agronaukaАграрная наука Евро-Северо-ВостокаAgricultural Science Euro-North-East2072-90812500-1396FARC North-East10.30766/2072-9081.2025.26.2.223-238agronauka-1983Research ArticleОБЗОРНЫЕ СТАТЬИREVIEWПрименение разбавителей спермы быков: современное состояние (обзор)Use of bull sperm extenders: current status (review)https://orcid.org/0000-0001-5324-4090СтариковаД. А.StarikovaD. A.

Старикова Дарья Андреевна, кандидат биол. наук, научный сотрудник

д. 55а, Московское шоссе, г. Пушкин, г. Санкт-Петербург, 196601

Daria A. Starikova, PhD in Biological Science, researcher

Moskovskoe Shosse, 55a, Pushkin, St. Petersburg, 196601

live8avis@mail.ruhttps://orcid.org/0000-0002-0963-905XБарковаО. Ю.BarkovaO. Yu.

Баркова Ольга Юрьевна, кандидат биол. наук, старший научный сотрудник

д. 55а, Московское шоссе, г. Пушкин, г. Санкт-Петербург, 196601

Olga Yu. Barkova, PhD in Biological Science, senior researcher

Moskovskoe Shosse, 55a, Pushkin, St. Petersburg, 196601

spbvniigen@mail.ruhttps://orcid.org/0000-0001-7229-5766ЧистяковаИ. В.ChistyakovaI. V.

Чистякова Ирэна Валерьевна, кандидат биол. наук, научный сотрудник

д. 55а, Московское шоссе, г. Пушкин, г. Санкт-Петербург, 196601

Irena V. Chistyakova, PhD in Biological Science, researcher

Moskovskoe Shosse, 55a, Pushkin, St. Petersburg, 196601

spbvniigen@mail.ruhttps://orcid.org/0000-0002-4634-7515ПлешановН. В.PleshanovN. V.

Плешанов Николай Вячеславович, научный сотрудник

д. 55а, Московское шоссе, г. Пушкин, г. Санкт-Петербург, 196601

Nikolay V. Pleshanov, researcher

Moskovskoe Shosse, 55a, Pushkin, St. Petersburg, 196601

spbvniigen@mail.ruhttps://orcid.org/0009-0007-6039-6958ГабоваА. В.GabovaA. V.

Габова Алина Валерьевна, младший научный сотрудник

д. 55а, Московское шоссе, г. Пушкин, г. Санкт-Петербург, 196601

Alina V. Gabova, junior researcher

Moskovskoe Shosse, 55a, Pushkin, St. Petersburg, 196601

spbvniigen@mail.ruВсероссийский научно-исследовательский институт генетики и разведения сельскохозяйственных животных – филиал ФГБНУ «Федеральный исследовательский центр животноводства – ВИЖ имени академика Л. К. Эрнста»РоссияRussian Research Institute of Farm Animal Genetics and Breeding – Branch of the L. K. Ernst Federal Research Center for Animal HusbandryRussian Federation202525042025262223–238Copyright © Старикова Д.А., Баркова О.Ю., Чистякова И.В., Плешанов Н.В., Габова А.В., 20252025Старикова Д.А., Баркова О.Ю., Чистякова И.В., Плешанов Н.В., Габова А.В.Starikova D.A., Barkova O.Y., Chistyakova I.V., Pleshanov N.V., Gabova A.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.agronauka-sv.ru/jour/article/view/1983

В последние годы вспомогательные репродуктивные технологии в животноводстве включают искусственное оплодотворение, нуждающееся в высококачественной криоконсервированной сперме быков. Качественная заморозка зависит от оптимально подобранных параметров разбавителя спермы. Цель данного обзора – распространение знаний об использовании различных типов разбавителей и добавок к ним при криоконсервации спермы быков для улучшения её качества. В данной статье приведен обзор 64 научных работ отечественных и зарубежных исследователей по моделированию разбавителей для обеспечения максимального выхода качественной спермы после разморозки. Разработки создания оптимального разбавителя спермы для защиты сперматозоидов и сохранения качественной спермы от криоповреждения в течение длительного времени, которая может сохранять свои свойства в условиях жидкого азота продолжительное время, ведутся с середины ХХ века. В связи с этим для разбавления спермы быков перед криоконсервацией применяются как классические среды с известным составом (ЛЖГ, Tris, MOPS и др.), так и коммерческие среды (Andromed, BullXcell, OptiXcell и др.). Использование разнообразных вариантов разбавителей спермы быков исследователями разных стран предоставляет важную информацию о влиянии составов разбавителей на показатели качества мужских гамет, таких как подвижность, жизнеспособность, процент аномальных сперматозоидов и другие, после длительной криоконсервации. Кроме того,  в последнее время широко применяется внесение в состав разбавителей для оптимизации их свойств добавок, имеющих природное или синтетическое происхождение и, соответственно, различное воздействие на сперму и сперматозоиды. Данный обзор может быть полезен для организаций и лабораторий, занимающихся разработкой технологий создания разбавителей спермы быков.

In recent years, assisted reproductive technologies in animal production have included artificial insemination in need of high-quality cryopreserved bull sperm. High-quality freezing depends on the optimally selected parameters of the semen diluent. In this connection, the purpose of this review is to spread the knowledge about the use of various types of diluents and additives to them in the cryopreservation of bull semen to improve its quality. This article provides an overview of 64 scientific papers of domestic and foreign researchers on modeling diluents in order to ensure the maximum yield of high-quality sperm after thawing. Research into the creation of an optimal sperm diluent to ensure sperm protection and save high-quality sperm from cryoinjury during a long time, which can long preserve its properties in liquid nitrogen has been carried out since the mid-20th century. Classic media with a known composition (LPG, Tris, MOPS, etc.) and commercial media (Andromed, BullXcell, OptiXcell, etc.) are used to dilute bull sperm before cryopreservation. The use of different variants of bull sperm extenders by researchers from different countries provides important information on the effect of extender compositions on sperm quality parameters, such as motility, viability, percentage of abnormal sperm, etc., after long-term cryopreservation. Also, recently, the inclusion of additives of natural or synthetic origin in extenders, which exhibit different effects on sperm and spermatozoa, has been widely used. This review may be useful for organizations and laboratories engaged in the development of technologies for creating bull sperm extenders.

сперматозоидкриоконсервация спермывитрификациякрупный рогатый скотBos taurusspermiumsperm cryopreservationdiluents vitrificationbovineBos taurusработа выполнена за счет гранта Российского научного фонда № 22-76-10041 (https://rscf.ru/project/22-76-10041/). Авторы благодарят рецензентов за их вклад в экспертную оценку этой работы.the research was funded by the Russian Science Foundation No. 22-76-10041 (https://rscf.ru/project/22-76-10041/). The authors thank the reviewers for their contribution to the expert assessment of this workReferencesZinovieva N. A., Pozyabin S. V., Chinarov R. Yu. Assisted reproductive technologies: the history and role in the development of genetic technologies in cattle (review). Sel'skokhozyaystvennaya biologiya = Agricultural Biology. 2020;55(2):225–242. (In Russ.). DOI: https://doi.org/10.15389/agrobiology.2020.2.225rusZinovieva N. A., Pozyabin S. V., Chinarov R. Yu. Assisted reproductive technologies: the history and role in the development of genetic technologies in cattle (review). Sel'skokhozyaystvennaya biologiya = Agricultural Biology. 2020;55(2):225–242. (In Russ.). DOI: https://doi.org/10.15389/agrobiology.2020.2.225rusKorochkina E. A., Moroz A. I. The importance of sperm diluents of different types of farm animals in the process of the cryopreservation. Genetika i razvedenie zhivotnykh = Genetics and breeding of animals. 2022;(4):108–113. (In Russ.). DOI: https://doi.org/10.31043/2410-2733-2022-4-108-113Korochkina E. A., Moroz A. I. The importance of sperm diluents of different types of farm animals in the process of the cryopreservation. Genetika i razvedenie zhivotnykh = Genetics and breeding of animals. 2022;(4):108–113. (In Russ.). DOI: https://doi.org/10.31043/2410-2733-2022-4-108-113Lonergan P. Review: Historical and futuristic developments in bovine semen technology. Animal. 2018;12(s1):s4–s18. DOI: https://doi.org/10.1017/S175173111800071XLonergan P. Review: Historical and futuristic developments in bovine semen technology. Animal. 2018;12(s1):s4–s18. DOI: https://doi.org/10.1017/S175173111800071XMilovanov V. K. Reproduction biology and artificial insemination of animals: biol.-zootechnical. the monograph. Moscow, 1962. 696 p.Milovanov V. K. Reproduction biology and artificial insemination of animals: biol.-zootechnical. the monograph. Moscow, 1962. 696 p.Phillips P. H., Lardy H. A. A Yolk-Buffer Pabulum for the Preservation of Bull Semen. Journal of Dairy Science. 1940;23:399–404. DOI: https://doi.org/10.3168/jds.S0022-0302(40)95541Phillips P. H., Lardy H. A. A Yolk-Buffer Pabulum for the Preservation of Bull Semen. Journal of Dairy Science. 1940;23:399–404. DOI: https://doi.org/10.3168/jds.S0022-0302(40)95541Polge C., Smith A., Parkes A. Revival of Spermatozoa after Vitrification and Dehydration at Low Temperatures. Nature. 1949;164:666. DOI: https://doi.org/10.1038/164666a0Polge C., Smith A., Parkes A. Revival of Spermatozoa after Vitrification and Dehydration at Low Temperatures. Nature. 1949;164:666. DOI: https://doi.org/10.1038/164666a0Smith A. U., Polge C. Storage of bull spermatozoa at low temperatures. Veterinary Record. 1950;62:115–116.Smith A. U., Polge C. Storage of bull spermatozoa at low temperatures. Veterinary Record. 1950;62:115–116.Pleshakov A. V. Fertilizing ability of sperm stored for a long time in liquid nitrogen. Vestnik Altayskogo gosudarstvennogo agrarnogo universiteta = Bulletin of Altai State Agricultural University. 2004;(3):319–321. (In Russ.). URL: https://elibrary.ru/item.asp?id=17993255Pleshakov A. V. Fertilizing ability of sperm stored for a long time in liquid nitrogen. Vestnik Altayskogo gosudarstvennogo agrarnogo universiteta = Bulletin of Altai State Agricultural University. 2004;(3):319–321. (In Russ.). URL: https://elibrary.ru/item.asp?id=17993255Naryshkina E. N. Changes in the indicators of own productivity of holstein bulls depending on the season and age. Agrarnyy vestnik Urala = Agrarian Bulletin of the Urals. 2020;(S14):40–48. (In Russ.). DOI: https://doi.org/10.32417/1997-4868-2021-14-40-48Naryshkina E. N. Changes in the indicators of own productivity of holstein bulls depending on the season and age. Agrarnyy vestnik Urala = Agrarian Bulletin of the Urals. 2020;(S14):40–48. (In Russ.). DOI: https://doi.org/10.32417/1997-4868-2021-14-40-48Baust J. G., Gao D., Baust J. M. Cryopreservation: An emerging paradigm change. Organogenesis. 2009;5(3):90–96. DOI: https://doi.org/10.4161/org.5.3.10021Baust J. G., Gao D., Baust J. M. Cryopreservation: An emerging paradigm change. Organogenesis. 2009;5(3):90–96. DOI: https://doi.org/10.4161/org.5.3.10021Hezavehei M., Sharafi M., Kouchesfahani H. M., Henkel R., Agarwal A., Esmaeili V., Shahverdi A. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reproductive BioMedicine Online. 2018;37(3):327–339. DOI: https://doi.org/10.1016/j.rbmo.2018.05.012Hezavehei M., Sharafi M., Kouchesfahani H. M., Henkel R., Agarwal A., Esmaeili V., Shahverdi A. Sperm cryopreservation: A review on current molecular cryobiology and advanced approaches. Reproductive BioMedicine Online. 2018;37(3):327–339. DOI: https://doi.org/10.1016/j.rbmo.2018.05.012Ozimic S., Ban-Frangez H., Stimpfel M. Sperm Cryopreservation Today: Approaches, Efficiency, and Pitfalls. Current Issues in Molecular Biology. 2023;45(6):4716–4734. DOI: https://doi.org/10.3390/cimb45060300Ozimic S., Ban-Frangez H., Stimpfel M. Sperm Cryopreservation Today: Approaches, Efficiency, and Pitfalls. Current Issues in Molecular Biology. 2023;45(6):4716–4734. DOI: https://doi.org/10.3390/cimb45060300Hu J. H., Li Q. W., Chen Y. L., Jiang Z. L., Jia Y. H., Wang L. Q., Ou B. B. Effects of addition of vitamin B12 to the extender on post-thaw motility, acrosome morphology, and plasma membrane integrity in bull semen. The Turkish Journal of Veterinary and Animal Sciences. 2009;33(5):379–384. DOI: https://doi.org/10.3906/vet-0712-19Hu J. H., Li Q. W., Chen Y. L., Jiang Z. L., Jia Y. H., Wang L. Q., Ou B. B. Effects of addition of vitamin B12 to the extender on post-thaw motility, acrosome morphology, and plasma membrane integrity in bull semen. The Turkish Journal of Veterinary and Animal Sciences. 2009;33(5):379–384. DOI: https://doi.org/10.3906/vet-0712-19Moussa M., Martinet V., Trimeche A., Tainturier D., Anton M. Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen–thawed bull semen. Theriogenology. 2002;57(6):1695–1706. DOI: https://doi.org/10.1016/S0093-691X(02)00682-9Moussa M., Martinet V., Trimeche A., Tainturier D., Anton M. Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen–thawed bull semen. Theriogenology. 2002;57(6):1695–1706. DOI: https://doi.org/10.1016/S0093-691X(02)00682-9Arif A. A., Maulana T., Kaiin E. M., Purwantara B., Arifiantini R. I. The Quality of Frozen Semen of Limousin Bull in Various Semen Diluents. Tropical Animal Science Journal. 2022;45(3):284–290. DOI: https://doi.org/10.5398/tasj.2022.45.3.284Arif A. A., Maulana T., Kaiin E. M., Purwantara B., Arifiantini R. I. The Quality of Frozen Semen of Limousin Bull in Various Semen Diluents. Tropical Animal Science Journal. 2022;45(3):284–290. DOI: https://doi.org/10.5398/tasj.2022.45.3.284Mayer D. T., Lesley F. The factor in egg yolk affecting the resistance, storage potentialities and fertilization capacity of mammalian spermatozoa. The Journal of Animal Science. 1945;4(3):261–269. DOI: https://doi.org/10.2527/jas1945.43261xMayer D. T., Lesley F. The factor in egg yolk affecting the resistance, storage potentialities and fertilization capacity of mammalian spermatozoa. The Journal of Animal Science. 1945;4(3):261–269. DOI: https://doi.org/10.2527/jas1945.43261xBlackshaw A. W. The prevention of temperature shock of bull and ram spermatozoa. The Australian Journal of Biological Sciences.1954;7(4):573–582. DOI: https://doi.org/10.1071/BI9540573Blackshaw A. W. The prevention of temperature shock of bull and ram spermatozoa. The Australian Journal of Biological Sciences.1954;7(4):573–582. DOI: https://doi.org/10.1071/BI9540573Martin I. C. A. Effects of lecithin, egg yolk, fructose and period of storage at 5 °C on bull spermatozoa frozen to -79 °C. Journals of Reproduction and Fertility. 1963;6(3):441–449. DOI: https://doi.org/10.1530/jrf.0.0060441Martin I. C. A. Effects of lecithin, egg yolk, fructose and period of storage at 5 °C on bull spermatozoa frozen to -79 °C. Journals of Reproduction and Fertility. 1963;6(3):441–449. DOI: https://doi.org/10.1530/jrf.0.0060441Lanz R. N., Pickett B. W., Komarek R. J. Effect of lipid additives on pre and post-freeze survival of bovine spermatozoa. Journal of Dairy Science. 1965;48(12):1692–1697. DOI: https://doi.org/10.3168/jds.S0022-0302(65)88553-8Lanz R. N., Pickett B. W., Komarek R. J. Effect of lipid additives on pre and post-freeze survival of bovine spermatozoa. Journal of Dairy Science. 1965;48(12):1692–1697. DOI: https://doi.org/10.3168/jds.S0022-0302(65)88553-8Gebauer M. R., Pickett B. W., Komarek R. J., Gaunya W. S. Motility of bovine spermatozoa extended in “defined” diluents. Journal of Dairy Science. 1970;53(6):817–823. DOI: https://doi.org/10.3168/jds.S0022-0302(70)86297-XGebauer M. R., Pickett B. W., Komarek R. J., Gaunya W. S. Motility of bovine spermatozoa extended in “defined” diluents. Journal of Dairy Science. 1970;53(6):817–823. DOI: https://doi.org/10.3168/jds.S0022-0302(70)86297-XBollwein H., Fuchs I., Koess C. Interrelationship between plasma membrane integrity, mitochondrial membranę potential and DNA fragmentation in cryopreserved bovine spermatozoa. Reproduction Domestic Animal. 2008;43(2):189–195. DOI: https://doi.org/10.1111/j.1439-0531.2007.00876.xBollwein H., Fuchs I., Koess C. Interrelationship between plasma membrane integrity, mitochondrial membranę potential and DNA fragmentation in cryopreserved bovine spermatozoa. Reproduction Domestic Animal. 2008;43(2):189–195. DOI: https://doi.org/10.1111/j.1439-0531.2007.00876.xAmirat L., Tainturier D., Jeanneau L., Thorin C., Gérard O., Courtens J. L., Anton M. Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl®, a commercial egg yolk extender. Theriogenology. 2004;61(5):895–907. DOI: https://doi.org/10.1016/S0093-691X(03)00259-0Amirat L., Tainturier D., Jeanneau L., Thorin C., Gérard O., Courtens J. L., Anton M. Bull semen in vitro fertility after cryopreservation using egg yolk LDL: a comparison with Optidyl®, a commercial egg yolk extender. Theriogenology. 2004;61(5):895–907. DOI: https://doi.org/10.1016/S0093-691X(03)00259-0Khan I. M., Cao Z., Liu H., Khan A., Rahman S. U., Khan M. Z., Sathanawongs A., Zhang Y. Impact of Cryopreservation on Spermatozoa Freeze-Thawed Traits and Relevance OMICS to Assess Sperm Cryo-Tolerance in Farm Animals. Frontiers In Veterinary Science. 2021;8:609180. DOI: https://doi.org/10.3389/fvets.2021.609180Khan I. M., Cao Z., Liu H., Khan A., Rahman S. U., Khan M. Z., Sathanawongs A., Zhang Y. Impact of Cryopreservation on Spermatozoa Freeze-Thawed Traits and Relevance OMICS to Assess Sperm Cryo-Tolerance in Farm Animals. Frontiers In Veterinary Science. 2021;8:609180. DOI: https://doi.org/10.3389/fvets.2021.609180Behnam M., Asadpour R., Topraggaleh T. R., Hamali H. Improvement of post-thaw quality and fertilizing ability of bull spermatozoa using Rho kinase inhibitor in freezing extender. Frontiers in Veterinary Science. 2023;10:1155048. DOI: https://doi.org/10.3389/fvets.2023.1155048Behnam M., Asadpour R., Topraggaleh T. R., Hamali H. Improvement of post-thaw quality and fertilizing ability of bull spermatozoa using Rho kinase inhibitor in freezing extender. Frontiers in Veterinary Science. 2023;10:1155048. DOI: https://doi.org/10.3389/fvets.2023.1155048Will M. A., Clark N. A., Swain J. E. Biological pH buffers in IVF: help or hindrance to success. The Journal of Assisted Reproduction and Genetics. 2011;28(8):711–724. DOI: https://doi.org/10.1007/s10815-011-9582-0Will M. A., Clark N. A., Swain J. E. Biological pH buffers in IVF: help or hindrance to success. The Journal of Assisted Reproduction and Genetics. 2011;28(8):711–724. DOI: https://doi.org/10.1007/s10815-011-9582-0Crespilho A. M., Filho M. F., Dell'Aqua J. A., Nichi M., Monteiro G. A., Avanzi B. R., Martins A., Papa F. O. Comparison of in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or new lecithin based extenders. Livestock Science. 2012;149(1-2):1–6. DOI: https://doi.org/10.1016/j.livsci.2012.05.011Crespilho A. M., Filho M. F., Dell'Aqua J. A., Nichi M., Monteiro G. A., Avanzi B. R., Martins A., Papa F. O. Comparison of in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or new lecithin based extenders. Livestock Science. 2012;149(1-2):1–6. DOI: https://doi.org/10.1016/j.livsci.2012.05.011Benko F., Mohammadi-Sangcheshmeh A., Ďuračka M., Lukáč N., Tvrdá E. In vitro versus cryo-induced capacitation of bovine spermatozoa, part 1: Structural, functional, and oxidative similarities and differences. PLoS One. 2022;17(10):e0276683. DOI: https://doi.org/10.1371/journal.pone.0276683Benko F., Mohammadi-Sangcheshmeh A., Ďuračka M., Lukáč N., Tvrdá E. In vitro versus cryo-induced capacitation of bovine spermatozoa, part 1: Structural, functional, and oxidative similarities and differences. PLoS One. 2022;17(10):e0276683. DOI: https://doi.org/10.1371/journal.pone.0276683Murphy E. M., O'Meara C., Eivers B., Lonergan P., Fair S. 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. 2018;191:70–75. DOI: https://doi.org/10.1016/j.anireprosci.2018.02.010Murphy E. M., O'Meara C., Eivers B., Lonergan P., Fair S. 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. 2018;191:70–75. DOI: https://doi.org/10.1016/j.anireprosci.2018.02.010Medvedev G. F., Anikevich L. P. Properties of milk media of various compositions for diluting bull semen. Izvestiya Natsional'noy akademii nauk Belarusi. Seriya agrarnykh nauk = Proceedings of the National Academy of Sciences of Belarus. Agrarian Series. 2001;(2):65–69. (In Belarus).Medvedev G. F., Anikevich L. P. Properties of milk media of various compositions for diluting bull semen. Izvestiya Natsional'noy akademii nauk Belarusi. Seriya agrarnykh nauk = Proceedings of the National Academy of Sciences of Belarus. Agrarian Series. 2001;(2):65–69. (In Belarus).Graham E. F., Crabo B. G., Brown K. I. Effect of some zwitter ion buffers on the freezing and storage of spermatozoa. Journal of Dairy Science. 1972;55(3):372–378. DOI: https://doi.org/10.3168/jds.S0022-0302(72)85499-7Graham E. F., Crabo B. G., Brown K. I. Effect of some zwitter ion buffers on the freezing and storage of spermatozoa. Journal of Dairy Science. 1972;55(3):372–378. DOI: https://doi.org/10.3168/jds.S0022-0302(72)85499-7Partyka A., Niaski W., Ochot M. Methods of assessment of cryopreserved semen. Current Frontiers in Cryobiology. Edited by Igor I. Katkov. 2012. pp. 547–574. DOI: https://doi.org/10.5772/33565Partyka A., Niaski W., Ochot M. Methods of assessment of cryopreserved semen. Current Frontiers in Cryobiology. Edited by Igor I. Katkov. 2012. pp. 547–574. DOI: https://doi.org/10.5772/33565Bathgate R., Maxwell W. M. C., Evans G. Studies on the effect of supplementing boar semen cryopreservation media with different avian egg yolk types on in vitro post-thaw sperm quality. Reproduction Domestic Animals. 2006;41(1):68–73. DOI: https://doi.org/10.1111/j.1439-0531.2006.00623.xBathgate R., Maxwell W. M. C., Evans G. Studies on the effect of supplementing boar semen cryopreservation media with different avian egg yolk types on in vitro post-thaw sperm quality. Reproduction Domestic Animals. 2006;41(1):68–73. DOI: https://doi.org/10.1111/j.1439-0531.2006.00623.xAires V. A., Hinsch K.-D., Mueller-Schloesser F., Bogner K., Mueller-Schloesser S., Hinsch E. In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen. Theriogenology. 2003;60(2):269–279. DOI: https://doi.org/10.1016/S0093-691X(02)01369-9Aires V. A., Hinsch K.-D., Mueller-Schloesser F., Bogner K., Mueller-Schloesser S., Hinsch E. In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen. Theriogenology. 2003;60(2):269–279. DOI: https://doi.org/10.1016/S0093-691X(02)01369-9Muiño R., Fernández M., Peña A. Post-thaw survival and longevity of bull spermatozoa frozen with an egg yolk-based or two egg yolk-free extenders after an equilibration period of 18°h. Reproduction Domestic Animals. 2007;42(3):305–311. DOI: https://doi.org/10.1111/j.1439-0531.2006.00784.xMuiño R., Fernández M., Peña A. Post-thaw survival and longevity of bull spermatozoa frozen with an egg yolk-based or two egg yolk-free extenders after an equilibration period of 18°h. Reproduction Domestic Animals. 2007;42(3):305–311. DOI: https://doi.org/10.1111/j.1439-0531.2006.00784.xBansal A. K., Bilaspuri G. Impacts of oxidative stress and antioxidants on semen functions. Veterinary Medicine International. 2010;23:21–28. DOI: https://doi.org/10.4061/2011/686137Bansal A. K., Bilaspuri G. Impacts of oxidative stress and antioxidants on semen functions. Veterinary Medicine International. 2010;23:21–28. DOI: https://doi.org/10.4061/2011/686137Gamidov S. I., Shatylko T. V., Popova A. Y., Gasanov N. G., Gamidov R. S. Sperm oxidative stress: clinical significance and management. Meditsinskiy sovet = Medical Council. 2021;(3):19–27. (In Russ.). DOI: https://doi.org/10.21518/2079-701X-2021-3-19-27Gamidov S. I., Shatylko T. V., Popova A. Y., Gasanov N. G., Gamidov R. S. Sperm oxidative stress: clinical significance and management. Meditsinskiy sovet = Medical Council. 2021;(3):19–27. (In Russ.). DOI: https://doi.org/10.21518/2079-701X-2021-3-19-27Agnieszka M. M., Magdalena B. N., Mariola S., Andrzej C. PRDX5 and PRDX6 translocation and oligomerization in bull sperm: a response to cryopreservation-induced oxidative stress. Cell Commun Signal. 2025;23(1):15. DOI: https://doi.org/10.1186/s12964-024-02015-9Agnieszka M. M., Magdalena B. N., Mariola S., Andrzej C. PRDX5 and PRDX6 translocation and oligomerization in bull sperm: a response to cryopreservation-induced oxidative stress. Cell Commun Signal. 2025;23(1):15. DOI: https://doi.org/10.1186/s12964-024-02015-9Pompella A., Visvikis A., Paolicchi A., Tata V. D., Casini A. F. The changing faces of glutathione, a cellular protagonist. Biochemical Pharmacology. 2003;66(8):1499–1503. DOI: https://doi.org/10.1016/s0006-2952(03)00504-5Pompella A., Visvikis A., Paolicchi A., Tata V. D., Casini A. F. The changing faces of glutathione, a cellular protagonist. Biochemical Pharmacology. 2003;66(8):1499–1503. DOI: https://doi.org/10.1016/s0006-2952(03)00504-5Llavanera M., Delgado-Bermúdez A., Olives S., Mateo-Otero Y., Recuero S., Bonet S., Fernández-Fuertes B., Yeste M., Barranco I. Glutathione S-Transferases Play a Crucial Role in Mitochondrial Function, Plasma Membrane Stability and Oxidative Regulation of Mammalian Sperm. Antioxidants (Basel). 2020;9(2):100. DOI: https://doi.org/10.3390/antiox9020100Llavanera M., Delgado-Bermúdez A., Olives S., Mateo-Otero Y., Recuero S., Bonet S., FernándezFuertes B., Yeste M., Barranco I. Glutathione S-Transferases Play a Crucial Role in Mitochondrial Function, Plasma Membrane Stability and Oxidative Regulation of Mammalian Sperm. Antioxidants (Basel). 2020;9(2):100. DOI: https://doi.org/10.3390/antiox9020100Gangwar C., Saxena A., Patel A., Singh S. P., Yadav S., Kumar R., Singh V. Effect of reduced glutathione supplementation on cryopreservation induced sperm cryoinjuries in Murrah bull semen. Animal Reproduction Science. 2018;192:171–178. DOI: https://doi.org/10.1016/j.anireprosci.2018.03.005Gangwar C., Saxena A., Patel A., Singh S. P., Yadav S., Kumar R., Singh V. Effect of reduced glutathione supplementation on cryopreservation induced sperm cryoinjuries in Murrah bull semen. Animal Reproduction Science. 2018;192:171–178. DOI: https://doi.org/10.1016/j.anireprosci.2018.03.005Yin J., Ren W., Yang G., Duan J., Huang X., Fang R., et al. Cysteine metabolism and its nutritional implications. Molecular Nutrition & Food Research. 2016;60(1):134–146. DOI: https://doi.org/10.1002/mnfr.201500031Yin J., Ren W., Yang G., Duan J., Huang X., Fang R., et al. Cysteine metabolism and its nutritional implications. Molecular Nutrition & Food Research. 2016;60(1):134–146. DOI: https://doi.org/10.1002/mnfr.201500031Tuncer P. B., Bucak M. N., Büyükleblebici S., Sarıözkan S., Yeni D., Eken A., et al. The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen. Cryobiology. 2010;61(3):303–307. DOI: https://doi.org/10.1016/j.cryobiol.2010.09.009Tuncer P. B., Bucak M. N., Büyükleblebici S., Sarıözkan S., Yeni D., Eken A., et al. The effect of cysteine and glutathione on sperm and oxidative stress parameters of post-thawed bull semen. Cryobiology. 2010;61(3):303–307. DOI: https://doi.org/10.1016/j.cryobiol.2010.09.009Bilodeau J. F., Chatterjee S., Sirard M. A., Gagnon C. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Molecular Reproduction and Development. 2000;55(3):282–288. DOI: https://doi.org/10.1002/(SICI)1098-2795(200003)55:3<282::AID-MRD6>3.0.CO;2-7Bilodeau J. F., Chatterjee S., Sirard M. A., Gagnon C. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Molecular Reproduction and Development. 2000;55(3):282–288. DOI: https://doi.org/10.1002/(SICI)1098-2795(200003)55:3<282::AID-MRD6>3.0.CO;2-7Hayanti S. Y., Sholikin M. M., Jayanegara A., Ulum M. F., da Costa M. A., Fitriawaty F., et al. Effect of supplementing L-cysteine and its group analogs on frozen semen quality of bulls: A meta-analysis. Veterinary World. 2022;15(11):2517–2524. DOI: https://doi.org/10.14202/vetworld.2022.2517-2524Hayanti S. Y., Sholikin M. M., Jayanegara A., Ulum M. F., da Costa M. A., Fitriawaty F., et al. Effect of supplementing L-cysteine and its group analogs on frozen semen quality of bulls: A meta-analysis. Veterinary World. 2022;15(11):2517–2524. DOI: https://doi.org/10.14202/vetworld.2022.2517-2524Gabr A. A., El Basuini M. F. Effect of tonophosphan, zinc oxide, and ascorbic acid on semen, sexual desire, and the fertility rate of Egyptian buffalo bulls. Annals of Agricultural Sciences. 2018;63(2):215–221. DOI: https://doi.org/10.1016/j.aoas.2018.12.001Gabr A. A., El Basuini M. F. Effect of tonophosphan, zinc oxide, and ascorbic acid on semen, sexual desire, and the fertility rate of Egyptian buffalo bulls. Annals of Agricultural Sciences. 2018;63(2):215–221. DOI: https://doi.org/10.1016/j.aoas.2018.12.001Bustani G. S., Baiee F. H. Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders. Veterinary World. 2021;14(5):1220–1233. DOI: https://doi.org/10.14202/vetworld.2021.1220-1233Bustani G. S., Baiee F. H. Semen extenders: An evaluative overview of preservative mechanisms of semen and semen extenders. Veterinary World. 2021;14(5):1220–1233. DOI: https://doi.org/10.14202/vetworld.2021.1220-1233Fariz H., Yon O., Daud S. Effect of vitamin E addition to frozen Simmental bull semen extender on postthawing quality. E3S Web of Conferences. 2020;142:02002. DOI: https://doi.org/10.1051/e3sconf/202014202002Fariz H., Yon O., Daud S. Effect of vitamin E addition to frozen Simmental bull semen extender on postthawing quality. E3S Web of Conferences. 2020;142:02002. DOI: https://doi.org/10.1051/e3sconf/202014202002Hassani-Bafrani H., Tavalaee M., Arbabian M., Dattilo M., Nasr-Esfahani M. H. The Effect of Vitamin E & Vitamin B on Sperm Function in Rat Varicocele Model. Andrologia. 2019;51(11):e13429. DOI: https://doi.org/10.1111/and.13429Hassani-Bafrani H., Tavalaee M., Arbabian M., Dattilo M., Nasr-Esfahani M. H. The Effect of Vitamin E & Vitamin B on Sperm Function in Rat Varicocele Model. Andrologia. 2019;51(11):e13429. DOI: https://doi.org/10.1111/and.13429Suharyati S., Hartono M., Siswanto S., Wahid A. N., Saputra H. R., Sari F. N. The effectiveness of vitamin c and e addition in various diluents of Brahman cattle spermatozoa after freezing. Jurnal Kedokteran Hewan – Indonesian Journal of Veterinary Sciences. 2024;18(3):88–92. DOI: https://doi.org/10.21157/j.ked.hewan.v18i3.37128Suharyati S., Hartono M., Siswanto S., Wahid A. N., Saputra H. R., Sari F. N. The effectiveness of vitamin c and e addition in various diluents of Brahman cattle spermatozoa after freezing. Jurnal Kedokteran Hewan – Indonesian Journal of Veterinary Sciences. 2024;18(3):88–92. DOI: https://doi.org/10.21157/j.ked.hewan.v18i3.37128Ahmed H., Jahan S., Riaz M., Ijaz M. U., Wahab A. Improving the quality and in vitro fertilization rate of frozen-thawed semen of buffalo (Bubalus bubalis) bulls with the inclusion of vitamin B12 in the cryopreservation medium. Animal Reproduction Science. 2021;229:106761. DOI: https://doi.org/10.1016/j.anireprosci.2021.106761Ahmed H., Jahan S., Riaz M., Ijaz M. U., Wahab A. Improving the quality and in vitro fertilization rate of frozen-thawed semen of buffalo (Bubalus bubalis) bulls with the inclusion of vitamin B12 in the cryopreservation medium. Animal Reproduction Science. 2021;229:106761. DOI: https://doi.org/10.1016/j.anireprosci.2021.106761Pérez-Marín C. C., Quevedo L., Salas M., Arando A. Ultra-Rapid Freezing Using Droplets Immersed into Liquid Nitrogen in Bull Sperm: Evaluation of Two Cryoprotective Disaccharides and Two Warming Temperatures. Biopreservation and Biobanking. 2023;21(6):554–560. DOI: https://doi.org/10.1089/bio.2022.0075Pérez-Marín C. C., Quevedo L., Salas M., Arando A. Ultra-Rapid Freezing Using Droplets Immersed into Liquid Nitrogen in Bull Sperm: Evaluation of Two Cryoprotective Disaccharides and Two Warming Temperatures. Biopreservation and Biobanking. 2023;21(6):554–560. DOI: https://doi.org/10.1089/bio.2022.0075Tuncer P. B., Sarıözkan S., Bucak M. N., Ulutaş P. A., Akalın P. P., Büyükleblebici S., Canturk F. Effect of glutamine and sugars after bull spermatozoa cryopreservation. Theriogenology. 2011;75(8):1459–1465. DOI: https://doi.org/10.1016/j.theriogenology.2010.12.006Tuncer P. B., Sarıözkan S., Bucak M. N., Ulutaş P. A., Akalın P. P., Büyükleblebici S., Canturk F. Effect of glutamine and sugars after bull spermatozoa cryopreservation. Theriogenology. 2011;75(8):1459–1465. DOI: https://doi.org/10.1016/j.theriogenology.2010.12.006Correia L. F. L., Alves B. R. C., Batista R. I. T. P., Mermillod P., Souza-Fabjan J. M. G. Antifreeze proteins for low-temperature preservation in reproductive medicine: A systematic review over the last three decades. Theriogenology. 2021;176:94–103. DOI: https://doi.org/10.1016/j.theriogenology.2021.09.025Correia L. F. L., Alves B. R. C., Batista R. I. T. P., Mermillod P., Souza-Fabjan J. M. G. Antifreeze proteins for low-temperature preservation in reproductive medicine: A systematic review over the last three decades. Theriogenology. 2021;176:94–103. DOI: https://doi.org/10.1016/j.theriogenology.2021.09.025Jang H., Kwon H. J., Sun W. S., Hwang S., Hwang I. S., Kim S., et al. Effects of Leucosporidium-derived ice-binding protein (LeIBP) on bull semen cryopreservation. Veterinary Medicine and Science. 2020;6(3):447–453. DOI: https://doi.org/10.1002/vms3.269Jang H., Kwon H. J., Sun W. S., Hwang S., Hwang I. S., Kim S., et al. Effects of Leucosporidium-derived ice-binding protein (LeIBP) on bull semen cryopreservation. Veterinary Medicine and Science. 2020;6(3):447–453. DOI: https://doi.org/10.1002/vms3.269Prathalingam N. S., Holt W. V., Revell S. G., Mirczuk S., Fleck R. A., Watson P. F. Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw. Theriogenology. 2006;66(8):1894–1900. DOI: https://doi.org/10.1016/j.theriogenology.2006.04.041Prathalingam N. S., Holt W. V., Revell S. G., Mirczuk S., Fleck R. A., Watson P. F. Impact of antifreeze proteins and antifreeze glycoproteins on bovine sperm during freeze-thaw. Theriogenology. 2006;66(8):1894–1900. DOI: https://doi.org/10.1016/j.theriogenology.2006.04.041Karaşör Ö. F., Bucak M. N., Cenariu M., Bodu M., Taşpınar M., Taşpınar F. The effects of different doses of ROCK inhibitor, antifreeze protein III, and boron added to semen extender on semen Freezeability of Ankara bucks. Molecules. 2022;27(22):8070. DOI: https://doi.org/10.3390/molecules27228070Karaşör Ö. F., Bucak M. N., Cenariu M., Bodu M., Taşpınar M., Taşpınar F. The effects of different doses of ROCK inhibitor, antifreeze protein III, and boron added to semen extender on semen Freezeability of Ankara bucks. Molecules. 2022;27(22):8070. DOI: https://doi.org/10.3390/molecules27228070Prastiya R. A., Sardjito T., Saputro A. L., Budi D. S., Maxdhameta S. I., Sulistiyawati E., et al. Quality and kinematic characteristics of Bali bulls frozen semen with purified green tea extract epigallocatechin-3-gallate antioxidant addition in diluent. Open Veterinary Journal. 2024;14(8):2040–2048. DOI: https://doi.org/10.5455/OVJ.2024.v14.i8.33Prastiya R. A., Sardjito T., Saputro A. L., Budi D. S., Maxdhameta S. I., Sulistiyawati E., et al. Quality and kinematic characteristics of Bali bulls frozen semen with purified green tea extract epigallocatechin-3-gallate antioxidant addition in diluent. Open Veterinary Journal. 2024;14(8):2040–2048. DOI: https://doi.org/10.5455/OVJ.2024.v14.i8.33Ahmed H., Jahan S., Ullah H., Ullah F., Salman M. M. The addition of resveratrol in tris citric acid extender ameliorates post-thaw quality parameters, antioxidant enzymes levels, and fertilizing capability of buffalo (Bubalus bubalis) bull spermatozoa. Theriogenology. 2020;152:106–113. DOI: https://doi.org/10.1016/j.theriogenology.2020.04.034Ahmed H., Jahan S., Ullah H., Ullah F., Salman M. M. The addition of resveratrol in tris citric acid extender ameliorates post-thaw quality parameters, antioxidant enzymes levels, and fertilizing capability of buffalo (Bubalus bubalis) bull spermatozoa. Theriogenology. 2020;152:106–113. DOI: https://doi.org/10.1016/j.theriogenology.2020.04.034Lebedev S. G. The use of biologically active substances in the freezing of sperm from breeding bulls. Uchenye zapiski uchrezhdeniya obrazovaniya Vitebskaya ordena Znak pocheta gosudarstvennaya akademiya veterinarnoy meditsiny. 2005;41(1):88–91. (In Belarus). URL: https://elibrary.ru/item.asp?id=25054554Lebedev S. G. The use of biologically active substances in the freezing of sperm from breeding bulls. Uchenye zapiski uchrezhdeniya obrazovaniya Vitebskaya ordena Znak pocheta gosudarstvennaya akademiya veterinarnoy meditsiny. 2005;41(1):88–91. (In Belarus). URL: https://elibrary.ru/item.asp?id=25054554Watson P. F. The preservation of semen in mammals. Oxford Reviews of Reproductive Biology. 1979;1:283–350.Watson P. F. The preservation of semen in mammals. Oxford Reviews of Reproductive Biology. 1979;1:283–350.Kolossa M., Seibert H. A chemically 'defined' diluent for cryopreservation of bovine spermatozoa. Andrologia. 1990;22(5):445–454. DOI: https://doi.org/10.1111/j.1439-0272.1990.tb02026.xKolossa M., Seibert H. A chemically 'defined' diluent for cryopreservation of bovine spermatozoa. Andrologia. 1990;22(5):445–454. DOI: https://doi.org/10.1111/j.1439-0272.1990.tb02026.xGadea J. Semen extenders used in the artificial insemination of swine. Spanish Journal of Agricultural Research. 2003;1(2):17–27. DOI: https://doi.org/10.5424/sjar/2003012-17Gadea J. Semen extenders used in the artificial insemination of swine. Spanish Journal of Agricultural Research. 2003;1(2):17–27. DOI: https://doi.org/10.5424/sjar/2003012-17Spinosa H. S., Górniak S. L., Bernardi M. M. Farmacologiaaplicada à medicinaveterinária. Rio de Janeiro: Guanabara Koogan. 2006. URL: https://repositorio.usp.br/item/001523488Spinosa H. S., Górniak S. L., Bernardi M. M. Farmacologiaaplicada à medicinaveterinária. Rio de Janeiro: Guanabara Koogan. 2006. URL: https://repositorio.usp.br/item/001523488Santos C. S., Silva A. R. Current and alternative trends in antibacterial agents used in mammalian semen technology. Animal Reproduction. 2020;17(1):e20190111. DOI: https://doi.org/10.21451/1984-3143-AR2019-0111Santos C. S., Silva A. R. Current and alternative trends in antibacterial agents used in mammalian semen technology. Animal Reproduction. 2020;17(1):e20190111. DOI: https://doi.org/10.21451/1984-3143-AR2019-0111Abd-Allah S. M. Effect of royal jelly on bovine sperm characteristics during post-thaw incubation in vitro. Revista Electronica de Veterinaria. 2010;21(2):81–85. DOI: https://doi.org/10.30972/vet.2121747Abd-Allah S. M. Effect of royal jelly on bovine sperm characteristics during post-thaw incubation in vitro. Revista Electronica de Veterinaria. 2010;21(2):81–85. DOI: https://doi.org/10.30972/vet.2121747Cojkic A., Hansson I., Johannisson A., Morrell J. M. Effect of Some Plant-Based Substances on Microbial Content and Sperm Quality Parameters of Bull Semen. International Journal of Molecular Sciences. 2023;24(4):3435. DOI: https://doi.org/10.3390/ijms24043435Cojkic A., Hansson I., Johannisson A., Morrell J. M. Effect of Some Plant-Based Substances on Microbial Content and Sperm Quality Parameters of Bull Semen. International Journal of Molecular Sciences. 2023;24(4):3435. DOI: https://doi.org/10.3390/ijms24043435

The authors declare that there are no conflicts of interest present.