Farklı Ekilibrasyon Ortamlarının Çözüm Sonu Boğa Sperması Kalitesi Üzerine Etkisi

Abstract

Ekilibrasyon, spermanın sıcaklığının düşürüldüğü ve faz değişimlerinin meydana geldiği aşama olarak, sperma dondurulmasında en önemli basamaklardan biridir. Ekilibrasyonun gerçekleştirildiği ortam çoğu zaman göz ardı edilmekte, ekilibrasyon koşullarının başarısı ampirik tecrübelere dayanmaktadır. Bu çalışmanın amacı farklı ekilibrasyon ortamlarının toplam motilite (TMOT), progresif motilite (PMOT), spermatozoa hareket parametleri (VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF), plazma membran ve akrozom bütünlüğü (PMAI) ve DNA fragmentasyon indeksi (DFI) üzerine etkisinin araştırılmasıdır. Çalışmada 4 baş boğadan iki kez sperma alınarak üç eşit hacme bölündü. Andromed® ile sulandırılan spermalar 24 saat 4°C’de payet içinde (Deneme 1), kapta (Deneme 2) ve çalkalayıcıda (Deneme 3) ekilibrasyona bırakıldı. Çözüm sonu sperma kalitesi TMOT, PMOT, VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF, PMAI ve DFI parametreleri değerlendirilerek belirlendi. Farklı ekilibrasyon koşulları arasında, hareket özellikleri olan TMOT, PMOT, VSL, VAP, BCF ve morfolojik özellikler olan PMAI ve DFI açısından istatistiki bir farklılık bulunamadı (p>0,05). En yüksek VCL değeri Deneme 3’te, en yüksek LIN, STR, WOB ve ALH değerleri Deneme 2’de gözlendi (p<0,05). Bu sonuçlar farklı kolullarda yapılan ekilibrasyonun çözüm sonu spermatolojik değerler üzerinde herhangi bir zararlı etkisi olmadığını göstermektedir. Ayrıca boğa spermasının çalkalayıcıda ekilibre edilmesinin kap ve payet içinde ekilibre edilmesine göre spermatozoa hareket özellikleri açısından daha yararlı olduğu söylenebilir.

Keywords

• Boğa Sperması,CASA,Ekilibrasyon,Eklibrasyon Ortamı,Flow Sitometri

Effects of Different Equilibration Conditions on Cryopreserved Bovine Sperm Quality

Abstract

Equilibration is the one of the important steps of semen cryopreservation and it is the stage in which semen is cooled and phase changes occur. The condition of equilibration is mostly regarded; and success of the equilibration conditions depend on empirical experiences. The aim of this study was to investigate the effects of three different equilibration methods on post-thaw total motility (TMOT), progressive motility (PMOT), kinetic parameters of spermatozoa (VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF), plasma membrane and acrosome integrity (PMAI) and DNA fragmentation index (DFI). In each of 4 bulls; two ejaculates were split into three aliquots, diluted by Andromed® and equilibrated for 24h at 4°C in Drawed Straw (Experiment 1), Cups (Experiment 2) and Shaker (Experiment 3). After thawing, sperm quality was determined by examination of TMOT, PMOT, VCL, VSL, VAP, LIN, STR, WOB, ALH, BCF, PMAI and DFI. There were no differences (p>0.05) depending on equilibration conditions for TMOT, PMOT, VSL, VAP, BCF as kinetic parameters and for PMAI and DFI as morphological parameters. The highest VCL value was observed in experiment 3, when the highest LIN, STR, WOB and ALH values were observed in experiment 2 (p<0.05). The results show that changes in equilibrating condition have no detrimental effect on post-thaw bull semen quality. In addition, it can be said that equilibrating bull semen in a shaker presents some kinetic benefits in contrast with cup or straw equilibration methods.

Keywords

• Bull Semen,CASA,Equilibration,Equilibration Condition,Flow Cytometry

References

1. Ahmad M, Ahmad N, Riaz A, Anzar M. Sperm survival kinetics in different types of bull semen: progressive motility, plasma membrane integrity, acrosomal status and reactive oxygen species generation. Reprod Fert Develop. 2015; 27: 784-793.
2. Aires VA, Hinsch KD, Mueller-Schloesser F, Bogner K, Mueller-Schloesser S, Hinsch E. In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen. Theriogenology. 2003; 60(2): 269-279.
3. Almadaly E, Farrag F, Shukry M, Murase T. Plasma membrane integrity and morphology of frozen-thawed bull spermatozoa supplemented with desalted and lyophilized seminal plasma. Global Veterinaria. 2014; 13: 753-766.
4. Amirat L, Tainturier D, Jeanneau L, Thorin C, Gérard O, Courtens JL, Anton M. Bull semen in vitro fertility after cryopreservation using egg yolk LDL: A comparison with Optidyl®, a commercial egg yolk extender. Theriogenology. 2004; 61: 895-907.
5. Anzar M, Kroetsch T, Boswall L. Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes. Animal Reproduction Science. 2011; 126: 23-31.
6. Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals: A damaging and capacitating phenomenon. Journal of Andrology. 2000; 21(1): 1-7.
7. Bochenek M, Smorag Z, Pilch J. Sperm chromatin structure assay of bulls qualified for artificial insemination. Theriogenology. 2001; 56: 557-567.
8. D’Occhio MJ, Hengstberger KJ, Johnston SD. Biology of sperm chromatin structure and relationship to male fertility and embryonic survival. Anim Reprod Sci. 2007; 101: 1-17.

9. Evenson D, Jost L. Sperm chromatin structure assay for fertility assessment, In: Current protocols in cytometry, Ed; Robinson JP, John Wiley & Sons, Inc., New York. 2001; pp. 1-7.
10. Fetterolf PM, Rogers BJ. Prediction of human sperm penetrating ability using computerized motion parameters. Mol Reprod Dev, 1990; 27: 326-331.
11. Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of mammalian sperm: What we ask them to survive. Journal of Andrology. 1990; 11(1): 73-88.
12. Hering DM, Lecewicz M, Kordan W, Majewska A, Kaminski S. Missense mutation in glutathione-S-transferase M1 gene is associated with sperm motility and ATP content in frozen-thawed semen of Holstein- Friesian bulls. Animal Reproduction Science. 2015; 159: 94-97.
13. Karabinus DS, Evenson DP, Jost LK, Baer RK. Comparison of semen quality in young and mature Holstein bulls measured by light microscopy and flow cytometry. J Dairy Sci. 1990; 73: 2364-2371.
14. Karoui S, Díaz C, González-Marín C, Amenabar ME, Serrano M, Ugarte E, Gosálvez J, Roy R, López-Fernández C, M. J. Carabaño MJ. Is sperm DNA fragmentation a good marker for field AI bull fertility? J Anim Sci. 2012; 90: 2437-2449.
15. Kumar S, Millar JD, Watson PF. The effect of cooling rate on the survival of cryopreserved bull, ram, and boar spermatozoa: a comparison of two controlled-rate cooling machines. Cryobiology. 2003; 46(3): 246-253.
16. Layek SS, Mohanty TK, Kumaresan A, Parks JE. Cryopreservation of bull semen: Evolution from egg yolk based to soybean based extenders. Animal Reproduction Science. 2016; 172: 1-9.
17. Leite TG, Filho VRV, Arruda RP, Andrade AFC, Emerick LL, Zaffalon FG, Martins JAM. Effects of extender and equilibration time on post-thaw motility and membrane integrity of cryopreserved Gyr bull semen evaluated by CASA and flow cytometry. Animal Reproduction Science. 2010; 120(1-4): 31-38.
18. Liu DY, Clarke GN, Baker HG. Relationship between sperm motility assessed with the Hamilton‐Thorn motility analyzer and fertilization rates in vitro. J Androl. 1991; 12: 231-239.
19. Moallem U, Neta N, Zeron Y, Zachut M, Roth Z. Dietary a-linolenic acid from flaxseed oil or eicosapentaenoic and docosahexaenoic acids from fish oil differentially alter fatty acid composition and characteristics of fresh and frozen-thawed bull semen. Theriogenology. 2015; 30: 1-11.
20. Murphy EM, Murphy C, O'Meara C, Dunne G, Eivers B, Lonergan P, Fair S. A comparison of semen diluents on the in vitro and in vivo fertility of liquid bull semen. Journal of Dairy Science. 2017; 100(2): 1541- 1551.
21. Nagy S, Jansen J, Topper EK, Gadella BM. A triple-stain flow cytometric method to assess plasma-and acrosome-membrane integrity of cryopreserved bovine sperm immediately after thawing in presence of egg-yolk particles. Biol Reprod. 2003; 68: 1828-1835.
22. Stadnik L, Rajmon R, Beran J, Simonik O, Dolezalova M, Sichtar J, Stupka R, Folkova P. Influence of selected factors on bovine spermatozoa cold shock resistance. Acta Vet Brno. 2015; 84: 125-131.
23. Sukcharoen N, Keith J, Irvine DS, Aitken RJ. Prediction of the in-vitro fertilization (IVF) potential of human spermatozoa using sperm function tests: the effect of the delay between testing and IVF. Hum Reprod. 1996; 11: 1030-1034.
24. Sukcharoen N, Sithipravej T, Promviengchai S, Chinpilas V, Boonkasemsanti W. Sperm morphology evaluated by computer (IVOS) cannot predict the fertilization rate in vitro after intracytoplasmic sperm injection. Fertil Steril. 1998; 69: 564-568.
25. Tartaglionea CM, Ritta MN. Prognostic value of spermatological parameters as predictors of in vitro fertility of frozen-thawed bull semen. 2004; 62(7): 1245-1252.
26. Thun R, Hurtado M, Janett F. Comparison of Biociphos-Plus® and TRIS-egg yolk extender for cryopreservation of bull semen. Theriogenology. 2002; 57(3): 1087-1094.
27. Watson PF. The causes of reduced fertility with cryopreserved semen. Animal Reproduction Science. 2000; 60-61: 481-492.