The aim of our study was to examine effects of the length of semen equilibration as well as two freezing techniques on ram sperm post-thaw quality. The ejaculates of Wallachian sheep rams (n = 12) were collected by an electro-ejaculation, equilibrated in a Triladyl® (0, 2, 4, 6, and 8 h) containing glycerol and egg yolk and frozen by programmable freezing (PF) or manual freezing (MF). After thawing, sperm samples were subjected to the motility (computer-assisted sperm analysis [CASA]), viability (SYBR-14/PI), and fertilizing ability (FA) (in vitro penetration/fertilization test on bovine oocytes) assays. It was found that the equilibration of 6 h (E-6) ensured higher post-thaw sperm motility and progressive movement compared with other lengths tested, irrespective of a freezing technique. The E-6 sperm viability did not differ between PF and MF but was lower (P < 0.05) than control. Sperm FA (E-6) was similar in PF (60.44%) and MF (62%) but slightly lower than in fresh (72.8%). Our data demonstrate that the use of MF was comparable with PF, which can be applied in the field conditions without need in a piece of cost-expensive equipment, which can greatly benefit the gene bank of animal genetic resources.

Department of Animal Morphology Physiology and Genetics Faculty of AgriSciences Mendel University in Brno Zemědělská 1 1665 Brno 613 00 Czech Republic

Institute of Biotechnology Faculty of Biotechnology and Food Science Slovak University of Agriculture in Nitra Tr A Hlinku 2 Nitra 949 76 Slovak Republic

NPPC Research Institute for Animal Production in Nitra Hlohovecka 2 Lužianky 951 41 Slovak Republic

Zobrazit více v PubMed

Ahmad, M., Nasrullah, R., & Ahmad, N. (2015). Effect of cooling rate and equilibration time on pre-freeze and post-thaw survival of buck sperm. Cryobiology, 70, 233-238. https://doi.org/10.1016/J.CRYOBIOL.2015.03.002

Alipour, P., Daghigh Kia, H., Moghaddam, G., & Ebrahimi, M. (2018). Evaluating caffeine antioxidant properties on Ghezel ram sperm quality after freeze-thawing. Revue de Médecine Vétérinaire, 169, 233-240.

Álvarez, M., Tamayo-Canul, J., Martínez-Rodríguez, C., López-Urueña, E., Gomes-Alves, S., Anel, L., Martinez-Pastor, F., & De Paz, P. (2012). Specificity of the extender used for freezing ram sperm depends of the spermatozoa source (ejaculate, electroejaculate or epididymis). Animal Reproduction Science, 132, 145-154. https://doi.org/10.1016/J.ANIREPROSCI.2012.05.006

Anzar, M., Kroetsch, T., & Boswall, L. (2011). Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes. Animal Reproduction Science, 126, 23-31. https://doi.org/10.1016/j.anireprosci.2011.04.018

Aurich, J., Kuhl, J., Tichy, A., & Aurich, C. (2020). Efficiency of semen cryopreservation in stallions. Animals, 10, 1-13. https://doi.org/10.3390/ani10061033

Blackshaw, A. W. (1960). The effects of milk diluents on the viability of ram spermatozoa and their revival after freezing. Australian Veterinary Journal, 36, 432-435. https://doi.org/10.1111/j.1751-0813.1960.tb03729.x

Bohlool, Z., Mohammadi, M., Mehr, M. R. A., & Hossein-Zadeh, N. G. (2015). Effect of different concentrations of trehalose and glycerol on the freezability of ram semen using soybean lecithin-based diluents. Animal Production Science, 55, 666-671. https://doi.org/10.1071/AN13431

Câmara, D. R., Silva, S. V., Almeida, F. C., Nunes, J. F., & Guerra, M. M. P. (2011). Effects of antioxidants and duration of pre-freezing equilibration on frozen-thawed ram semen. Theriogenology, 76, 342-350. https://doi.org/10.1016/j.theriogenology.2011.02.013

Chaveiro, A., Liu, J., Mullen, S., Woelders, H., & Critser, J. K. (2004). Determination of bull sperm membrane permeability to water and cryoprotectants using a concentration-dependent self-quenching fluorophore. Cryobiology, 48, 72-80. https://doi.org/10.1016/j.cryobiol.2003.12.005

Cofré-Narbona, E. J., Peralta-Troncoso, O. A., Urquieta-Mangiola, B. E., Raggi-Saini, L. A., Benavides-Aguila, N., & Parraguez-Gamboa, V. H. (2016). Improvement of antioxidant status and semen quality by oral supplementation with vitamins c and e in rams. Revista Científica, 26, 156-163.

Curry, M. R., Kleinhans, F. W., & Watson, P. F. (2000). Measurement of the water permeability of the membranes of boar, ram, and rabbit spermatozoa using concentration-dependent self-quenching of an entrapped fluorophore. Cryobiology, 41, 167-173. https://doi.org/10.1006/cryo.2000.2277

D'Alessandro, A., & Martemucci, G. (2003). Evaluation of seasonal variations of semen freezability in Leccese ram. Animal Reproduction Science, 79, 93-102. https://doi.org/10.1016/S0378-4320(03)00113-1

Emamverdi, M., Zhandi, M., Zare Shahneh, A., Sharafi, M., & Akbari-Sharif, A. (2013). Optimization of ram semen cryopreservation using a chemically defined soybean lecithin-based extender. Reproduction in Domestic Animals, 48, 899-904. https://doi.org/10.1111/rda.12183

Eriani, K., Sari, N., Ihdina, M., & Rosnizar, R. (2017). The effect of equilibration time on semen freezing of local swamp buffalo (Bubalus bubalis) with combination extender of lactose and glycerol. Nusantara Bioscience, 9, 77-82. https://doi.org/10.13057/nusbiosci/n090113

Esmaeili, V., Shahverdi, A. H., Alizadeh, A. R., Alipour, H., & Chehrazi, M. (2014). Saturated, omega-6 and omega-3 dietary fatty acid effects on the characteristics of fresh, frozen-thawed semen and blood parameters in rams. Andrologia, 46, 42-49. https://doi.org/10.1111/and.12040

Esteso, M. C., Toledano-Díaz, A., Castaño, C., Pradiee, J., Lopez-Sebastián, A., & Santiago-Moreno, J. (2018). Effect of two cooling protocols on the post-thaw characteristics of Iberian ibex sperms. Cryobiology, 80, 12-17. https://doi.org/10.1016/j.cryobiol.2018.01.003

Fang, Y., Zhao, C., Xiang, H., Jia, G., & Zhong, R. (2020). Melatonin improves cryopreservation of ram sperm by inhibiting mitochondrial permeability transition pore opening. Reproduction in Domestic Animals, 55, 1240-1249. https://doi.org/10.1111/rda.13771

Fleisch, A., Malama, E., Witschi, U., Leiding, C., Siuda, M., Janett, F., & Bollwein, H. (2017). Effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen. Theriogenology, 89, 255-262. https://doi.org/10.1016/j.theriogenology.2016.10.018

Forouzanfar, M., Sharafi, M., Hosseini, S. M., Ostadhosseini, S., Hajian, M., Hosseini, L., Abedi, P., Nili, N., Rahmani, H. R., & Nasr-Esfahani, M. H. (2010). In vitro comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of ram semen. Theriogenology, 73, 480-487. https://doi.org/10.1016/J.THERIOGENOLOGY.2009.10.005

Galarza, D. A., López-Sebastián, A., Woelders, H., Blesbois, E., & Santiago-Moreno, J. (2019). Two-step accelerating freezing protocol yields a better motility, membranes and DNA integrities of thawed ram sperm than three-steps freezing protocols. Cryobiology, 91, 84-89. https://doi.org/10.1016/j.cryobiol.2019.10.007

Garner, D. L., & Johnson, L. A. (1995). Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biology of Reproduction, 53, 276-284. https://doi.org/10.1095/biolreprod53.2.276

Gilmore, J. A., McGann, L. E., Liu, J., Gao, D. Y., Peter, A. T., Kleinhans, F. W., & Critser, J. K. (1995). Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biology of Reproduction, 53, 985-995. https://doi.org/10.1095/biolreprod53.5.985

Hill, J., Godley, W., & Hurst, V. (1959). Effect of glycerol equilibration time, glycerol level and rate of temperature descent on the freezing of ram spermatozoa. Journal of Animal Science, 18, 614-621. https://doi.org/10.2527/jas1959.182614x

Jerez, R., González, N., Olaciregui, M., Luño, V., de Blas, I., & Gil, L. (2016). Use of soy milk combined with different cryoprotectants for the ram semen cryopreservation. Small Ruminant Research, 134, 34-38. https://doi.org/10.1016/j.smallrumres.2015.12.003

Jiménez-Rabadán, P., Soler, A. J., Ramón, M., García-Álvarez, O., Maroto-Morales, A., Iniesta-Cuerda, M., Fernández-Santos, M. R., Montoro, V., Pérez-Guzmán, M. D., & Garde, J. J. (2016). Influence of semen collection method on sperm cryoresistance in small ruminants. Animal Reproduction Science, 167, 103-108. https://doi.org/10.1016/j.anireprosci.2016.02.013

Jones, R. C., & Martin, I. C. (1965). Deep-freezing ram spermatozoa: The effects of milk, yolk-citrate and synthetic diluents containing sugar. Journal of Reproduction and Fertility, 10, 413-423. https://doi.org/10.1530/jrf.0.0100413

Küçük, N., Aksoy, M., Uçan, U., Ahmad, E., Naseer, Z., Ceylan, A., & Serin, İ. (2014). Comparison of two different cryopreservation protocols for freezing goat semen. Cryobiology, 68, 327-331. https://doi.org/10.1016/J.CRYOBIOL.2014.04.009

Ledesma, A., Manes, J., Ríos, G., Aller, J., Cesari, A., Alberio, R., & Hozbor, F. (2015). Effect of seminal plasma on post-thaw quality and functionality of Corriedale ram sperm obtained by electroejaculation and artificial vagina. Reproduction in Domestic Animals, 50, 386-392. https://doi.org/10.1111/RDA.12500

Leite, T. G., do Vale Filho, V. R., de Arruda, R. P., de Andrade, A. F. C., Emerick, L. L., Zaffalon, F. G., Martins, J. A. M., & de Andrade, V. J. (2010). Effects of extender and equilibration time on post-thaw motility and membrane integrity of cryopreserved Gyr bull semen evaluated by CASA and flow cytometry. Animal Reproduction Science, 120, 31-38. https://doi.org/10.1016/j.anireprosci.2010.04.005

Luna-Orozco, J. R., González-Ramos, M. A., Calderón-Leyva, G., Gaytán-Alemán, L. R., Arellano-Rodríguez, F., Ángel-García, O., & Véliz-Deras, F. G. (2019). Comparison of different diluents based on liposomes and egg yolk for ram semen cooling and cryopreservation. Iranian Journal of Veterinary Research, 20, 126-130.

Lv, C., Wu, G., Hong, Q., & Quan, G. (2019). Spermatozoa cryopreservation: State of art and future in small ruminants. Biopreservation and Biobanking, 17, 171-182. https://doi.org/10.1089/BIO.2018.0113

Makarevich, A., Kubovicova, E., Sirotkin, A., Pivko, J. (2010). Demonstration of the effect of epidermal growth factor on ram sperm parameters using two fluorescent assays. Veterinární Medicína, 55, 581-589. https://doi.org/10.17221/2944-VETMED, DOI: https://doi.org/10.17221/2944-VETMED

Masoudi, R., Sharafi, M., Zare Shahneh, A., Towhidi, A., Kohram, H., Zhandi, M., Esmaeili, V., & Shahverdi, A. (2016). Effect of dietary fish oil supplementation on ram semen freeze ability and fertility using soybean lecithin- and egg yolk-based extenders. Theriogenology, 86, 1583-1588. https://doi.org/10.1016/J.THERIOGENOLOGY.2016.05.018

Matini Behzad, A., Ebrahimi, B., Alizadeh, A. R., Esmaeili, V., Dalman, A., Rashki, L., & Shahverdi, A. H. (2014). Improvement in in vitro fertilization rate, decrease in reactive oxygen species and spermatozoa death incidence in rams by dietary fish oil. Reproduction in Domestic Animals, 49, 599-605. https://doi.org/10.1111/rda.12328

Maxwell, W. M. C., Evans, G., Mortimer, S. T., Gillan, L., Gellatly, E. S., & McPhie, C. A. (1999). Normal fertility in ewes after cervical insemination with frozen-thawed spermatozoa supplemented with seminal plasma. Reproduction, Fertility and Development, 11, 123-126. https://doi.org/10.1071/RD99046

Memon, A., Wahid, H., Rosnina, Y., Goh, Y., Ebrahimi, M., & Nadia, F. (2013). Effect of ascorbic acid concentrations, methods of cooling and freezing on Boer goat semen cryopreservation. Reproduction in Domestic Animals, 48, 325-330. https://doi.org/10.1111/J.1439-0531.2012.02155.X

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. https://doi.org/10.1016/J.ANIREPROSCI.2018.02.010

Najafi, A., Najafi, M. H., Zanganeh, Z., Sharafi, M., Martinez-Pastor, F., & Adeldust, H. (2014). Cryopreservation of ram semen in extenders containing soybean lecithin as cryoprotectant and hyaluronic acid as antioxidant. Reproduction in Domestic Animals, 49, 934-940. https://doi.org/10.1111/rda.12405

Ntemka, A., Tsakmakidis, I. A., Kiossis, E., Milovanović, A., & Boscos, C. M. (2018). Current status and advances in ram semen cryopreservation. Journal of the Hellenic Veterinary Medical Society, 69, 911-924. https://doi.org/10.12681/JHVMS.18014

O'Hara, L., Hanrahan, J. P., Richardson, L., Donovan, A., Fair, S., Evans, A. C. O., & Lonergan, P. (2010). Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm. Theriogenology, 73, 541-549. https://doi.org/10.1016/J.THERIOGENOLOGY.2009.10.009

Patt, J. A., & Nath, J. (1969). Effects of diluents, equilibration time, and freezing rates on the storage of ram semen. Cryobiology, 5, 385-392. https://doi.org/10.1016/S0011-2240(69)80102-1

Paul, R. K., Balaganur, K., Kumar, D., & Singh, R. (2020). Pre-freezing equilibration for 22 h improves post-thaw sperm functions in cryopreserved ram semen by reducing cholesterol efflux. Cryobiology, 96, 76-84. https://doi.org/10.1016/j.cryobiol.2020.07.013

Ptáček, M., Stádníková, M., Savvulidi, F., & Stádník, L. (2019). Ram semen cryopreservation using egg yolk or egg yolk-free extenders: Preliminary results. Scientia Agriculturae Bohemica, 50, 96-103. https://doi.org/10.2478/sab-2019-0014

Purdy, P. H. (2006a). The post-thaw quality of ram sperm held for 0 to 48 h at 5 °C prior to cryopreservation. Animal Reproduction Science, 93, 114-123. https://doi.org/10.1016/J.ANIREPROSCI.2005.07.002

Purdy, P. H. (2006b). A review on goat sperm cryopreservation. Small Ruminant Research, 63, 215-225. https://doi.org/10.1016/J.SMALLRUMRES.2005.02.015

Purdy, P. H., Mocé, E., Stobart, R., Murdoch, W. J., Moss, G. E., Larson, B., Ramsey, S., Graham, J. K., & Blackburn, H. D. (2010). The fertility of ram sperm held for 24 h at 5 °C prior to cryopreservation. Animal Reproduction Science, 118, 231-235. https://doi.org/10.1016/J.ANIREPROSCI.2009.06.014

Rateb, S. A., Khalifa, M. A., Abd El-Hamid, I. S., & Shedeed, H. A. (2020). Enhancing liquid-chilled storage and cryopreservation capacities of ram spermatozoa by supplementing the diluent with different additives. Asian-Australasian Journal of Animal Sciences, 33, 1068-1076. https://doi.org/10.5713/ajas.19.0338

Salamon, S. (1968). Deep freezing of ram semen: Recovery of spermatozoa after pelleting and comparison with other methods of freezing. Australian Journal of Biological Sciences, 21, 351-360. https://doi.org/10.1071/BI9680351

Salamon, S., & Maxwell, W. M. C. (2000). Storage of ram semen. Animal Reproduction Science, 62, 77-111. https://doi.org/10.1016/S0378-4320(00)00155-X

Sharafi, M., Zhandi, M., & Sharif, A. A. (2014). Supplementation of soybean lecithin-based semen extender by antioxidants: Complementary flowcytometric study on post-thawed ram spermatozoa. Cell and Tissue Banking, 16(2), 261-269. https://doi.org/10.1007/S10561-014-9458-5

Šterbenc, N., Morrell, J. M., Kosec, M., Rath, D., Klein, S., & Klinc, P. (2019). Single layer colloid centrifugation technique improves motility, viability and chromatin integrity of ram spermatozoa after thawing. Cryobiology, 86, 77-83. https://doi.org/10.1016/j.cryobiol.2018.12.001

Tibary, A., & Manar, S. (2018). Cryo-preservation of sperm and embryos in small ruminants. Revue Marocaine Des Sciences Agronomiques et Vétérinaires., 6, 195-210.

Toker, M. B., Alcay, S., Gokce, E., & Ustuner, B. (2016). Cryopreservation of ram semen with antioxidant supplemented soybean lecithin-based extenders and impacts on incubation resilience. Cryobiology, 72, 205-209. https://doi.org/10.1016/J.CRYOBIOL.2016.05.001

Üstüner, B., Alçay, S., Nur, Z., Saǧirkaya, H., & Kemal Soylu, M. (2014). Effect of egg yolk and soybean lecithin on tris-based extender in post-thaw ram semen quality and in vitro fertility. Kafkas Universitesi Veteriner Fakultesi Dergisi, 20, 393-398. https://doi.org/10.9775/kvfd.2013.10248

Vishwanath, R., & Shannon, P. (2000). Storage of bovine semen in liquid and frozen state. Animal Reproduction Science, 62, 23-53. https://doi.org/10.1016/S0378-4320(00)00153-6

Glycerol-Free Equilibration with the Addition of Glycerol Shortly before the Freezing Procedure: A Perspective Strategy for Cryopreservation of Wallachian Ram Sperm

Comprehensive Flow-Cytometric Quality Assessment of Ram Sperm Intended for Gene Banking Using Standard and Novel Fertility Biomarkers

The Cryopreserved Sperm Traits of Various Ram Breeds: Towards Biodiversity Conservation