Burcu ÜSTÜNER, Sema BİRLER, Berrin ZIK, Selim ALÇAY, Gül BAKIRER ÖZTÜRK, Sabire GULER, Hakan SAĞIRKAYA, Zekariya NUR, Serhat PABUCCUOĞLU
Effect of Freeze-Drying Extenders on Ram Sperm DNA Integrity
DNA hasarının belirlenebilmesi, sperma saklama yöntemlerinin geliştirilmesi açısından önemlidir. Biz çalışmamızda farklı liyofilizasyon medyumları kullanılarak saklanan sperm hücrelerinin DNA bütünlüğünün değerlendirilmesinde akridin oranj (AO) ve therminal deoxynucleotidyl transferase-medited dUDP nick and labelling (TUNEL) florasan boyama yöntemlerini kullandık. Koçlardan alınan sperma dört gruba bölündü ve her bir grup: I) %10 fötal buzağı serumu (FCS) içeren TCM 199 solüsyonu II) %10 FCS ve 0.2 mol/L trehaloz içeren TCM 199 solüsyonu III) 50 mmol/L NaCl ve EGTA [Ethylen glycol-bis (β-aminoethyl ether)-N, N, N, N,-tetraacetic acid] 10 mmol/L Tris solüsyonu ve IV) %20 yumurta sarısı ve %7 gliserol içeren Tris bazlı solüsyonlardan biri ile konsantrasyonu 10 x 106 spermatozoa/100 μL olacak şekilde sulandırıldı.
Taze alınan spermada DNA fragmentasyon oranı AO ve TUNEL yöntemlerinde sırasıyla %2 ve %4 olarak tespit edildi (P>0.05). AO ve TUNEL yöntemiyle değerlendirilen liyofilizasyon sonrası DNA bütünlüğüne sahip spermatozoa oranlarının, sulandırıcı farklılığına göre etkilenmediği tespit edildi (P>0.05). Aynı şekilde her iki boyama yöntemi karşılaştırıldığında, liyofilizasyon sonrası elde edilen DNA bütünlüğünün değerlendirilmesinde AO ve TUNEL sonuçlarının istatistiksel olarak farklı olmadığı belirlendi (P>0.05).
Keywords:
koç sperması, liyofilizasyon, DNA bütünlüğü, TUNEL, AO,
___
- Referans1 Alcay S, Toker B, Ustuner B, Nur Z, Sagirkaya H, Soylu MK. Investigation of relationships between DNA integrity and fresh semen parameters in rams. Kafkas Üniversitesi Veteriner Fakültesi Dergisi, 20, (5): 793-798, 2014.
- Referans2 Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl, 21: 1–7, 2000.
- Referans3 Barroso G, Morshedi M, Oehninger S. Analysis of DNA fragmentation, plasma membrane translocation of phosphatidylserine and oxidative stress in human spermatozoa. Hum Reprod, 15 (6): 1338-1344, 2000.
- Referans4 Benchaib M, Braun V, Lornage J, Hadj S, Salle B, Lejeune H, Guerin JF. Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique. Hum Reprod, 18: 1023-1028, 2003.
- Referans5 Choi YH, Varner D, Love CC, Hartman DL, Hinrichs K. Production of live foals via intracytoplasmic injection of lyophilized sperm and sperm extract in the horse. Reproduction, 142 (4): 529-538, 2011.
- Referans6 Duran EH, Gurgan T, Gunalp S, Enginsu ME, Yarali H, Ayhan A. A logistic regression model including DNA status and morphology of spermatozoa for prediction of fertilization in vitro. Hum Reprod, 13: 1235-1239, 1998.
- Referans7 Erenpreiss J, Jepson K, Giwercman A, Tsarev I, Erenpreisa J, Spano M. Toluidine blue cytometry test for sperm DNA conformation: comparison with the flow cytometric sperm chromatin structure and TUNEL assays. Hum Reprod, 19 (10): 2277-2282, 2004.
- Referans8 Erenpreiss J, Spano M, Erenpreisa J, Bungum M, Giwercman A. Sperm chromatin structure and male fertility: biological and clinical aspects. Asian J Androl, 8: 11-29, 2006.
- Referans9 Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K, de Angelis P, Claussen OP. Utility of the sperm chromatin structure assay as a diagnostic and prognostic tool in the human fertility clinic. Hum Reprod, 14 (4): 1039-1049, 1999.
- Referans10 Garcia de Castro A, Lapinski J, Tunnacliffe A. Anhydrobiotic engineering. Nature Biotechnology, 18: 473, 2000.
- Referans11 Gil L, Olaciregui M, Luño V, Malo C, González N, Martínez F. Current status of freeze-drying technology to preserve domestic animals sperm. Reproduction in Domestic Animals, 49: 72–81, 2014.
- Referans12 Hara H, Tagiri M, Hwang IS, Takahashi, M, Hirabayasi M. Adverse effect of cake collapse on the functional integrity of freeze‐dried bull spermatozoa. Cryobiology, 68: 354– 360, 2014.
- Referaans13 Kaneko T, Whittingham DG, Yanagimachi R. Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa. Biol Reprod, 68: 136-139, 2003.
- Referans14 Kaneko T, Nakagata N. Improvement in the long-term stability of freeze-dried mouse spermatozoa by adding of a chelating agent. Cryobiology, 53: 279–282, 2006.
- Referans15 Keskintepe L, Pacholczyk G, Machnicka A, Norris K, Curuk MA, Khan I, Brackett BG. Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa. Biol Reprod, 67, 409-415, 2002.
- Referans16 Koppers AJ, De Iuliis GN, Finnie JM, McLaughlin EA, Aitken RJ. Significance of mitochondrial reactive oxygen species in the generation of oxidative stress in spermatozoa. J Clin Endocrinol Metab, 93: 3199–3207, 2008.
- Referans17 Kusakabe H, Szczygiel MA, Whittingham DG, Yanagimachi R. Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proc Natl Acad Sci USA, 98: 13501–13506, 2001.
- Referans18 Liu, JL, Kusakabe H, Chang CC, Suzuki H, Schmidt DW, Julian M, Pfeffer R, Bormann CL, Tian XC, Yanagimachi R, Yang X. Freeze-dried sperm fertilization leads to full-term development in rabbits. Biol Reprod, 70: 1776-1781, 2004.
- Referans 19 Martins CF, Dode MN, Báo, SN, Rumpf R. The use of the acridine orange test and the TUNEL assay to assess the integrity of; freeze-dried bovine spermatozoa DNA. Genet Mol Res, 6: 94-104, 2007.
- Referans 20 Matsukawa, K., Wagyu calf born after conception with freeze-dried semen in Japan, 2020. https://english.kyodonews.net/news/2020/04/61b68b6dbd2a-wagyu-calf-born-after-conception-with-freeze-dried-semen-in-japan.html?phrase=postwords=&words=
- Referans21 McGinnis LK, Zhu L, Lawitts JA, Bhowmickm S, Toner M, Biggers JD. Mouse sperm desiccated and stored in trehalose medium without freezing. Biology of Reproduction, 73: 627–633, 2005.
- Referans22 Molina MC, Armstrong TK, Zhang Y, Patel MM, Lentaz YK, Anchordoquy TJ. The stability of lyophilized lipid/DNA complexes during prolonged storage. J Pharm Sci, 93, 2259–2273, 2004.
- Referans23 Nakai M, Kashiwazaki N, Takizawa A, Maedomari N, Ozawa M, Noguchi J, Kaneko H, Shino M, Kikuchi K. Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection. Zygote, 15 (1): 15-24, 2007.
- Referans24 Nanassy L, Leeb K, Javora A, Machatyb Z. Changes in MPF and MAPK activities in porcine oocytes activated by different methods. Theriogenology, 68: 146 – 152, 2007.
- Referans25 Nur Z, Birler S, Ustuner B, Zık B, Ozdas OB, Ozguden Akkoc CG. Koç spermasının liyofilizasyonu ve intra-stoplazmik sperm enjeksiyonu yöntemi ile embriyo üretimi, TEBAG- 108R018, 2009.
- Referans26 Nur Z, Zik B, Ustuner B, Sagirkaya H, Ozguden CG. Effects of different cryoprotective agents on ram sperm morphology and DNA integrity. Theriogenology, 73 (9):1267-1275, 2010.
- Referans27 Olaciregui M, Luño V, Marti JI, Aramayona J, Gil L. Freeze-dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays. Andrologia, 48: 988–994, 2016.
- Referans28 Olaciregui M, Gil L. Freeze-dried spermatozoa: a future tool? Reprod Dom Anim, 52 (2): 248–254, 2017.
- Referans29 Rigler R. Microfluorometric characterization of intracellular nucleic acids and nucleoproteins by acridine orange. Acta Physiol Scand, 67 (267): 1-122, 1966.
- Referans30 Takeda K, Uchiyama K, Kinukawa M, Tagami T, Kaneda M, Watanabe S. Evaluation of sperm DNA damage in bulls by TUNEL assay as a parameter of semen quality. J Reprod Dev, 61(3): 185–190, 2015. Referans31 Üstüner B, Pabuccuoglu S, Demir K, Nur Z, Soylu MK, Sağırkaya H, Birler S. Liyofilize koç sperm plazma membranının embriyonik gelişim üzerine etkileri, TOVAG-(113O593), 2013.
- Referans32 Wakayama T, Yanagimachi R. Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat Biotechnol, 16: 639-641, 1998.
- Başlangıç: 1981
- Yayıncı: Bursa Uludağ Üniversitesi
Sayıdaki Diğer Makaleler
Sena ARDICLI, Hakan ÜSTÜNER, Öznur ARSLAN
Enver ÇAVUŞOĞLU, Roshan RİAZ, Mohamed Yusuf OMAR, Mehmet DEMİR, Abdülkadir ORMAN
Nurşen ÖZTÜRK, Halil KILIÇ, Bülent EKİZ
Sevil ERDENLİĞ GÜRBİLEK, Osman Yaşar TEL, Ayfer GÜLLÜ YÜCETEPE, Oktay KESKİN
Türkiye'de Ceylanda (Gazella subgutturosa) ilk Teladorsagiozis Vakası
Burcu ÜSTÜNER, Sema BİRLER, Berrin ZIK, Selim ALÇAY, Gül BAKIRER ÖZTÜRK, Sabire GULER, Hakan SAĞIRKAYA, Zekariya NUR, Serhat PABUCCUOĞLU
Widya PBPİNTAKA BAYU PUTRA, Roshan RİAZ, Andrean Amar GUNAWAN, Abdülkadir ORMAN
Fatma İŞBİLİR, Ayşe Burcu ÇELİK, Kadri ARSLAN, Betül BULCA, Bahri YILDIZ