Ankara Üniversitesi Veteriner Fakültesi Dergisi
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Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C

Yıl 2017, Cilt: 64 Sayı: 4, 313 - 320, 01.12.2017

Elif GÖKÇE Selim ALÇAY Zülfiye GÜL

https://doi.org/10.1501/Vetfak_0000002815

Öz

The aim of the study was to evaluate different BSA doses in soybean-lecithin based extenders for liquid storage of ram semen. Pooled semen was divided into four equal volumes and diluted in a single-step dilution method with BSA supplemented (BSA5, BSA7.5, BSA10 groups supplemented 5mg/mL, 7.5mg/mL, 10mg/mL BSA respectively) and BSA-free (control) extenders. Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypo-osmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) at 0, 24, 48 and 72h of storage at 5 °C. Antioxidant activities of groups at 0 and 72h were measured with glutathionine (GSH) concentration, membrane lipid peroxidation status was analyzed using the malondialdehyde (MDA) concentration. Antioxidant capacity of extender also was measured with 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assays. In the study, BSA (5mg/mL) supplementation has beneficial effect on ram sperm parameters at all evaluation times. In addition, the increasing doses of BSA supplementation has a negative effect on sperm parameters especially 48 and 72h of storage

Anahtar Kelimeler

BSA, lecithin, ram semen, short-term storage

Kaynakça

  • Agarwal A, Makker K, Sharma R (2008): Clinical relevance of oxidative stress in male factor infertility: An update. J Reprod Immunol, 59, 2-11.
  • Akhter S, Rakha BA, Iqbal R, et al. (2014): Effect of bovine serum albumin on motility, plasmalemma, viability and chromatin integrity of buffalo bull spermatozoa and chromatin integrity of buffalo bull spermatozoa. Pakistan J Zool, 46, 115-120.
  • Alçay S, Gökçe E, Toker MB, et al. (2016): Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa. Cryobiology, 72, 269-273.
  • Alçay S, Toker MB, Üstüner B, et al. (2014): Investigation of relationships between DNA integrity and fresh semen parameters in rams. Kafkas Univ Vet Fak Derg, 20, 793- 798.
  • Alçay S, Toker MB, Gökçe E, et al. (2015): Successful ram semen cryopreservation with lyophilized egg yolk-based extender. Cryobiology, 71, 329-333.
  • Amidi F, Farshad A, Khor AK (2010): Effects of cholesterol-loaded cyclodextrin during freezing step of cryopreservation with TCGY extender containing bovine serum albumin on quality of goat spermatozoa. Cryobiology, 61, 94-99.
  • Anghel A, Zamfirescu S, Dragomir C, et al. (2010): The effects of antioxidants on the cytological parameters of cryopreserved buck semen. Rom Biotech Lett, 15, 26-32.
  • Bakst MR, Cecil HC (1992): Effect of bovine serum albumin on motility and fecundity of turkey spermatozoa before and after storage. J Reprod Fert, 94, 287-293.
  • Ball BA, Medina V, Gravance CG, et al. (2001): Effect of antioxidants on preservation of motility, viability and acrosomal integrity of equine spermatozoa during storage at 5C. Theriogenology, 56, 577-589.
  • Bouayed J, Bohn T (2010): Exogenous antioxidants- double-edged swords in cellular redox state health beneficial effects at physiologic doses versus deleterious effects at high doses. Oxid Med Cell Longev, 3, 228-237.
  • Bucak MN, Çoyan K, Öztürk C, et al. (2012): Methionine supplementation improves ram sperm parameters during liquid storage at 5 C. Cryobiology, 65, 335-337.
  • Bucak MN, Tekin N (2007): Protective effect of taurine, glutathione and trehalose on the liquid storage of ram semen. Small Rum Res, 73, 103-108.
  • Bucak MN, Tuncer PB, Sarıözkan S, et al. (2009): Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters, lipid peroxidation and anti-oxidant activities. Small Rum Res, 81, 13-17.
  • Çoyan K, Başpınar N, Bucak MN, et al. (2010): Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen. Res Vet Sci, 89, 426-431.
  • Davis BK, Byrne R, Hun B (1979): Studies on the mechanism of capacitation. II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro. Biochim Biophys Acta, 558, 257-266.
  • De Paz P, Esteso MC, Alvarez M, et al. (2010): Development of extender based on soybean lecithin for its application in liquid ram semen. Theriogenology, 74, 663- 671.
  • Demircan C, Gül Z, Büyükuysal RL (2014): High glutamate attenuates S100B and LDH outputs from rat cortical slices enhanced by either oxygen-glucose deprivation or menadione. Neurochem Res, 39, 1232-1244.
  • Dorman HJD, Peltoketo A, Hiltunen R, et al. (2003): Characterization of antioxidant properties of de-odourized aqueous extracts from selected Lamiaceae herbs. Food Chem, 83, 255-262.
  • El-Kon I (2011): Testing usability of bovine serum albumin (BSA) for preservation of Egyptian Buffalo semen. Am Eurasian J Agric Environ Sci, 11, 495-502.
  • Ellman GL (1959): Tissue sulfhydryl groups. Arch Biochem Biophys, 82, 70-77.
  • Gaspar J, Rodrigues A, Laires A, et al. (1994): On the mechanisms of genotoxicity and metabolism of quercetin. Mutagenesis, 9, 445-449.
  • Gündoğan M (2009): Short term preservation of ram semen with different extenders. Kafkas Univ Vet Fak Derg, 15, 429-435.
  • Gündoğan M, Tekerli M, Uçar M, et al. (2003): Effect of diluents on motility of ram sperm during storage at 5 C. Arch Androl, 49, 69-75.
  • Gündoğan M, Yeni D, Avdatek F, et al. (2010): Influence of sperm concentration on the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm during liquid storage. Anim Reprod Sci, 122, 200-207.
  • Hodnick WF, Kung FS, Roettger WJ, et al. (1986): Inhibition of mitochondrial respiration and production of toxic oxygen radicals by flavonoids. A structure-activity study. Biochem Pharmacol, 35, 2345-2357.
  • Hossain MS, Hyeong LJ, Miah AG, et al. (2007): Effect of fatty acids bound to bovine serum albumin-V on acrosome reaction and utilization of glucose in boar spermatozoa. Reprod Med Biol, 6, 109-115.
  • Kankofer M, Kolm G, Aurich J (2005): Activity of glutathione peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen during storage at 5 C. Theriogenology, 63, 1354-1365.
  • Matsuoka T, Imai H, Kohno H, et al. (2006): Effect of bovine serum albumin and trehalose in semen diluents for improvement of frozen-thawed ram semen. J Reprod Dev, 52, 675-683.
  • Maxwell WMC, Salamon S (1993): Liquid storage of ram semen: A review. Reprod Fertil Dev, 5, 613-638.
  • Metodiewa D, Jaiswal AK, Cenas N, et al. (1999): Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radic Biol Med, 26,107-116.
  • Naijian HR, Kohram H, Zare SA, et al. (2013): Effects of various concentrations of BSA on microscopic and oxidative parameters of Mahabadi goat semen following the freeze-thaw process. Small Rum Res, 113, 371-375.
  • Ochiai M, Nagao M, Wakabayashi K, et al. (1984): Superoxide dismutase acts as an enhancing factor for quercetin mutagenesis in rat-liver cytosol by preventing its decomposition. Mutat Res, 129, 19-24.
  • Sarıözkan S, Tuncer PB, Bucak MN, et al. (2009): Influence of various antioxidants on microscopic-oxidative stress indicators and fertilizing ability of frozen-thawed bull semen. Acta Vet Brno, 78, 463-469.
  • Sarıözkan S, Türk G, Cantürk F, et al. (2013): The effect of bovine serum albumin and fetal calf serum on sperm quality, DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen. Cryobiology, 67, 1-6.
  • Toker MB Alçay S, Gökçe E, et al. (2016): Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience. Cryobiology, 72, 205- 209.
  • Uysal O, Bucak MN (2007): Effect of oxidized serum albumin, cysteine and lycopene on the quality of frozen thawed ram semen. Acta Vet Brno, 76, 383-390.
  • Uysal O, Bucak MN, Yavaş İ, et al. (2007): Effect of various antioxidants on the quality of frozen-thawed bull semen. J Anim Vet Adv, 6, 1362-1366.
  • Üstüner B, Alçay S, Nur Z, et al. (2014): Effect of egg yolk and soybean lecithin on tris-based extender in post-thaw ram semen quality and in vitro fertility. Kafkas Univ Vet Fak Derg, 20, 393-398.
  • Watjen W, Michels G, Steffan B, et al. (2005): Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis. J Nutr, 135, 525-531.
  • Xia J, Ren D (2009): The BSa-induced Ca(2+) influx during sperm capacitation is CATSPER channel-dependent. Reprod Biol Endocrinol, 7, 119-127.

BSA ilave edilmiş lesitin bazlı sulandırıcının koç spermasının 5 C sıcaklıkta muhafaza edilmesineolumlu etkisi

Yıl 2017, Cilt: 64 Sayı: 4, 313 - 320, 01.12.2017

Elif GÖKÇE Selim ALÇAY Zülfiye GÜL

https://doi.org/10.1501/Vetfak_0000002815

Öz

Bu çalışmada, farklı dozlarda BSA ilave edilmiş lesitin bazlı sulandırıcının koç spermasının 5°C’ta kısa sureli saklanması üzerindeki etkisinin değerlendirilmesi amaçlandı. Çalışma amacıyla alınan spermalar pooling yapılarak dört eşit hacme bölündü ve 5mg/mL, 7.5mg/mL, 10mg/mL BSA ilave edilmiş (BSA5, BSA7.5, BSA10 sırasıyla) ve edilmemiş (kontrol) sulandırıcılarla sulandırıldı. Sulandırılmış spermanın sıcaklığı 5°C’a düştükten sonra 0, 24, 48 ve 72. saatlerde; motilite, plazma membran fonksiyonel bütünük [hipoozmotik şişme testi (HOST)] ve akrozomal hasar [FITC-Pisum sativum agglutinin (PSA-FITC)] muayeneleri yapıldı. Çalışmanın başlangıcı ve sonunda antioksidan aktivitenin ve lipid peroksidasyonun değerlendirilmesi için malondialdehyde (MDA) ve glutathionine (GSH) konsantrasyonları ölçüldü. Ayrıca 2,2-Diphenyl-1-picrylhydrazyl (DPPH) ve Dichloro-dihydro-fluorescein diacetate (DCFH-DA) yöntemleri ile her bir sulandırıcının antioksidan kapasiteleri değerlendirildi. Yapılan muayenelerin sonucunda; 5mg/mL BSA ilavesinin koç spermasının kısa süreli muhafazasında etkili olduğu gözlemlendi. Ayrıca artan BSA konsantrasyonunun spermanın soğutularak saklanmasında olumsuz etki oluşturduğu belirlendi

Anahtar Kelimeler

BSA, kısa süreli muhafaza, koç sperması, lesitin

Kaynakça

  • Agarwal A, Makker K, Sharma R (2008): Clinical relevance of oxidative stress in male factor infertility: An update. J Reprod Immunol, 59, 2-11.
  • Akhter S, Rakha BA, Iqbal R, et al. (2014): Effect of bovine serum albumin on motility, plasmalemma, viability and chromatin integrity of buffalo bull spermatozoa and chromatin integrity of buffalo bull spermatozoa. Pakistan J Zool, 46, 115-120.
  • Alçay S, Gökçe E, Toker MB, et al. (2016): Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa. Cryobiology, 72, 269-273.
  • Alçay S, Toker MB, Üstüner B, et al. (2014): Investigation of relationships between DNA integrity and fresh semen parameters in rams. Kafkas Univ Vet Fak Derg, 20, 793- 798.
  • Alçay S, Toker MB, Gökçe E, et al. (2015): Successful ram semen cryopreservation with lyophilized egg yolk-based extender. Cryobiology, 71, 329-333.
  • Amidi F, Farshad A, Khor AK (2010): Effects of cholesterol-loaded cyclodextrin during freezing step of cryopreservation with TCGY extender containing bovine serum albumin on quality of goat spermatozoa. Cryobiology, 61, 94-99.
  • Anghel A, Zamfirescu S, Dragomir C, et al. (2010): The effects of antioxidants on the cytological parameters of cryopreserved buck semen. Rom Biotech Lett, 15, 26-32.
  • Bakst MR, Cecil HC (1992): Effect of bovine serum albumin on motility and fecundity of turkey spermatozoa before and after storage. J Reprod Fert, 94, 287-293.
  • Ball BA, Medina V, Gravance CG, et al. (2001): Effect of antioxidants on preservation of motility, viability and acrosomal integrity of equine spermatozoa during storage at 5C. Theriogenology, 56, 577-589.
  • Bouayed J, Bohn T (2010): Exogenous antioxidants- double-edged swords in cellular redox state health beneficial effects at physiologic doses versus deleterious effects at high doses. Oxid Med Cell Longev, 3, 228-237.
  • Bucak MN, Çoyan K, Öztürk C, et al. (2012): Methionine supplementation improves ram sperm parameters during liquid storage at 5 C. Cryobiology, 65, 335-337.
  • Bucak MN, Tekin N (2007): Protective effect of taurine, glutathione and trehalose on the liquid storage of ram semen. Small Rum Res, 73, 103-108.
  • Bucak MN, Tuncer PB, Sarıözkan S, et al. (2009): Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters, lipid peroxidation and anti-oxidant activities. Small Rum Res, 81, 13-17.
  • Çoyan K, Başpınar N, Bucak MN, et al. (2010): Influence of methionine and dithioerythritol on sperm motility, lipid peroxidation and antioxidant capacities during liquid storage of ram semen. Res Vet Sci, 89, 426-431.
  • Davis BK, Byrne R, Hun B (1979): Studies on the mechanism of capacitation. II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro. Biochim Biophys Acta, 558, 257-266.
  • De Paz P, Esteso MC, Alvarez M, et al. (2010): Development of extender based on soybean lecithin for its application in liquid ram semen. Theriogenology, 74, 663- 671.
  • Demircan C, Gül Z, Büyükuysal RL (2014): High glutamate attenuates S100B and LDH outputs from rat cortical slices enhanced by either oxygen-glucose deprivation or menadione. Neurochem Res, 39, 1232-1244.
  • Dorman HJD, Peltoketo A, Hiltunen R, et al. (2003): Characterization of antioxidant properties of de-odourized aqueous extracts from selected Lamiaceae herbs. Food Chem, 83, 255-262.
  • El-Kon I (2011): Testing usability of bovine serum albumin (BSA) for preservation of Egyptian Buffalo semen. Am Eurasian J Agric Environ Sci, 11, 495-502.
  • Ellman GL (1959): Tissue sulfhydryl groups. Arch Biochem Biophys, 82, 70-77.
  • Gaspar J, Rodrigues A, Laires A, et al. (1994): On the mechanisms of genotoxicity and metabolism of quercetin. Mutagenesis, 9, 445-449.
  • Gündoğan M (2009): Short term preservation of ram semen with different extenders. Kafkas Univ Vet Fak Derg, 15, 429-435.
  • Gündoğan M, Tekerli M, Uçar M, et al. (2003): Effect of diluents on motility of ram sperm during storage at 5 C. Arch Androl, 49, 69-75.
  • Gündoğan M, Yeni D, Avdatek F, et al. (2010): Influence of sperm concentration on the motility, morphology, membrane and DNA integrity along with oxidative stress parameters of ram sperm during liquid storage. Anim Reprod Sci, 122, 200-207.
  • Hodnick WF, Kung FS, Roettger WJ, et al. (1986): Inhibition of mitochondrial respiration and production of toxic oxygen radicals by flavonoids. A structure-activity study. Biochem Pharmacol, 35, 2345-2357.
  • Hossain MS, Hyeong LJ, Miah AG, et al. (2007): Effect of fatty acids bound to bovine serum albumin-V on acrosome reaction and utilization of glucose in boar spermatozoa. Reprod Med Biol, 6, 109-115.
  • Kankofer M, Kolm G, Aurich J (2005): Activity of glutathione peroxidase, superoxide dismutase and catalase and lipid peroxidation intensity in stallion semen during storage at 5 C. Theriogenology, 63, 1354-1365.
  • Matsuoka T, Imai H, Kohno H, et al. (2006): Effect of bovine serum albumin and trehalose in semen diluents for improvement of frozen-thawed ram semen. J Reprod Dev, 52, 675-683.
  • Maxwell WMC, Salamon S (1993): Liquid storage of ram semen: A review. Reprod Fertil Dev, 5, 613-638.
  • Metodiewa D, Jaiswal AK, Cenas N, et al. (1999): Quercetin may act as a cytotoxic prooxidant after its metabolic activation to semiquinone and quinoidal product. Free Radic Biol Med, 26,107-116.
  • Naijian HR, Kohram H, Zare SA, et al. (2013): Effects of various concentrations of BSA on microscopic and oxidative parameters of Mahabadi goat semen following the freeze-thaw process. Small Rum Res, 113, 371-375.
  • Ochiai M, Nagao M, Wakabayashi K, et al. (1984): Superoxide dismutase acts as an enhancing factor for quercetin mutagenesis in rat-liver cytosol by preventing its decomposition. Mutat Res, 129, 19-24.
  • Sarıözkan S, Tuncer PB, Bucak MN, et al. (2009): Influence of various antioxidants on microscopic-oxidative stress indicators and fertilizing ability of frozen-thawed bull semen. Acta Vet Brno, 78, 463-469.
  • Sarıözkan S, Türk G, Cantürk F, et al. (2013): The effect of bovine serum albumin and fetal calf serum on sperm quality, DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen. Cryobiology, 67, 1-6.
  • Toker MB Alçay S, Gökçe E, et al. (2016): Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience. Cryobiology, 72, 205- 209.
  • Uysal O, Bucak MN (2007): Effect of oxidized serum albumin, cysteine and lycopene on the quality of frozen thawed ram semen. Acta Vet Brno, 76, 383-390.
  • Uysal O, Bucak MN, Yavaş İ, et al. (2007): Effect of various antioxidants on the quality of frozen-thawed bull semen. J Anim Vet Adv, 6, 1362-1366.
  • Üstüner B, Alçay S, Nur Z, et al. (2014): Effect of egg yolk and soybean lecithin on tris-based extender in post-thaw ram semen quality and in vitro fertility. Kafkas Univ Vet Fak Derg, 20, 393-398.
  • Watjen W, Michels G, Steffan B, et al. (2005): Low concentrations of flavonoids are protective in rat H4IIE cells whereas high concentrations cause DNA damage and apoptosis. J Nutr, 135, 525-531.
  • Xia J, Ren D (2009): The BSa-induced Ca(2+) influx during sperm capacitation is CATSPER channel-dependent. Reprod Biol Endocrinol, 7, 119-127.

Ayrıntılar

Birincil Dil İngilizce
Konular Veteriner Cerrahi
Diğer ID JA57BD36YS
Bölüm Araştırma Makalesi
Yazarlar

Elif GÖKÇE
Uludağ University, Faculty of Veterinary Medicine

Selim ALÇAY
Uludağ University, Faculty of Veterinary Medicine

Zülfiye GÜL
Uludağ University, Faculty of Veterinary Medicine

Yayımlanma Tarihi 1 Aralık 2017
Yayınlandığı Sayı Yıl 2017Cilt: 64 Sayı: 4

Kaynak Göster

Bibtex @editöre mektup { auvfd525955, journal = {Ankara Üniversitesi Veteriner Fakültesi Dergisi}, issn = {1300-0861}, eissn = {1308-2817}, address = {}, publisher = {Ankara Üniversitesi}, year = {2017}, volume = {64}, number = {4}, pages = {313 - 320}, doi = {10.1501/Vetfak\_0000002815}, title = {Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C}, key = {cite}, author = {Gökçe, Elif and Alçay, Selim and Gül, Zülfiye} }
APA Gökçe, E. , Alçay, S. & Gül, Z. (2017). Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C . Ankara Üniversitesi Veteriner Fakültesi Dergisi , 64 (4) , 313-320 . DOI: 10.1501/Vetfak_0000002815
MLA Gökçe, E. , Alçay, S. , Gül, Z. "Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C" . Ankara Üniversitesi Veteriner Fakültesi Dergisi 64 (2017 ): 313-320 <http://vetjournal.ankara.edu.tr/tr/pub/issue/43284/525955>
Chicago Gökçe, E. , Alçay, S. , Gül, Z. "Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C". Ankara Üniversitesi Veteriner Fakültesi Dergisi 64 (2017 ): 313-320
RIS TY - JOUR T1 - BSA ilave edilmiş lesitin bazlı sulandırıcının koç spermasının 5 C sıcaklıkta muhafaza edilmesineolumlu etkisi AU - ElifGökçe, SelimAlçay, ZülfiyeGül Y1 - 2017 PY - 2017 N1 - doi: 10.1501/Vetfak_0000002815 DO - 10.1501/Vetfak_0000002815 T2 - Ankara Üniversitesi Veteriner Fakültesi Dergisi JF - Journal JO - JOR SP - 313 EP - 320 VL - 64 IS - 4 SN - 1300-0861-1308-2817 M3 - doi: 10.1501/Vetfak_0000002815 UR - https://doi.org/10.1501/Vetfak_0000002815 Y2 - 2023 ER -
EndNote %0 Ankara Üniversitesi Veteriner Fakültesi Dergisi Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C %A Elif Gökçe , Selim Alçay , Zülfiye Gül %T Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C %D 2017 %J Ankara Üniversitesi Veteriner Fakültesi Dergisi %P 1300-0861-1308-2817 %V 64 %N 4 %R doi: 10.1501/Vetfak_0000002815 %U 10.1501/Vetfak_0000002815
ISNAD Gökçe, Elif , Alçay, Selim , Gül, Zülfiye . "Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C". Ankara Üniversitesi Veteriner Fakültesi Dergisi 64 / 4 (Aralık 2017): 313-320 . https://doi.org/10.1501/Vetfak_0000002815
AMA Gökçe E. , Alçay S. , Gül Z. Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C. Ankara Univ Vet Fak Derg. 2017; 64(4): 313-320.
Vancouver Gökçe E. , Alçay S. , Gül Z. Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C. Ankara Üniversitesi Veteriner Fakültesi Dergisi. 2017; 64(4): 313-320.
IEEE E. Gökçe , S. Alçay ve Z. Gül , "Positive effect of BSA supplemented soybean lecithin based extender on liquid storage of ram semen at 5C", Ankara Üniversitesi Veteriner Fakültesi Dergisi, c. 64, sayı. 4, ss. 313-320, Ara. 2017, doi:10.1501/Vetfak_0000002815