi H. The effect of season on spermatozoa motility, plasma membrane and acrosome integrity in fresh and frozen-thawed semen from Xinong Saanen bucks. Reprod Domest Anim 2015; 50(1): 23-8.