Ülgen GÜNAY, ZEKARİYA NUR, M. Kemal SOYLU

Effect of glass wool filtration method on frozen-thawed dog semen

Bu çalışmada dondurulmuş-çözündürülmüş köpek spermasınını motilite ve morfolojisi üzerine glass wool filtrasyon yönteminin etkilerinin araştırılması amaçlanmıştır. Materyal olarak 2 adet Alman çoban köpeği kullanıldı. Ejakulatlar Tris-sitrik asit- yumurta sarısı ve Sodyum sitrat-yumurta sarısı sulandırıcıları ile finalde %4 gliserol içerecek şekilde 1.1 oranında sulandırıldılar. Sperma 0.5 ml.lik payetlerde donduruldu ve 37 C'lik su banyosunda 30 sn'de çözündürüldüler. Kolon olarak pasteur pipetleri kullanıldı. Her bir kolon 20 mg, 40 mg ve 80 mg ağırlığında ve sırasıyla 2, 3 ve 4 cm.lik genişlikten oluşturuldu. Filtratlar filtrasyon öncesi ve sonrasında motilite, akrozomal, diğer ve toplam defektler yönünden muayene edildiler. Filtrasyon yöntemleri karşılaştırıldığında, en iyi motilite ve ilerleme yüzdesi 40 mg.lık glass wool'dan oluşan kolonda bulundu. Diğer yandan sulandırıcıların motilite ve diğer morfolojik defektler üzerine herhangi bir etkisinin olmadığı saptandı. Fakat filtrasyon teknikleri ile spermatolojik özellikler arasında önemli ilişki bulundu. Sonuç olarak, donmuş-çözündürülmüş köpek spermasında glass wool filtrasyon tekniğinin başarılı bir şekilde kullanılabileceği sonucuna varılmıştır.

Anahtar Kelimeler:

motilite, donma, semen, çözülme, köpek, dondurarak koruma, filtrasyon, dondurulmuş semen, morfoloji, kalite, spermatozoa

Dondurulmuş-çözündürülmüş köpek sperması üzerine glass wool filtrasyon yönteminin etkisi

The aim of this study was to evaluate the effects of the glass wool filtration on motility and morphology of post-thaw quality of dog semen Two German Shepherd Dogs were used as material. Ejaculates were diluted with Tris-citric acid-egg yolk and Sodium citrate-egg yolk extenders at 1:1 ratio including a final 4% glycerol concentration. Semen were frozen in 0.5 ml straws and thawed in a water bath at 37°C for 30 sec. Pasteur pipettes were used as columns. Each column was constructed with 20, 40 and 80 mg glass wool and at 2, 3 and 4 cm heights, respectively. Filtrates were examined before and after filtration for motility, acrosomal, other and total defects. When the filtration methods were compared, the highest improvement percentage in motility was found in column filled with 40 mg glass wool. On the other hand extenders did not have any effect on motility and other morphological defects. But significantly interactions were found between spermatological parameters and filtration techniques. In conclusion, glass wool filtration techniques could be used successfully for filtration of frozen-thawed dog semen.

Keywords:

motility, freezing, semen, thawing, dogs, cryopreservation, filtration, frozen semen, morphology, quality, spermatozoa,

PDF

___

1. ANZAR M, GRAHAM EF. Filtration of bovine semen. 1. Development of a sephadex ionexchange filter. Anim Reprod Sci. 1993; 1: 187- 195. 2. ANZAR M, GRAHAM EF. Effect of filtration on post-thaw quality of bull semen. Theriogenology. 1995; 43:439-449. 3. ANZAR M, GRAHAM EF. Role of sperm motility and acrosome integrity in the filtration of bovine semen. Theriogenology. 1996; 45: 513-520. 4. CHANDRAHASAN C, PATTABIRAMAN SP, ENKATASWAMI V. Studies on the effect of wool column filtration on the quality of semen of crossbred bulls. Indian Vet J. 1986; 63: 913-918. 5. CHEN MJ, BONGSO A. Comparative evaluation of two density gradients preparations for sperm separation for medically assisted conception. Hum Reprod. 1999; 14:159-764. 6. CHRISTIANSEN IBJ. Reproduction in the dog and cat. W.B. Saunders, Bailliere Tindall, London, 1984. 7. CISALE HO, FISCHMAN ML, BLASI CD, FERNANDEZ HA, GLEDHILL BL.Enrichment of high quality spermatozoa in bovine semen: relative effectiveness of three filtration matrixes. Andrologia 2001; 33:143-150.

8. ENGEL S, WEBER H, PETZOLDT R, SEIDL B, WIEHE W, SPERL J An improved method of sperm selection by glass wool filtration. Andrologia 2001; 33: 223-230. 9. FERNANDEZ HA, CISALE H, AISEN E. Mecanismo de mejoramiento de semen bovino por filtracion a traves de Sephadex. Vet Argentina 1985 ; 2: 144-148. 10. .FOOTE RH. Inhibition of sperm motility does not affect live-dead separation of bull sperm by glass beads. Asian J Androl. 2001; 3: 193-198. 11. GOYAL RL, TULI RK, GEORGIE GC, CHAND D. Comparison of quality and freezability of water buffalo semen after washing or sephadex filtration. Theriogenology 1996; 46: 679-686. 12. HAMMEDEH ME, ZAVOS PM, ROSENBAUM P, SCHMIDT W. Comparison between the quality and function of sperm after semen processing with two different methods. Asian J Androl 2001; 3: 125-130. 13. JAROUDI KA, CARVER-WARD JA, HAMILTON CJCM, SIECK UV, SHETH KV. Percoll semen preparation enhances human oocyte fertilization in male-factor infertility as shown by a randomized cross-over study. Human Reprod 1993; 8: 1438-1442. 14. JOHNSTON, S.D. Performing a complete canine semen evalution in a small animal hospital. Vet. Clin. of N. Am. Small Anim. Pract. 1991; 21: 545- 550. 15. KHOLKUTE DS, RODRIGUEZ J, RAWLINNS RG, DUKELOW WR. Glass wool column filtration: effects on motility, viability and fertilising ability of epididimal spermatozoa from mice. Lab Anim Sci. 1994; 44: 537-539. 16. LINDE-FORSBERG C. Achieving canine pregnancy by using frozen or chilled extended semen. Small Animal Practice 1991; 21: 467-485. 17. LINDE-FORSBERG, C. Artificial insemination with fresh, chilled extended and frozen-thawed semen in the dog. Sem Vet Med Surg (Small Anim) 1995; 10: 48-58. 18. LINDEMANN CB, FISHER M, LIPTON M.. A comparative study of the effect of freezing and frozen storage on intact and demembranated bull spermatozoa. Cryobiology 1982; 19: 20-28. 19. MAKI- LAURILA M, GRAHAM EF. Separation of dead and live spermatozoa in bovine semen. J Dairy Sci 1968; 51: 965. 20. MAKLER A, STOLLER J, MAKLER-SHIRAN E. Dynamic aspects concerned with the mechanism of separating motile sperm from nonmotile sperm, leukocytes, and debris with use of high-density Percoll gradients. Fertility and Sterility 1998; 70: 961-966. 21. MOGAS T, RIGAU T, PIEDRAFITS J, BONET S, RODRIGUEZ-GILL JE. Effect of column filtration upon the quality parameters of fresh dog semen. Theriogenology 1998; 50: 1171-1189. 22. OETTLE EE. Changes in acrosome morphology during cooling and freezing of dog semen. Animal Reproduction Science 1986 ; 12: 145-150. 23. OETTLE E.E. Sperm morphology and fertility in the dog. J Reprod Fertil 1993; 47: 257-260. 24. JOHNSTON SD. Performing a complete canine semen evaluation in a small animal hospital. Canine Reproduction 1991; 21: 545-550. 25. PEREIRA RJTA, TULI RK, WALLENHORST S, HOLTZ W. The effect of heparin, caffeine and calcium ionophore A 23187 on in vitro induction of the acrosome reaction in frozen-thawed bovine and caprine spermatozoa. Theriogenoloy 2000; 54: 185-192. 26. PRAKASH P, LEYKIN L, CHEN Z, TOTH T, SAYEGH R, SCHIFF I, ISAACSON K. Preparation by differential gradient centrifugation is better than swim-up in selecting sperm with normal morphology (strict criteria). Reprod Biol 1998; 69: 722-726. 27. SAACKE RG, WHITE JM. Semen quality tests and their relationship to fertility. In: Proceedings of the 4 th NAAB Tech Conf Artif Insem Reprod. NAAB, USA, pp 22-27,1972. 28. SAACKE RG, VINSON WE, O’CONNOR ML, CANDLER JE, MULLINS J, AMANN RP, MARSHALL CE, WALLACE RA, VINCEL WN, KELLGREN HC. The relationship of semen quality and fertility: a heterospermic study. In: Proc 8 th NAAB Tech Conf Artif Insem Reprod. NAAB, USA, pp 71-78, 1980 29. SAMPER JC, HELLANDER JC, CRABO BG. Relationship between the fertility of fresh and frozen stallion semen and semen quality. J Reprod Fertil 1991; 44: 107-114. 30. SAMPER JC, CRABO BG. Assay of capacitated, freeze-damaged and extended stallion spermatozoa by filtration. Theriogenology 1993; 39: 1209-1220. 31. SHANNON P, CURSON B. Toxic effect and action of dead sperm on diluted bovine semen. J Dairy Sci 1972; 55: 615-620. 32. STUBBINGS RB, WOSIK CP. Glass wool versus swim up separation of bovine spermatozoa for in vitro fertilization. Theriogenology 1991; 35: 276. 33. TRENTALANCE GM, BEORLEGUI NB. Sperm evaluation in cryopreserved bovine semen recovered by two selection methods. Andrologia 2002; 34: 397-403. 34. VYAS S, MOHAN G, DAHMI AJ, SAHNI KL. Effect of Sephadex and glass-wool column filtra tion on the quality and storage (+5°C) of crossbred bull semen. Indian J Anim Sci 1991 ; 61: 702-704. 35. VYAS S, DAHMI AJ, SAHNI KL. Effect of filtration through Sephadex and glasswool on the quality and freezability of semen of crossbred bulls. Indian J Animal Sciences 1992 ;62: 341-343. 36. VYAS S, MOHAN G, DAHMI AJ, SAHNI KL. Comparative evaluation of different filtration techniques for improving the semen quality of crossbred bulls. Indian J Dairy Sci 1992; 45: 237-240.