The Effect of Different Additives on Semen Freezing of Aksaray Malaklı Shepherd Dog

Abstract

The aim of this study was to investigate the spermatological parameters of Aksaray Malakli shepherd dog after freeze-thawing semen with various additives. Study groups were set up as Tris-based extender (TBE)+trehalose (10mM), TBE+methionine (2mM) andcontrol. Sperm was collected by bulbus glandis massage (Digital Manipulation). Diluted semen samples were aspirated into 0,25 ml French straws and equilibrated at 5 ºC for 1,5 h. After equilibration, the straws were frozen in liquid nitrogen vapor, plunged into liquid nitrogen for storage. For determination the sperm volume, grade semen collecting tubes were used and the results were recorded in milliliters. The sperm concentration was determined with hemocytometric method as mm³. Eosin-nigrosine staining method was used for live sperm percentages. The HOS-test was performed to determine the ratio of intact spermatozoa membrane. When the freeze-thawing results are statistically examined with one-way analysis of variance, methionine group, motility (52,5%±2,7) and HOS test analysis (43,1%±2,9), (41,5%±3,1;33,2%±2,9) shown significant differences compared control (41,5%±3,1; 33,2%±2,9) group (p <0,05). The best result in the live sperm percentages was observed in methionine group but there was no statistically significant difference between the groups (p> 0.05).In the trehalose group, motility, HOS test and live sperm percentages results were 50.0% ±3,9;60,6%±3,7;38,4±3,6%, respectively. As a result of the study, it was determined that methionine 2 mM added to tris-based diluent in Aksaray Malaklı shepherd dog sperm was beneficial on spermatological parameters. The cryoprotective effect of methionine was found to be stronger than trehalose and control group.

Keywords

• Dog,methionine,sperm, trehalose

Project Number

1919B011602713

Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi

Abstract

Bu çalışmanın amacı, Aksaray Malaklı çoban köpeği spermasının farklı katkı maddeleri içeren sulandırıcılar ile dondurulup çözüm sonu spermatolojik parametrelerin araştırılmasıdır. Çalışma grupları, Tris ana stok (TAS)+trehaloz (10 mM), TAS+metiyonin (2 mM) ve kontrol olarak oluşturuldu. Köpeklerden sperma digital maniplasyon yöntemi ile alındı.  Sulandırma işlemi tamamlanan sperma örnekleri 0.25 ml lik payetlere çekildi. Payetlenen spermalar 1.5 saat +5˚C’ta ekilibrasyona tabi tutuldu. Ekilibrasyon sonrası payetler sıvı azot buharında donduruldu ve sıvı azot içerisinde saklandı. Sperma hacminin belirlenmesinde dereceli sperma toplama tüpleri kullanıldı ve sonuçları mililitre olarak kaydedildi. Hemositometrik yöntem ile spermatozoon konsantrasyonu belirlenerek mm³ olarak kaydedildi. Ölü canlı spermatozoa muayenesi için eosin-nigrosin boyama yöntemi kullanıldı. Spermatozoa plazma membran bütünlüğü oranını belirlemek için Hipo osmotik şişirme (HOS) testi yapıldı. Sonuçlar istatistiksel açıdan tek yönlü varyans analizi ile incelendiğinde çözüm sonu metiyonin grubunda motilite (%52,5±2,7) ve HOS test parametreleri açısından (%43,1±2,9), kontrol grubuna (%41,5±3,1;%33,2±2,9) göre istatistiksel farklılık gözlendi (p<0,05). Çözüm sonu canlı spermatozoa oranına en yüksek metiyonin grubunda ulaşılmasına rağmen gruplar arası istatistiksel farklılık bulunamadı (p>0,05). Trehaloz grubunda ise motilite, HOS test ve canlı spermatozoa oranı sırasıyla %50,0±3,9;%60,6±3,7;%38,4±3,6 olarak bulundu. Çalışmanın sonucunda Aksaray Malaklı çoban köpeği spermasında tris bazlı sulandırıcıya eklenen metiyonin 2mM dozunun spermatolojik parametreler üzerine faydalı olduğu belirlenmiştir. Metiyoninin kriyoprotektif etkinliğinin trehaloz ve kontrol grubuna göre daha güçlü olduğu kanısına varıldı.

Keywords

• Köpek,metiyonin,sperma,trehaloz

Supporting Institution

TÜBİTAK

Project Number

1919B011602713

Thanks

Bu çalışma Tübitak 2209-A Üniversite Ögrencileri Arastırma Projeleri tarafından (Proje no: 1919B011602713) desteklenmiştir.

References

1. Abdelhakeam AA, Graham EF, Vazquez IA, Chaloner KM. Studies on the absence of glycerol in unfrozen and frozen ram semen: Development of an extender for freezing: Effects of osmotic pressure, egg yolk levels, type of sugars, and the method of dilution, Cryobiology, 1991; 28: 43-49.
2. Agarwal A, Cocuzza M, Abdelrazik H, Sharma RK. “Oxidative stress measurement in patients with male or female factor infertility”, Handbook of Chemiluminescent Methods in Oxidative Stress Assessment, 2008; 195-218.
3. Agarwal A, Durairajanayagam D, Halabi J, Peng J, Vazquez-Levin M. Proteomics, oxidative stress and male İnfertility. Reproductive BioMedicine Online, 2014; 29: 32-58.
4. Akalın PP, Başpınar N, Çoyan K, Bucak MN, Ataman MB, Ömür AD, Bilgili A, Güngör Ş, Sarıözkan S, Öztürk C, Bodu M, Acibaeva B. Metiyonin ve Trehaloz İçeren Sulandırıcıların Koç Spermasının Dondurma-Çözdürme Sonrası Sperm Akrozom Bütünlüğü ve Biyokimyasal Parametreleri Üzerine Etkileri. Bahri Dağdaş Hayvancılık Araştırma Dergisi, 2015; 3(1): 7-15.
5. Álamo D, Batista M, González F, Rodrı́guez N, Cruz G, Cabrera F, Gracia A. Cryopreservation of semen in the dog: use of ultra-freezers of -152° C as a viable alternative to liquid nitrogen. Theriogenology, 2005; 63(1): 72-82.
6. Atasoy F, Erdoğan M, Özarslan B, Yüceer B, Kocakaya A, Akçapınar H. Malaklı Karabaş köpeklerde bazı morfolojik ve genetik özellikler, Ankara Üniv Vet Fak Derg, 2014; 61: 125-132.
7. Badr MR, Abd El- Malak MG, Hassan HM. Effect of trehalose on cryopreservation, oxidative stress and DNA integrity of buffalo spermatozoa, J Reprod Infertil, 2010; 1: 50-57.
8. Bencharif D, Amirat-Briand L, Garand A, Anton M, Schmitt E, Desherces S, Delhomme G, Langlois M, Barriere P, Destrumelle S, Vera-Munoz O, Tainturier D. The advantages of using a combination of LDL and glutamine in comparison with TRIS egg yolk and Equex STAMP extenders in the cryopreservation of canine sperm, Research in Vet Science, 2012; 93: 440-447.

9. Bilodeau JF, Blanchette S, Gagnon IC, Sirard MA. Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen, Theriogenology, 2001; 56: 275-286.
10. Bouyeh M. Ann Biol Res, 2012; 3(7): 3218-3224.
11. Bucak MN, Çoyan K, Öztürk C, Güngör Ş, Ömür AD. Methionine supplementation improves ram sperm parameters during liquid storage at 5 C, Cryobiology, 2012; 65(3): 335-337.
12. Bucci D, Cunto M, Gadani B, Spinaci M, Zambelli D, Galeati G. Epigallocatechin-3-gallate added after thawing to frozen dog semen: Effect on sperm parameters and ability to bind to oocytes’ zona pellucida, Reproductive biology, 2019; 19(1): 83-88.
13. Câmara DR, Silva SV, Almeida FC, Nunes JF, Guerra MM. Effects of antioxidants and duration of pre-freezing equilibration on frozenthawed ram semen, Theriogenology, 2011; 76: 342-350.
14. Çoyan K, Başpınar N, Bucak MN, Akalın PP, Ataman MB, Ömür AD, Güngör Ş, Küçükgünay S, Özkalp B, Sarıözkan S. Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen. Research in veterinary science, 2010; 89(3): 426-431.
15. Demirtaş A, Üntan İ. Seminal sıvı ve spermde oksidatif stres ve antioksidanlar, Türk Ürol Sem, 2011; 2: 24-30.
16. El-Alamy MA, Foote RH. Freezability of spermatozoa from Finn and Dorset rams in multiple semen extenders, Anim Reprod Sci, 2001; 65: 245-254.
17. El-Badry DA, El-Maaty AMA, Gamal A. The effect of trehalose supplementation of INRA-82 extender on quality and fertility of cooled and frozen-thawed stallion spermatozoa, Journal of equine veterinary science, 2017; 48: 86-92.
18. El-Sheshtawy RI, Sisy GA, El-Nattat WS. Effects of different concentrations of sucrose or trehalose on the post-thawing quality of cattle bull semen, Asian Pacific Journal of Reproduction, 2015; 4(1): 26-31.
19. England GC. Cryopreservation of dog semen: a review. Journal of reproduction and fertility. Supplement, 1992; 47: 243-255.
20. Flores JA, Fernández A, Huamán U, Ruiz G, Santiani A. Refrigeration of canine semen using glucose, fructose, trehalose or sucrose to extend sperm survival, Rev Inv Vet Perú, 2010; 21: 26-34.
21. Garcia MA, Graham EF. Development of a buffer system for dialysis of bovine spermatozoa before freezing. II. Effect of sugars and sugar alcohols on posthaw motility, Theriogenology, 1989; 31: 1029-1237.
22. Gungor S, Bucak MN. Basal medium eagle solution may improve the post-thaw parameters of Kangal dog semen, Eurasian J Vet Sci, 2016; 32: 193-199.
23. Gungor S, Ata A, Inanc ME. Effects of trehalose and catalase on the viability and kinetic parameters of cryopreserved ram sperm, Acta Scientiae Veterinariae 2018; 46: 1.
24. Guthrie HD, Welch GR. Effects of reactive oxygen species on sperm function, Theriogenology, 2012; 78: 1700-1708.
25. Inanc ME, Tekin K, Olgac KT, Yilmaz B, Cil B, Tasdemir U, Tuncer BP, Buyukleblebici S, Durmaz E, Uysal O. Effect of cholesterol loaded cyclodextrin on semen cryopreservation of Aksaray Malakli shepherd dogs of different ages, Animal Reproduction Science, 2018; 193: 191-200.
26. Iqbal S, Andrabi SMH, Riaz A, Durrani AZ, Ahmad N. Trehalose improves semen antioxidant enzymes activity, post-thaw quality, and fertility in Nili Ravi buffaloes (Bubalus bubalis). Theriogenology, 2016; 85(5): 954-959.
27. Kim JG, Parthasarathy S. Oxidation and the spermatozoa, In Seminars in reproductive endocrinology, 1998; 16: 235-339.
28. Kulaksız R, Daşkın A. Farklı antioksidanlarla dondurulan Saanen teke spermasının in vitro ve in vivo değerlendirilmesi, Ankara Üniv Vet Fak Derg, 2009; 56: 201-205.
29. Kulaksız R. Farklı antioksidanlar eklenmiş sulandırıcılarla dondurulmuş Saanen teke spermasının in vitro değerlendirilmesi. Doktora tezi, Ankara Üniversitesi Sağlık Bilimleri Enstitüsü, Ankara, 2009
30. Liu Z, Foote RH, Brockett CC. Survival of bull sperm frozen at different rates in media varying in osmolarity, Cryobiology, 1998; 37(3): 219-230.
31. Livine RL, Berlett BS, Moskovitz J, Mosoni L, Stadtman ER. Methionine residues may protect proteins from critical oxidative damage, Mech Ageing Dev, 1999; 107: 323–332.
32. Malo C, Gil L, Gonzalez N, Cano R, Blas I, Espinosa E. Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender, Cryobiology, 2010; 61(1): 17-21.
33. Martins-Besa A, Rocha A, Maenco-Aguirre A. Incorporation of taurine and hypotaurine did not improve the efficiency of the Uppsala Equex II extender for dog semen freezing, Theriogenology, 2007; 68: 1088-1096.
34. Michael AJ, Alexopoulos C, Pontiki EA, Hadjipavlou-Litina DJ, Saratis P, Ververidis HN, Boscos CM. Effect of antioxidant supplementation in semen extenders on semen quality and reactive oxygen species of chilled canine spermatozoa, Animal Rep Science, 2009; 112: 119-135.
35. Molinia FC, Evans G, Casares PI, Maxwell WMC. Effect of monosaccharides and disaccharides in tris based diluents on motility, acrosome integrity and fertility of pellet frozen ram spermatozoa, Animal Reproduction Science, 1994; 36: 113-122.
36. Mosca F, Madeddu M, Sayed AA, Zaniboni L, Iaffaldano N, Cerolini S. Data on the positive synergic action of dimethylacetamide and trehalose on quality of cryopreserved chicken sperm. Data in brief, 2016; 9: 1118-1121.
37. Neagu VR, García BM, Sandoval CS, Rodríguez AM, Ferrusola CO, Fernández LG, Tapia JA, Peña, FJ. Freezing dog semen in presence of the antioxidant butylated hydroxytoluene improves postthaw sperm membrane integrity, Theriogenology, 2010; 73(5); 645-650.
38. Ogata K, Sasaki A, Kato Y, Takeda A, Wakabayashi M, Sarentonglaga B. Glutathione supplementation to semen extender improves the quality of frozenthawed canine spermatozoa for transcervical insemination, J Reprod Dev, 2015; 61; 116–122.
39. Ortega Ferrusola C, González Fernández L, Morrell JM, Salazar Sandoval C, Macías García B, Rodríguez-Martinez H. Lipid peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion dependent and is related to apoptotic-like changes. Reproduction, 2009; 138; 55-63.
40. Patra RC, Swarup D, Dwivedi SK. Antioxidant effects of alpha-tocopherol, ascorbic acid and L-methionine on lead induced oxidative stress to the liver, kidney and brain in rats. Toxicology, 2001; 162; 81-88.
41. Peña FJ, Núñez‐Martínez I, Morán JM. Semen technologies in dog breeding: an update, Reproduction in Domestic Animals, 2006; 41(s2); 21-29.
42. Reddy NSS, Mohanarao GJ, Atreja SK. Effects of adding taurine and trehalose to a tris-based egg yolk extender on buffalo (Bubalus bubalis) sperm quality following cryopreservation, Animal Reproduction Science, 2010; 119(3): 183-190.
43. Reed DJ. Glutathione: toxicological implications, Annu Rev Pharmacol Toxicol, 1990; 30: 603–631.
44. Sabuncuoğlu S, Özgüneş H. Kemoterapi, serbest radikaller ve oksidatif stres, Hacettepe Üniversitesi Eczacılık Fakültesi Dergisi, 2011; 31(2): 137-50.
45. Sharawy SM, Saleh NH, Attalah SA, Absy GM, Doaa HK. Studies on the effect of added ascorbic acid and trehalose to tris buffered egg yolk extender on chilled ram semen, MENA Sci J, 2015; 1: 1–4.
46. Singh VK, Atreja SK, Kumar R, Chhillar S. Singh AK. Assessment of intracellular Ca2+, cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender, Reprod Dom Anim, 2012; 47: 584-590.
47. Storey BT, Noiles EE, Thompson KA. Comparison of glycerol other polyols, trehalose, and raffinose to provide a defined cryoprotectant medium for mouse sperm cryopreservation. Cryobiology, 1998; 37: 46-58.
48. Tekcan M. Oksidatif stres-antioksidan sistemler ve testis, Androloji Bülteni, 2009; 37: 131-136.
49. Tekin N. Spermanın muayenesi ve değerlendirilmesi, Evcil Hayvanlarda Reprodüksiyon, Sun’i Tohumlama, Doğum ve İnfertilite. Ed. E. Alaçam, Dizgievi, Konya. 1994; pp 69-70.
50. Tosun H, Uysal O. Köpek spermasının dondurulmasında farklı sulandırıcıların ve bireylerin etkisi, Ankara Üniv Vet Fak Derg, 2007; 54(1): 23.
51. Tuncer PB, Bucak MN, Sarıözkan S, Sakin F, Yeni D, Çiğerci İH, Büyükleblebici O. The effect of raffinose and methionine on frozen/thawed Angora buck (Capra hircus ancryrensis) semen quality, lipid peroxidation and antioxidant enzyme activities, Cryobiology, 2010; 61(1), 89-93.
52. Uysal O, Bucak MN, Yavaş İ, Varışlı Ö, Gurcan IS. Evaluation of ram Sperm frozen with various taurine concentrations. Indian Vet J, 2005; 82: 1059-1061.
53. Walczak-Jedrzejowska R, Wolski JK, Slowikowska-Hilczer J. The role of oxidative stress and antioxidants in male fertility, Central Eur J Urology, 2012; 66: 60-67.
54. Watson PF. The Causes of Reduced Fertility with Cryopreserved Semen, Anim Reprod Sci, 2000; 60: 481-492.
55. Watson PF. Use of Giemsa stain to detect changes in the acrosome of frozen ram spermatozoa, Vet Rec, 1975; 97(1): 12-15.
56. Woelders H, Matthijs A, Engel B. Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing, Cryobiology, 1997; 35: 93-105.