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Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi

Year 2019, Volume: 12 Issue: 3, 329 - 335, 30.09.2019
https://doi.org/10.30607/kvj.594063

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.

Supporting Institution

TÜBİTAK

Project Number

1919B011602713

Thanks

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

References

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  • 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.
  • Agarwal A, Durairajanayagam D, Halabi J, Peng J, Vazquez-Levin M. Proteomics, oxidative stress and male İnfertility. Reproductive BioMedicine Online, 2014; 29: 32-58.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Bouyeh M. Ann Biol Res, 2012; 3(7): 3218-3224.
  • 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.
  • 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.
  • 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.
  • 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.
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  • El-Alamy MA, Foote RH. Freezability of spermatozoa from Finn and Dorset rams in multiple semen extenders, Anim Reprod Sci, 2001; 65: 245-254.
  • 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.
  • 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.
  • England GC. Cryopreservation of dog semen: a review. Journal of reproduction and fertility. Supplement, 1992; 47: 243-255.
  • 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.
  • 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.
  • 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.
  • 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.
  • Guthrie HD, Welch GR. Effects of reactive oxygen species on sperm function, Theriogenology, 2012; 78: 1700-1708.
  • 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.
  • 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.
  • Kim JG, Parthasarathy S. Oxidation and the spermatozoa, In Seminars in reproductive endocrinology, 1998; 16: 235-339.
  • 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.
  • Kulaksız R. Farklı antioksidanlar eklenmiş sulandırıcılarla dondurulmuş Saanen teke spermasının in vitro değerlendirilmesi. Doktora tezi, Ankara Üniversitesi Sağlık Bilimleri Enstitüsü, Ankara, 2009
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
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The Effect of Different Additives on Semen Freezing of Aksaray Malaklı Shepherd Dog

Year 2019, Volume: 12 Issue: 3, 329 - 335, 30.09.2019
https://doi.org/10.30607/kvj.594063

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.

Project Number

1919B011602713

References

  • 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.
  • 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.
  • Agarwal A, Durairajanayagam D, Halabi J, Peng J, Vazquez-Levin M. Proteomics, oxidative stress and male İnfertility. Reproductive BioMedicine Online, 2014; 29: 32-58.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Bouyeh M. Ann Biol Res, 2012; 3(7): 3218-3224.
  • 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.
  • 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.
  • 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.
  • 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.
  • Demirtaş A, Üntan İ. Seminal sıvı ve spermde oksidatif stres ve antioksidanlar, Türk Ürol Sem, 2011; 2: 24-30.
  • El-Alamy MA, Foote RH. Freezability of spermatozoa from Finn and Dorset rams in multiple semen extenders, Anim Reprod Sci, 2001; 65: 245-254.
  • 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.
  • 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.
  • England GC. Cryopreservation of dog semen: a review. Journal of reproduction and fertility. Supplement, 1992; 47: 243-255.
  • 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.
  • 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.
  • 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.
  • 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.
  • Guthrie HD, Welch GR. Effects of reactive oxygen species on sperm function, Theriogenology, 2012; 78: 1700-1708.
  • 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.
  • 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.
  • Kim JG, Parthasarathy S. Oxidation and the spermatozoa, In Seminars in reproductive endocrinology, 1998; 16: 235-339.
  • 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.
  • Kulaksız R. Farklı antioksidanlar eklenmiş sulandırıcılarla dondurulmuş Saanen teke spermasının in vitro değerlendirilmesi. Doktora tezi, Ankara Üniversitesi Sağlık Bilimleri Enstitüsü, Ankara, 2009
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
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There are 56 citations in total.

Details

Primary Language Turkish
Subjects Veterinary Sciences
Journal Section RESEARCH ARTICLE
Authors

Caner Öztürk 0000-0003-0566-0684

Şükrü Güngör This is me 0000-0003-3460-522X

Halit Furkan Demir This is me 0000-0002-6437-2183

Project Number 1919B011602713
Publication Date September 30, 2019
Acceptance Date August 23, 2019
Published in Issue Year 2019 Volume: 12 Issue: 3

Cite

APA Öztürk, C., Güngör, Ş., & Demir, H. F. (2019). Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi. Kocatepe Veterinary Journal, 12(3), 329-335. https://doi.org/10.30607/kvj.594063
AMA Öztürk C, Güngör Ş, Demir HF. Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi. kvj. September 2019;12(3):329-335. doi:10.30607/kvj.594063
Chicago Öztürk, Caner, Şükrü Güngör, and Halit Furkan Demir. “Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi”. Kocatepe Veterinary Journal 12, no. 3 (September 2019): 329-35. https://doi.org/10.30607/kvj.594063.
EndNote Öztürk C, Güngör Ş, Demir HF (September 1, 2019) Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi. Kocatepe Veterinary Journal 12 3 329–335.
IEEE C. Öztürk, Ş. Güngör, and H. F. Demir, “Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi”, kvj, vol. 12, no. 3, pp. 329–335, 2019, doi: 10.30607/kvj.594063.
ISNAD Öztürk, Caner et al. “Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi”. Kocatepe Veterinary Journal 12/3 (September 2019), 329-335. https://doi.org/10.30607/kvj.594063.
JAMA Öztürk C, Güngör Ş, Demir HF. Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi. kvj. 2019;12:329–335.
MLA Öztürk, Caner et al. “Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi”. Kocatepe Veterinary Journal, vol. 12, no. 3, 2019, pp. 329-35, doi:10.30607/kvj.594063.
Vancouver Öztürk C, Güngör Ş, Demir HF. Farklı Katkı Maddelerinin Aksaray Malaklı Çoban Köpeği Spermasının Dondurulmasına Etkisi. kvj. 2019;12(3):329-35.

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