Evaluation of Bull Semen Frozen with Different Antioxidants

Year 2025, Volume: 65 Issue: 1, 40 - 48, 30.06.2025

Derya Şahin İlktan Baştan , Seher Şimşek , Fırat Korkmaz İdil Şerbetçi Mustafa Numan Bucak

https://doi.org/10.46897/livestockstudies.1731736

Abstract

In this project, ejaculates (n=34) were collected from three Brown Swiss bulls using the artificial vagina method, then divided into seven equal parts and diluted to 60×10⁶ spermatozoa/mL using the following extenders: Control, Control+1mM carnosic acid (CR1), Control+3mM carnosic acid (CR3), Control+2.5mM glutamine (G2.5), Control+7.5mM glutamine (G7.5), Control+CR1+G7.5, and Control+CR3+G2.5. All diluted aliquots were frozen after 3 hours of equilibration. Thawed samples were analyzed for motility, plasma membrane and acrosome integrity (PMAI), mitochondrial membrane potential (MMP), and DNA integrity. The Control, G7.5, and G2.5 groups showed the highest total and progressive motility. The highest PMAI value was observed in the Control group, while the lowest was in the CR3 group. The PMAI values of G2.5, G7.5, and G7.5+CR1 were significantly higher than those of CR1, CR3, and CR3+G2.5. There were no statistically significant differences in MMP among the groups. The lowest DNA damage was observed in the Control group and the highest in CR3+G2.5. Control, G2.5, G7.5, and CR1+G7.5 groups had statistically similar kinematic values (VAP, VSL, VCL) and outperformed the others. Although 2.5 mM and 7.5 mM glutamine showed a synergistic interaction, they did not significantly improve spermatological parameters. Carnosic acid appeared to act as an antagonistic antioxidant.

Keywords

Bull semen , Carnosic acid , Glutamine

References

• Aitken, R. J., & Baker, M. A. (2004). Oxidative stress and male reproductive biology. Reproduction, Fertility and development, 16(5), 581-588.
• Aitken, R. J., Lambourne, S., & Gibb, Z. (2014). The John Hughes Memorial Lecture: aspects of sperm physiology—oxidative stress and the functionality of stallion spermatozoa. Journal of Equine Veterinary Science, 34(1), 17-27.
• Aksoy, A., & Yavuz, F. (2012). Büyükbaş hayvan üreticilerinin suni tohumlama yaptirma kararlarini belirleyiciler üzerine bir analiz: doğu anadolu bölgesi örneği. Atatürk Üniversitesi İktisadi ve İdari Bilimler Dergisi, 25.
• Amirat-Briand, L., Bencharif, D., Vera-Munoz, O., Ali, H. B. H., Destrumelle, S., Desherces, S., Schmidt, E., Anton, M., & Tainturier, D. (2009). Effect of glutamine on post-thaw motility of bull spermatozoa after association with LDL (low density lipoproteins) extender: preliminary results. Theriogenology, 71(8), 1209-1214.
• Ansari, M. S., Rakha, B. A., & Akhter, S. (2017). Cryopreservation of bull semen in OptiXcell® and conventional extenders: Comparison of semen quality and fertility. Animal Science Papers & Reports, 35(3).
• Arı, U., & Öztürkler, Y. (2015). Spermanın Kısa ve Uzun Süreli Saklanmasında Oksidatif Stres ve Antioksidan Kullanımı. Turkiye Klinikleri J Reprod Artif Insemin-Special Topics, 1(3), 16-21.
• Aytekin, Ö. (2011). Saha Şartlarında Gerçekleştirilen Suni Tohumlama Uygulamalarının Retrospektif Analizi. Dicle Üniversitesi Veteriner Fakültesi Dergisi(2), 44-49.
• Bottrel, M., Acha, D., Ortiz, I., Hidalgo, M., Gósalvez, J., Camisão, J., & Dorado, J. (2018). Cryoprotective effect of glutamine, taurine, and proline on post-thaw semen quality and DNA integrity of donkey spermatozoa. Animal reproduction science, 189, 128-135.
• Bousseau, S., Brillard, J., Marquant-Le Guienne, B., Guerin, B., Camus, A., & Lechat, M. (1998). Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology, 50(5), 699-706.
• Bucak, M. N., Sarıözkan, S., Tuncer, P. B., Ulutaş, P. A., & Akçadağ, H. İ. (2009a). Effect of antioxidants on microscopic semen parameters, lipid peroxidation and antioxidant activities in Angora goat semen following cryopreservation. Small Ruminant Research, 81(2-3), 90-95.
• Bucak, M. N., Tuncer, P. B., Sarıözkan, S., & Ulutaş, P. A. (2009b). Comparison of the effects of glutamine and an amino acid solution on post-thawed ram sperm parameters, lipid peroxidation and anti-oxidant activities. Small Ruminant Research, 81(1), 13-17.
• Crespilho, A., Sá Filho, M., Dell'Aqua Jr, J., Nichi, M., Monteiro, G., Avanzi, B., Martins, A., & Papa, F. O. (2012). Comparison of in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or new lecithin based extenders. Livestock Science, 149(1-2), 1-6.
• Daghigh-Kia, H., Olfati-Karaji, R., Hoseinkhani, A., & Ashrafi, I. (2014). Effect of rosemary (Rosmarinus officinalis) extracts and glutathione antioxidants on bull semen quality after cryopreservation. Spanish Journal of Agricultural Research, 12(1), 98-105.
• Gavin-Plagne, L., Commin, L., Bruyere, P., Buff, S., & Joly, T. (2019). Comparison between an animal-derived product medium and a chemically defined medium for ram sperm cryopreservation. Biopreservation and Biobanking, 17(1), 11-17.
• Gungor, S., Inanc, M. E., Ozturk, C., Korkmaz, F., Bastan, I., Cil, B., & Kastelic, J. P. (2019). Gallic and carnosic acids improve quality of frozen‐thawed ram spermatozoa. Andrologia, 51(10), e13393.
• Gökçen H (2020). Süt sığırcılığında sürü yönetim ilkeleri. Bölüm 6. Erişim adresi: http://online.fliphtml5.com/kxfdj/jyvx/?fbclid=IwAR07OJ4vJAWN8sgp1vG4R4S63P7XQPvrlvhMZmDfEQcOe6K80APELSe6lO0.
• Gürler, H., Malama, E., Heppelmann, M., Calisici, O., Leiding, C., Kastelic, J., & Bollwein, H. (2016). Effects of cryopreservation on sperm viability, synthesis of reactive oxygen species, and DNA damage of bovine sperm. Theriogenology, 86(2), 562-571.
• HAYGEM. Hayvancılık Genel Müdürlüğü (2020). Erişim adresi: https://www.tarimorman.gov.tr/HAYGEM/Duyuru/135/Suni-Tohumlama-Tabii-Tohumlama-Sperma-Ovum-Ve-Embriyonun-Ithalati-Uretimi-Suni-Tohumlama-Uygulama-Talimati.
• Khlifaoui, M., Battut, I., Bruyas, J. F., Chatagnon, G., Trimeche, A., & Tainturier, D. (2005). Effects of glutamine on post-thaw motility of stallion spermatozoa: an approach of the mechanism of action at spermatozoa level. Theriogenology, 63(1), 138-149.
• Knowlton, S. M., Sadasivam, M., & Tasoglu, S. (2015). Microfluidics for sperm research. Trends in biotechnology, 33(4), 221-229.
• Korkmaz, F., & Çil, B. (2020). The use of flow cytometry in semen quality analyses Akış Sitometrisinin (Flow Cytometry) Sperma Kalite Analizlerinde Kullanımı. Ataturk Universitesi Veteriner Bilimleri Dergisi, 15(1).
• Layek, S., Mohanty, T., Kumaresan, A., & Parks, J. (2016). Cryopreservation of bull semen: Evolution from egg yolk based to soybean based extenders. Animal reproduction science, 172, 1-9.
• Luconi, M., Forti, G., & Baldi, E. (2006). Pathophysiology of sperm motility. Front Biosci, 11(1), 1433.
• Luño, V., Gil, L., Olaciregui, M., González, N., Jerez, R. A., & De Blas, I. (2014). Rosmarinic acid improves function and in vitro fertilising ability of boar sperm after cryopreservation. Cryobiology, 69(1), 157-162.
• Malo, C., Gil, L., Gonzalez, N., Martínez, F., Cano, R., De Blas, I., & Espinosa, E. (2010). Anti-oxidant supplementation improves boar sperm characteristics and fertility after cryopreservation: comparison between cysteine and rosemary (Rosmarinus officinalis). Cryobiology, 61(1), 142-147.
• Motlagh, M. K., Sharafi, M., Zhandi, M., Mohammadi-Sangcheshmeh, A., Shakeri, M., Soleimani, M., & Zeinoaldini, S. (2014). Antioxidant effect of rosemary (Rosmarinus officinalis L.) extract in soybean lecithin-based semen extender following freeze–thawing process of ram sperm. Cryobiology, 69(2), 217-222.
• Murphy, E. M., O’Meara, C., Eivers, B., Lonergan, P., & Fair, S. (2018). Comparison of plant-and egg yolk-based semen diluents on in vitro sperm kinematics and in vivo fertility of frozen-thawed bull semen. Animal reproduction science, 191, 70-75.
• Narud, B., Klinkenberg, G., Khezri, A., Zeremichael, T. T., Stenseth, E.-B., Nordborg, A., Haukaas, T. H., Morrell, J. M., Heringstad, B., & Myromslien, F. D. (2020). Differences in sperm functionality and intracellular metabolites in Norwegian Red bulls of contrasting fertility. Theriogenology, 157, 24-32.
• O'Flaherty, C., de Lamirande, E., & Gagnon, C. (2006). Positive role of reactive oxygen species in mammalian sperm capacitation: triggering and modulation of phosphorylation events. Free Radical Biology and Medicine, 41(4), 528-540.
• Önal, A., & Özder, M. (2008). Edirne ili damızlık sığır yetiştiricileri birliğine üye işletmelerin yapısal özellikleri. Tekirdağ Ziraat Fakültesi Dergisi, 5(2), 197-203.
• Perteghella, S., Gaviraghi, A., Cenadelli, S., Bornaghi, V., Galli, A., Crivelli, B., Vigani, B., Vigo, D., Chlapanidas, T., & Faustini, M. (2017). Alginate encapsulation preserves the quality and fertilizing ability of Mediterranean Italian water buffalo (Bubalus bubalis) and Holstein Friesian (Bos taurus) spermatozoa after cryopreservation. Journal of Veterinary Science, 18(1), 81-88.
• Pothmann, H., Nechanitzky, K., Sturmlechner, F., & Drillich, M. (2014). Consultancy to dairy farmers relating to animal health and herd health management on small-and medium-sized farms. Journal of dairy science, 97(2), 851-860.
• Roelofs, J., Lopez-Gatius, F., Hunter, R., Van Eerdenburg, F., & Hanzen, C. (2010). When is a cow in estrus? Clinical and practical aspects. Theriogenology, 74(3), 327-344.
• TAGEM. TARIMSAL ARAŞTIRMALAR VE POLİTİKALAR GENEL MÜDÜRLÜĞÜ (2019). Sektör politika belgeleri 2018-2022. Erişim adresi: https://www.tarimorman.gov.tr/TAGEM/Duyuru/145/Sektor-Politika-Belgeleri-2018-2022. Erişim tarihi: 29.10.2019.
• Thun, R., Hurtado, M., & Janett, F. (2002). Comparison of Biociphos-Plus® and TRIS-egg yolk extender for cryopreservation of bull semen. Theriogenology, 57(3), 1087-1094.
• Tuncer, P. B., Sarıözkan, S., Bucak, M. N., Ulutaş, P. A., Akalın, P. P., Büyükleblebici, S., & Canturk, F. (2011). Effect of glutamine and sugars after bull spermatozoa cryopreservation. Theriogenology, 75(8), 1459-1465.
• Tüzemen, N. (2015). Kastamonu ilinde sığır yetiştiriciliğinin durumu, sorunları ve çözüm önerileri. Kastamonu University Journal of Engineering and Sciences, 1(2), 33-51.
• Ugur, M. R., Saber Abdelrahman, A., Evans, H. C., Gilmore, A. A., Hitit, M., Arifiantini, R. I., Purwantara, B., Kaya, A., & Memili, E. (2019). Advances in cryopreservation of bull sperm. Frontiers in veterinary science, 6, 268.
• Wang, S., Sun, M., Wang, N., Yang, K., Guo, H., Wang, J., Zhang, Y., Yue, S., & Zhou, J. (2018). Effects of L-glutamine on boar sperm quality during liquid storage at 17 C. Animal reproduction science, 191, 76-84.
• Yener, A. (2017). Konya ilinde süt sığırcılığı yapan aile işletmelerinde yeniliklerin benimsenmesi ve yayılmasına etki eden faktörler. Yeni, D., & Avdatek, F. (2017). Effect of Carnosic Acid On The Short Term Storage Anatolian Buffalo Epididymal Sperm. Kocatepe Veterinary Journal, 10(3), 187-195.
• Yeni, D., İnanç, M. E., Avdatek, F., Tuncer, P. B., Türkmen, R., & Taşdemir, U. (2018). Supplementation of rosmarinic acid has reduced oxidative stress on bull spermatozoa following the freeze thawing process. CryoLetters, 39(2), 156-165.
• Zanganeh, Z., Zhandi, M., Zare-Shahneh, A., Najafi, A., Nabi, M. M., & Mohammadi-Sangcheshmeh, A. (2013). Does rosemary aqueous extract improve buck semen cryopreservation? Small Ruminant Research, 114(1), 120-125.