Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol

Muhammed Enes İnanç; Olgun Uysal; Ayhan Ata

doi:10.1501/Vetfak_0000002845

Letter to Editor

Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol

Year 2018, Volume: 65 Issue: 2, 187 - 192, 01.06.2018

Muhammed Enes İnanç Olgun Uysal Ayhan Ata

https://doi.org/10.1501/Vetfak_0000002845

Cited By: 11

Abstract

The experiment was conducted to determine the in vitro results of 7-dehydrocholesterol loaded cyclodextrin compound (7-DCLC) addition to tris extenders for freezing ram semen. Three Akkaraman rams, local species in Turkey, were used. Semen samples were extended with tris containing 0 mg (control), 1.5 mg, 2.5 mg and 3.5 mg/120x106 7-DCLC and they were equilibrated to 4°C and frozen in mini straws then stored in liquid nitrogen. Straws were thawed at 37°C for 30 s in water bath for spermatological examination. Statistically significant differences were observed between 7-DCLC and control groups in terms of acrosome integrity, CASA motility, HOS test and viability after thawing (P <0.001). In all these parameters, control group had the lowest percentages. There was no statistically significant difference between groups in terms of DNA fragmentation, abnormal spermatozoa ratio, mitochondrial activation, lipid peroxidation and apoptosis level (P> 0.05). In conclusion, it can be said that adding 7-DCLC to Akkaraman ram semen improved cell membrane cryosurvival. Besides, 7-DCLC can be used in tris extender instead of cholesterol loaded cyclodextrin (CLC) compounds for sperm cryopreservation

Keywords

Cholesterol loaded cyclodextrin, freezing-thawing, ram sperm, 7-dehydrocholesterol loaded cyclodextrin

References

Aisen EG, Medina VH, Venturino A (2002): Cryopreservation and post-thwed fertility of ram semen frozen Theriogenology, 57, 1801-1808. trehalose concentrations.
Amorim EAM, Graham JK, Spizziri B, et al. (2009): Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm. Cryobiology, 58, 210-214.
Award MM (2011): Effects of sub-optimal glycerol concentration and cholesterol-loaded cyclodextrin in a Tris-based diluent on cryopreserved ram sperm longevity and acrosomal integrity. Small Rumin Res, 100, 164-168.
Barrera-Compean MH, Purdy PH, Dzakuma JM et al. (2005): Cholesterol-loaded cyclodextrin improves post- thaw goat sperm motility. J Anim Sci, 83 (Suppl. 1), abstract T91.
Başpinar N, Çoyan K, Bucak MN, et al. (2011): Effects of dithioerythritol on ram semen after the freeze–thawing process. Cryobiology, 63, 152-156.
Bucak MN, Tekin N (2007): Kriyoprotektanlar ve gamet hücrelerinin dondurulmasında kryoprotektif etki. Ankara Üniv Vet Fak Derg, 54, 67-72.
Bucak MN, Keskin N, Taşpınar M, et al. (2013): Raffinose and hypotaurine improve the post-thawed Merino ram. Cryobiology, 67, 34-39.
Bucak MN, Ataman MB, Başpınar N, et al. (2015): Lycopene and resveratrol improve post-thaw bull sperm parameters: Sperm motility, mitochondrial activity and DNA integrity. Andrologia, 47, 545-552.
Comercio EA, Monachesi E, Loza ME, et al. (2013): Hypo-osmotic test in cat spermatozoa. Andrologia, 45, 310- 314.
Conell MO, McClure N, Lewis SEM (2002): The effect of cryopreservation on sperm morphology, motility and mitochondrial function. Human Reprod, 17, 704-709.
Çoyan K, Başpınar N, Bucak MN, et al. (2011): Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and biochemical parameters. Cryobiology, 63, 1-6.
Garner DL, Hafez ESE (1993): Reproduction in Farm Animals, Spermatozoa and seminal plasma. Lea & Febier, Philadelphia.
Garner DL, Johnson LA (1995): Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod, 53, 276-284.
Graaf De SP, Evans G, Gillan L, et al. (2007): The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and nonsorted ram spermatozoa. Theriogenology, 67, 217-227.
Holt WV (2000): Basic aspects of frozen storage of semen. Anim Reprod Sci, 62, 3-22.
Inoue K, Kubota S, Seyama Y (1999): Cholestanol induces apoptosis of cerebellar neuronal cells, biochemical and biophysical. Biochemical and Biophys Res Commun, 256, 198-203.
Jung L, Su S, Kye K, et al. (2008): Effect of cholesterol supplementation in freezing medium on the survival and integrity of human sperm after cryopreservation. Korean J Reprod Med, 35, 203-212.
Katanbegzadeh H, Farid B, Mohammadreza T (2014): Cryoprotectant-free freezing of the goat epididymal sperm. Cryoletters 35, 293-298.
Lopez-Revuelta A, Sanchez-Gallego JI, Garcia-Montero AC, et al. (2007): Membrane cholesterol in the regulation of aminophospholipid asymmetry and phagocytosis in oxidized erythrocytes. Free Radical Bio Med, 42, 1106-1118.
Mandal DK, Nagpaul PK, Gupta AK (2003): Motion characteristics of murrah buffalo bull spermatozoa in various seasons and its relationship with functional integrity of the plasmalemma. Theriogenology, 60, 349-358.
Moce E, Purdy PH, Graham JK (2010): Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival. Anim Reprod Sci, 118, 236-247.
Mohan R, Atreja SK (2014): Soya Milk Tris-based phytoextender reduces apoptosis in cryopreserved buffalo (Bubalus Bubalis) spermatozoa. Reprod Dom Anim, 49, 797-805.
Moraes EA, Graham JK, Torres CA, et al. (2010): Delivering cholesterol or cholestanol to bull sperm membranes improves cryosurvival. Anim Reprod Sci, 118, 148-54.
Nagy S, Jansen J, Topper EK, et al. (2003): 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, 68, 1828-1835.
Naseer Z, Ahmad E, Aksoy M, et al. (2015): Protective effect of cholesterol-loaded cyclodextrin pretreatment against hydrogen peroxide induced oxidative damage in ram sperm. Cryobiology, 71, 18-23.
Neild D, Chaves G, Flores M, et al. (2003): Hypo-osmotic test in equine characteristics and membrane integrity of live spermatozoa. Theriogenology, 51, 721-727.
Novotna B, Topinka J, Solansky I, et al. (2007): Impact of air pollution and genotype variability on DNA damage in Prague policemen. Toxicol Lett, 172, 37-47.
Nur Z, Zik B, Üstüner B, et al. (2010): Effects of different cryoprotective agents on ram sperm morphology and DNA integrity. Theriogenology, 73, 1267-1275.
Oliveira RR, Rates DM, Pugliesi G, et al. (2014): Use of cholesterol-loaded cryopreservation improves sperm viability but results in low fertility in mares. Reprod Dom Anim, 49, 845-850. semen
Ostling O, Johanson KJ (1984): Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun, 123, 291-298.
Parks JE, Lynch DV (1992): Lipid composition and thermotropic phase behaviour of boar, bull, stallion and rooster sperm membranes. Cryobiology, 29, 255-266.
Pena FJ, Johannisson A, Wallgren M, et al. (2003): Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: New method of evaluating sperm membrane integrity. Theriogenology, 60, 677-689.
Purdy PH, Graham JK (2004): Effect of cholesterol- loaded cyclodextrin on the cryosurvival of bull sperm. Cryobiology, 48, 36-45.
Purdy PH, Moce E, Stobart R, et al. (2010): The fertility of ram sperm held for 24h at 5 degrees C prior to cryopreservation. Anim Reprod Sci, 118, 231-235.
Sarıözkan S, Bucak MN, Tuncer PB, et al. (2014): Influence of various antioxidants added to TCM-199 on post-thaw bovine sperm parameters, DNA integrity and fertilizing ability. Cryobiology, 68, 129-133.
Silva ECB, Cajueiro JFP, Silva SV, et al. (2012): Effect of antioxidants resveratrol and quercetin on in vitro evaluation of frozen ram sperm. Theriogenology, 77, 1722- 1726.
Silvamaia M, Bicudo SD, Azevedo HC, et al. (2009): Motility and viability of ram sperm cryopreserved in a Tris- egg yolk extender supplemented with anti-oxidants. Small Rumin Res, 85, 85-90.
Spizziri BE, Fox MH, Bruemmer JE, et al. (2010): Cholesterol-loaded cyclodextrins and fertility potential of stallion spermatozoa. Anim Reprod Sci, 118, 255-264.
Stanger JD, Long V, Yovich JL (2010): Hypo-osmotic swelling test identifies individual spermatozoa with minimal DNA fragmentation. Reprod Bio Med, 21, 474-484.
Tırpan MB, Tekin N (2015): Effect of boron (sodium pentaborate), added instead of tris components, on freezing and post-thaw quality of Angora buck semen. Ankara Univ Vet Fak Derg, 62, 295-302.
Tomas A, Blanch E, Fazeli A, et al. (2013): Effect of a pre- freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics. Reprod Fertil Dev, 25, 935- 946.
Tuncer PB, Bucak MN, Büyükleblebici S, et al. (2010): The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen. Cryobiology, 61, 303-307.
Tuncer PB, Büyükleblebici S, Eken A, et al. (2014): Comparison of cryoprotective effects of lycopene and cysteamine in different cryoprotectants on bull semen and fertility results. Reprod Dom Anim, 49, 746-752.
Uysal O, Bucak MN (2009): Koç spermalarının dondurulmasında farklı trehaloz konsantrasyonu ve soğutma oranlarının rolü. Ankara Univ Vet Fak Derg, 56, 99-103.
Vanderhost G, Maree L (2009): SpermBlue®: A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis. Biotech Histochem, 84, 209-308.
Watson PF (2000): The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci, 60, 481-492.
White IG (1993): Lipids and calcium uptake of sperm in relation to cold shock and preservation. Reprod Fertil Dev, 5, 639-658.

Akkaraman koç spermasının 7-dehidrokolesterol ile dondurulması ve değerlendirilmesi

Year 2018, Volume: 65 Issue: 2, 187 - 192, 01.06.2018

Muhammed Enes İnanç Olgun Uysal Ayhan Ata

https://doi.org/10.1501/Vetfak_0000002845

Cited By: 11

Abstract

Bu çalışma, tris sulandırıcısına 7-dehidrokolesterol ile doyurulmuş siklodekstrin bileşimi (7-DCLC) eklenerek dondurmaçözdürme sonrası in vitro sonuçlarını değerlendirmek üzere yapıldı. Çalışmada yerli bir ırk olarak üç adet Akkaraman koç kullanıldı. Sperma örnekleri 0 mg (kontrol), 1.5, 2.5 ve 3.5 mg/120x106 7-DCLC içeren tris sulandırıcısı ile sulandırıldı, 4 C⁰’de alışıma bırakıldı ve mini payetlerde dondurularak sıvı azotta saklandı. Daha sonra payetler spermatolojik analizler için 37 C⁰’de 30 saniyede su banyosunda çözdürüldü. Çözüm sonu, 7-DCLC grupları ve kontrol grubu arasında CASA motilitesi, canlılık, akrozomal bütünlük ve HOS test yönünden istatiksel olarak farklılık önemli bulundu (P<0.001). Bütün bu parametrelerde kontrol gruplarında en düşük değerler tespit edildi. Anormal spermatozoa oranı, lipit peroksidasyon seviyesi, DNA hasarı, mitokondriyal aktivasyon, lipit peroksidasyon ve apoptozis seviyesi yönünden istatiksel açıdan bir farklılık bulunamadı (P>0.05). Çalışma sonucunda, 7-DCLC’nin Akkaraman koç spermasına eklenmesinin hücre membranlarını soğuktan koruduğu söylenebilir. Ayrıca, spermanın dondurulması için tris sulandırıcılarında kolesterol ile doyurulmuş siklodekstrin (CLC) bileşimi yerine 7-DCLC’nin kullanılabileceği tespit edildi

Keywords

Çözdürme, dondurma, koç sperması, kolesterol ile doyurulmuş siklodekstrin, 7-dehidrokolesterol ile doyurulmuş siklodekstrin

References

Aisen EG, Medina VH, Venturino A (2002): Cryopreservation and post-thwed fertility of ram semen frozen Theriogenology, 57, 1801-1808. trehalose concentrations.
Amorim EAM, Graham JK, Spizziri B, et al. (2009): Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm. Cryobiology, 58, 210-214.
Award MM (2011): Effects of sub-optimal glycerol concentration and cholesterol-loaded cyclodextrin in a Tris-based diluent on cryopreserved ram sperm longevity and acrosomal integrity. Small Rumin Res, 100, 164-168.
Barrera-Compean MH, Purdy PH, Dzakuma JM et al. (2005): Cholesterol-loaded cyclodextrin improves post- thaw goat sperm motility. J Anim Sci, 83 (Suppl. 1), abstract T91.
Başpinar N, Çoyan K, Bucak MN, et al. (2011): Effects of dithioerythritol on ram semen after the freeze–thawing process. Cryobiology, 63, 152-156.
Bucak MN, Tekin N (2007): Kriyoprotektanlar ve gamet hücrelerinin dondurulmasında kryoprotektif etki. Ankara Üniv Vet Fak Derg, 54, 67-72.
Bucak MN, Keskin N, Taşpınar M, et al. (2013): Raffinose and hypotaurine improve the post-thawed Merino ram. Cryobiology, 67, 34-39.
Bucak MN, Ataman MB, Başpınar N, et al. (2015): Lycopene and resveratrol improve post-thaw bull sperm parameters: Sperm motility, mitochondrial activity and DNA integrity. Andrologia, 47, 545-552.
Comercio EA, Monachesi E, Loza ME, et al. (2013): Hypo-osmotic test in cat spermatozoa. Andrologia, 45, 310- 314.
Conell MO, McClure N, Lewis SEM (2002): The effect of cryopreservation on sperm morphology, motility and mitochondrial function. Human Reprod, 17, 704-709.
Çoyan K, Başpınar N, Bucak MN, et al. (2011): Effects of cysteine and ergothioneine on post-thawed Merino ram sperm and biochemical parameters. Cryobiology, 63, 1-6.
Garner DL, Hafez ESE (1993): Reproduction in Farm Animals, Spermatozoa and seminal plasma. Lea & Febier, Philadelphia.
Garner DL, Johnson LA (1995): Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod, 53, 276-284.
Graaf De SP, Evans G, Gillan L, et al. (2007): The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and nonsorted ram spermatozoa. Theriogenology, 67, 217-227.
Holt WV (2000): Basic aspects of frozen storage of semen. Anim Reprod Sci, 62, 3-22.
Inoue K, Kubota S, Seyama Y (1999): Cholestanol induces apoptosis of cerebellar neuronal cells, biochemical and biophysical. Biochemical and Biophys Res Commun, 256, 198-203.
Jung L, Su S, Kye K, et al. (2008): Effect of cholesterol supplementation in freezing medium on the survival and integrity of human sperm after cryopreservation. Korean J Reprod Med, 35, 203-212.
Katanbegzadeh H, Farid B, Mohammadreza T (2014): Cryoprotectant-free freezing of the goat epididymal sperm. Cryoletters 35, 293-298.
Lopez-Revuelta A, Sanchez-Gallego JI, Garcia-Montero AC, et al. (2007): Membrane cholesterol in the regulation of aminophospholipid asymmetry and phagocytosis in oxidized erythrocytes. Free Radical Bio Med, 42, 1106-1118.
Mandal DK, Nagpaul PK, Gupta AK (2003): Motion characteristics of murrah buffalo bull spermatozoa in various seasons and its relationship with functional integrity of the plasmalemma. Theriogenology, 60, 349-358.
Moce E, Purdy PH, Graham JK (2010): Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival. Anim Reprod Sci, 118, 236-247.
Mohan R, Atreja SK (2014): Soya Milk Tris-based phytoextender reduces apoptosis in cryopreserved buffalo (Bubalus Bubalis) spermatozoa. Reprod Dom Anim, 49, 797-805.
Moraes EA, Graham JK, Torres CA, et al. (2010): Delivering cholesterol or cholestanol to bull sperm membranes improves cryosurvival. Anim Reprod Sci, 118, 148-54.
Nagy S, Jansen J, Topper EK, et al. (2003): 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, 68, 1828-1835.
Naseer Z, Ahmad E, Aksoy M, et al. (2015): Protective effect of cholesterol-loaded cyclodextrin pretreatment against hydrogen peroxide induced oxidative damage in ram sperm. Cryobiology, 71, 18-23.
Neild D, Chaves G, Flores M, et al. (2003): Hypo-osmotic test in equine characteristics and membrane integrity of live spermatozoa. Theriogenology, 51, 721-727.
Novotna B, Topinka J, Solansky I, et al. (2007): Impact of air pollution and genotype variability on DNA damage in Prague policemen. Toxicol Lett, 172, 37-47.
Nur Z, Zik B, Üstüner B, et al. (2010): Effects of different cryoprotective agents on ram sperm morphology and DNA integrity. Theriogenology, 73, 1267-1275.
Oliveira RR, Rates DM, Pugliesi G, et al. (2014): Use of cholesterol-loaded cryopreservation improves sperm viability but results in low fertility in mares. Reprod Dom Anim, 49, 845-850. semen
Ostling O, Johanson KJ (1984): Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun, 123, 291-298.
Parks JE, Lynch DV (1992): Lipid composition and thermotropic phase behaviour of boar, bull, stallion and rooster sperm membranes. Cryobiology, 29, 255-266.
Pena FJ, Johannisson A, Wallgren M, et al. (2003): Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: New method of evaluating sperm membrane integrity. Theriogenology, 60, 677-689.
Purdy PH, Graham JK (2004): Effect of cholesterol- loaded cyclodextrin on the cryosurvival of bull sperm. Cryobiology, 48, 36-45.
Purdy PH, Moce E, Stobart R, et al. (2010): The fertility of ram sperm held for 24h at 5 degrees C prior to cryopreservation. Anim Reprod Sci, 118, 231-235.
Sarıözkan S, Bucak MN, Tuncer PB, et al. (2014): Influence of various antioxidants added to TCM-199 on post-thaw bovine sperm parameters, DNA integrity and fertilizing ability. Cryobiology, 68, 129-133.
Silva ECB, Cajueiro JFP, Silva SV, et al. (2012): Effect of antioxidants resveratrol and quercetin on in vitro evaluation of frozen ram sperm. Theriogenology, 77, 1722- 1726.
Silvamaia M, Bicudo SD, Azevedo HC, et al. (2009): Motility and viability of ram sperm cryopreserved in a Tris- egg yolk extender supplemented with anti-oxidants. Small Rumin Res, 85, 85-90.
Spizziri BE, Fox MH, Bruemmer JE, et al. (2010): Cholesterol-loaded cyclodextrins and fertility potential of stallion spermatozoa. Anim Reprod Sci, 118, 255-264.
Stanger JD, Long V, Yovich JL (2010): Hypo-osmotic swelling test identifies individual spermatozoa with minimal DNA fragmentation. Reprod Bio Med, 21, 474-484.
Tırpan MB, Tekin N (2015): Effect of boron (sodium pentaborate), added instead of tris components, on freezing and post-thaw quality of Angora buck semen. Ankara Univ Vet Fak Derg, 62, 295-302.
Tomas A, Blanch E, Fazeli A, et al. (2013): Effect of a pre- freezing treatment with cholesterol-loaded cyclodextrins on boar sperm longevity, capacitation dynamics, ability to adhere to porcine oviductal epithelial cells in vitro and DNA fragmentation dynamics. Reprod Fertil Dev, 25, 935- 946.
Tuncer PB, Bucak MN, Büyükleblebici S, et al. (2010): The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen. Cryobiology, 61, 303-307.
Tuncer PB, Büyükleblebici S, Eken A, et al. (2014): Comparison of cryoprotective effects of lycopene and cysteamine in different cryoprotectants on bull semen and fertility results. Reprod Dom Anim, 49, 746-752.
Uysal O, Bucak MN (2009): Koç spermalarının dondurulmasında farklı trehaloz konsantrasyonu ve soğutma oranlarının rolü. Ankara Univ Vet Fak Derg, 56, 99-103.
Vanderhost G, Maree L (2009): SpermBlue®: A new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis. Biotech Histochem, 84, 209-308.
Watson PF (2000): The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci, 60, 481-492.
White IG (1993): Lipids and calcium uptake of sperm in relation to cold shock and preservation. Reprod Fertil Dev, 5, 639-658.

There are 47 citations in total.

Details

Primary Language	English
Subjects	Veterinary Surgery
Other ID	JA39UB67DH
Journal Section	Research Article
Authors	Muhammed Enes İnanç Olgun Uysal Ayhan Ata
Publication Date	June 1, 2018
Published in Issue	Year 2018Volume: 65 Issue: 2

Cite

APA	İnanç, M. E., Uysal, O., & Ata, A. (2018). Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol. Ankara Üniversitesi Veteriner Fakültesi Dergisi, 65(2), 187-192. https://doi.org/10.1501/Vetfak_0000002845
AMA	İnanç ME, Uysal O, Ata A. Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol. Ankara Univ Vet Fak Derg. June 2018;65(2):187-192. doi:10.1501/Vetfak_0000002845
Chicago	İnanç, Muhammed Enes, Olgun Uysal, and Ayhan Ata. “Cryopreservation and Evaluation of Akkaraman Ram Semen With 7-Dehydrocholesterol”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 65, no. 2 (June 2018): 187-92. https://doi.org/10.1501/Vetfak_0000002845.
EndNote	İnanç ME, Uysal O, Ata A (June 1, 2018) Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol. Ankara Üniversitesi Veteriner Fakültesi Dergisi 65 2 187–192.
IEEE	M. E. İnanç, O. Uysal, and A. Ata, “Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol”, Ankara Univ Vet Fak Derg, vol. 65, no. 2, pp. 187–192, 2018, doi: 10.1501/Vetfak_0000002845.
ISNAD	İnanç, Muhammed Enes et al. “Cryopreservation and Evaluation of Akkaraman Ram Semen With 7-Dehydrocholesterol”. Ankara Üniversitesi Veteriner Fakültesi Dergisi 65/2 (June 2018), 187-192. https://doi.org/10.1501/Vetfak_0000002845.
JAMA	İnanç ME, Uysal O, Ata A. Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol. Ankara Univ Vet Fak Derg. 2018;65:187–192.
MLA	İnanç, Muhammed Enes et al. “Cryopreservation and Evaluation of Akkaraman Ram Semen With 7-Dehydrocholesterol”. Ankara Üniversitesi Veteriner Fakültesi Dergisi, vol. 65, no. 2, 2018, pp. 187-92, doi:10.1501/Vetfak_0000002845.
Vancouver	İnanç ME, Uysal O, Ata A. Cryopreservation and evaluation of Akkaraman ram semen with 7-dehydrocholesterol. Ankara Univ Vet Fak Derg. 2018;65(2):187-92.