The aim of our research was to compare three Slovak sheep breeds in the quality parameters of cryopreserved sperm. The ejaculates of Slovak Dairy (SD), Native Wallachian (NW), and Improved Wallachian (IW) sheep rams (n = 12) were collected by electro-ejaculation. Heterospermic samples were created from suitable ejaculates, separately for each breed (at least 90% of total and 80% of progressive motility). Samples were equilibrated in a Triladyl® diluent and frozen by automated freezing. Sperm samples were subjected to the motility, morphology, (CASA), viability and apoptosis (DRAQ7/Yo-Pro-1), fertilizing capability (penetration/fertilization test (P/F) in vitro) and acrosomal status (transmission electron microscopy) assays before freezing and after thawing. It was found that there were no significant differences (p < 0.05) between the evaluated breeds in motility, viability, apoptosis, morphological properties, and fertilizing ability of cryopreserved sperm. Significant differences occurred in acrosomal status. Our results demonstrate that the use of the selected cryopreservation protocol is suitable for at least three different sheep breeds, which can greatly benefit the biodiversity protection and simplifies the creation of an animal genetic resources gene bank.

AgroBioTech Research Centre Slovak University of Agriculture in Nitra Tr A Hlinku 2 94901 Nitra Slovakia

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

Department of Botany and Genetics Constantine the Philosopher University in Nitra Trieda Andreja Hlinku 1 94974 Nitra Slovakia

Institute of Biotechnology Faculty of Biotechnology and Food Sciences Slovak University of Agriculture in Nitra Tr A Hlinku 2 94901 Nitra Slovakia

NPPC Research Institute for Animal Production Nitra Hlohovecka 2 95141 Lužianky Slovakia

See more in PubMed

Silva S., Soares A., Batista A., Almeida F., Nunes J., Peixoto C., Guerra M. In Vitro and In Vivo Evaluation of Ram Sperm Frozen in Tris Egg-Yolk and Supplemented with Superoxide Dismutase and Reduced Glutathione. Reprod. Domest. Anim. 2011;46:874–881. doi: 10.1111/j.1439-0531.2011.01758.x. PubMed DOI

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. Influence of Semen Collection Method on Sperm Cryoresistance in Small Ruminants. Anim. Reprod. Sci. 2016;167:103–108. doi: 10.1016/j.anireprosci.2016.02.013. PubMed DOI

Raspa M., Guan M., Paoletti R., Montoliu L., Ayadi A., Marschall S., Fray M., Scavizzi F. Dry Ice Is a Reliable Substrate for the Distribution of Frozen Mouse Spermatozoa: A Multi-Centric Study. Theriogenology. 2017;96:49–57. doi: 10.1016/j.theriogenology.2017.04.003. PubMed DOI

Gürler H., Malama E., Heppelmann M., Calisici O., Leiding C., Kastelic J.P., Bollwein H. Effects of Cryopreservation on Sperm Viability, Synthesis of Reactive Oxygen Species, and DNA Damage of Bovine Sperm. Theriogenology. 2016;86:562–571. doi: 10.1016/j.theriogenology.2016.02.007. PubMed DOI

Küçük N., Aksoy M., Uçan U., Ahmad E., Naseer Z., Ceylan A., Serin İ. Comparison of Two Different Cryopreservation Protocols for Freezing Goat Semen. Cryobiology. 2014;68:327–331. doi: 10.1016/j.cryobiol.2014.04.009. PubMed DOI

Bailey J.L., Blodeau J.-F., Cormier N. Semen Cryopreservation in Domestic Animals: A Damaging and Capacitating Phenomenon Minireview. J. Androl. 2000;21:1–7. doi: 10.1002/j.1939-4640.2000.tb03268.x. PubMed DOI

Alcay S., Berk Toker M., Gokce E., Ustuner B., Tekin Onder N., Sagirkaya H., Nur Z., Kemal Soylu M. Successful Ram Semen Cryopreservation with Lyophilized Egg Yolk-Based Extender. Cryobiology. 2015;71:329–333. doi: 10.1016/j.cryobiol.2015.08.008. PubMed DOI

Amirat 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;61:895–907. doi: 10.1016/S0093-691X(03)00259-0. PubMed DOI

Arando A., Gonzalez A., Delgado J.V., Arrebola F.A., Perez-Marín C.C. Storage Temperature and Sucrose Concentrations Affect Ram Sperm Quality after Vitrification. Anim. Reprod. Sci. 2017;181:175–185. doi: 10.1016/j.anireprosci.2017.04.008. PubMed DOI

Tibary A., Manar S. Cryo-Preservation of Sperm and Embryos in Small Ruminants. Rev. Maroc. Sci. Agron. Vétérinaires. 2018;6:195–210.

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. Comparison of Different Diluents Based on Liposomes and Egg Yolk for Ram Semen Cooling and Cryopreservation. Iran. J. Vet. Res. 2019;20:126–130. PubMed PMC

Bohlool Z., Mohammadi M., Mehr M.R.A., Hossein-Zadeh N.G. Effect of Different Concentrations of Trehalose and Glycerol on the Freezability of Ram Semen Using Soybean Lecithin-Based Diluents. Anim. Prod. Sci. 2015;55:666–671. doi: 10.1071/AN13431. DOI

Emamverdi M., Zhandi M., Zare Shahneh A., Sharafi M., Akbari‐Sharif A. Optimization of Ram Semen Cryopreservation Using a Chemically Defined Soybean Lecithin‐Based Extender. Reprod. Domest. Anim. 2013;48:899–904. doi: 10.1111/rda.12183. PubMed DOI

Forouzanfar M., Sharafi M., Hosseini S.M., Ostadhosseini S., Hajian M., Hosseini L., Abedi P., Nili N., Rahmani H.R., Nasr-Esfahani M.H. In Vitro Comparison of Egg Yolk–Based and Soybean Lecithin–Based Extenders for Cryopreservation of Ram Semen. Theriogenology. 2010;73:480–487. doi: 10.1016/j.theriogenology.2009.10.005. PubMed DOI

Masoudi R., Sharafi M., Zareh Shahneh A., Towhidi A., Kohram H., Zhandi M., Esmaeili V., Shahverdi A. Effect of Dietary Fish Oil Supplementation on Ram Semen Freeze Ability and Fertility Using Soybean Lecithin– and Egg Yolk–Based Extenders. Theriogenology. 2016;86:1583–1588. doi: 10.1016/j.theriogenology.2016.05.018. PubMed DOI

Najafi A., Najafi M.H., Zanganeh Z., Sharafi M., Martinez-Pastor F., Adeldust H. Cryopreservation of Ram Semen in Extenders Containing Soybean Lecithin as Cryoprotectant and Hyaluronic Acid as Antioxidant. Reprod. Domest. Anim. 2014;49:934–940. doi: 10.1111/rda.12405. PubMed DOI

Sharafi M., Zhandi M., Sharif A.A. Supplementation of Soybean Lecithin-Based Semen Extender by Antioxidants: Complementary Flowcytometric Study on Post-Thawed Ram Spermatozoa. Cell Tissue Bank. 2014;16:261–269. doi: 10.1007/s10561-014-9458-5. PubMed DOI

Toker M.B., Alcay S., Gokce E., Ustuner B. Cryopreservation of Ram Semen with Antioxidant Supplemented Soybean Lecithin-Based Extenders and Impacts on Incubation Resilience. Cryobiology. 2016;72:205–209. doi: 10.1016/j.cryobiol.2016.05.001. PubMed DOI

Üstüner B., Alçay S., Nur Z., Saǧirkaya H., Kemal Soylu M. 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. 2014;20:393–398. doi: 10.9775/kvfd.2013.10248. DOI

Vozaf J., Makarevich A.V., Balazi A., Vasicek J., Svoradova A., Olexikova L., Chrenek P. Cryopreservation of Ram Semen: Manual versus Programmable Freezing and Different Lengths of Equilibration. Anim. Sci. J. 2021;92:e13670. doi: 10.1111/asj.13670. PubMed DOI

Chrenek P., Makarevič A.V., Kubovičová E., Bulla J., Supuka P. Slovak National Animal Breeds. Slovak University of Agriculture; Nitra, Slovakia: 2019.

García-Álvarez O., Maroto-Morales A., Martínez-Pastor F., Fernández-Santos M.R., Esteso M.C., Pérez-Guzmán M.D., Soler A.J. Heterologous in Vitro Fertilization Is a Good Procedure to Assess the Fertility of Thawed Ram Spermatozoa. Theriogenology. 2009;71:643–650. doi: 10.1016/j.theriogenology.2008.09.036. PubMed DOI

Olexikova L., Miranda M., Kulikova B., Baláži A., Chrenek P. Cryodamage of Plasma Membrane and Acrosome Region in Chicken Sperm. Anat. Histol. Embryol. 2019;48:33–39. doi: 10.1111/ahe.12408. PubMed DOI

Joost S., Bruford M.W. The Genomic-Resources Consortium Editorial: Advances in Farm Animal Genomic Resources. Front. Genet. 2015;6:333. doi: 10.3389/fgene.2015.00333. PubMed DOI PMC

Mara L., Casu S., Carta A., Dattena M. Cryobanking of Farm Animal Gametes and Embryos as a Means of Conserving Livestock Genetics. Anim. Reprod. Sci. 2013;138:25–38. doi: 10.1016/j.anireprosci.2013.02.006. PubMed DOI

FAO In Vivo Conservation of Animal Genetic Resources 2013. FAO Animal Production and Health Guidelines. No. 14. Rome. [(accessed on 9 February 2022)]. Available online: https://portals.iucn.org/library/sites/library/files/documents/Bios-Cons-Gen-040.pdf.

Rakha B.A., Ansari M.S., Akhter S., Hussain I., Blesbois E. Cryopreservation of Indian Red Jungle Fowl (Gallus gallus murghi) Semen. Anim. Reprod. Sci. 2016;174:45–55. doi: 10.1016/j.anireprosci.2016.09.004. PubMed DOI

Svoradová A., Kuželová L., Vašíček J., Olexíková L., Chrenek P. Cryopreservation of Chicken Blastodermal Cells and Their Quality Assessment by Flow Cytometry and Transmission Electron Microscopy. Biotechnol. Prog. 2018;34:778–783. doi: 10.1002/btpr.2615. PubMed DOI

Curry M.R., Kleinhans F.W., Watson P.F. Measurement of the Water Permeability of the Membranes of Boar, Ram, and Rabbit Spermatozoa Using Concentration-Dependent Self-Quenching of an Entrapped Fluorophore. Cryobiology. 2000;41:167–173. doi: 10.1006/cryo.2000.2277. PubMed DOI

Gáspárdy A., Renkó E., Somoskői B., Bába A., Cseh S. Practical Experience with Artificial Insemination (AI) Using Fresh Chilled and Frozen Semen in Mares. Acta Vet. Hung. 2020;68:85–90. doi: 10.1556/004.2020.00007. PubMed DOI

Barbas J.P., Mascarenhas R.D. Cryopreservation of Domestic Animal Sperm Cells. Cell Tissue Bank. 2009;10:49–62. doi: 10.1007/s10561-008-9081-4. PubMed DOI

Kumaresan A., Elango K., Datta T.K., Morrell J.M. Cellular and Molecular Insights Into the Etiology of Subfertility/Infertility in Crossbred Bulls (Bos taurus × Bos indicus): A Review. Front. Cell Dev. Biol. 2021;9:696637. doi: 10.3389/fcell.2021.696637. PubMed DOI PMC

Amann R.P., Waberski D. Computer-Assisted Sperm Analysis (CASA): Capabilities and Potential Developments. Theriogenology. 2014;81:5–17.e3. doi: 10.1016/j.theriogenology.2013.09.004. PubMed DOI

Galarza D.A., López-Sebastián A., Woelders H., Blesbois E., Santiago-Moreno J. Two-Step Accelerating Freezing Protocol Yields a Better Motility, Membranes and DNA Integrities of Thawed Ram Sperm than Three-Steps Freezing Protocols. Cryobiology. 2019;91:84–89. doi: 10.1016/j.cryobiol.2019.10.007. PubMed DOI

Peris-Frau P., Martín-Maestro A., Iniesta-Cuerda M., Sánchez-Ajofrín I., Mateos-Hernández L., Garde J.J., Villar M., Soler A.J. Freezing–Thawing Procedures Remodel the Proteome of Ram Sperm before and after In Vitro Capacitation. Int. J. Mol. Sci. 2019;20:4596. doi: 10.3390/ijms20184596. PubMed DOI PMC

Masoudi R., Sharafi M., Zareh Shahneh A., Towhidi A., Kohram H., Esmaeili V., Shahverdi A., Davachi N.D. Fertility and Flow Cytometry Study of Frozen-Thawed Sperm in Cryopreservation Medium Supplemented with Soybean Lecithin. Cryobiology. 2016;73:69–72. doi: 10.1016/j.cryobiol.2016.05.010. PubMed DOI

Khawar M.B., Gao H., Li W. Mechanism of Acrosome Biogenesis in Mammals. Front. Cell Dev. Biol. 2019;7:195. doi: 10.3389/fcell.2019.00195. PubMed DOI PMC

Meyers S.A. Chapter 5—Sperm Physiology. In: Samper J.C., editor. Equine Breeding Management and Artificial Insemination. 2nd ed. W.B. Saunders; Saint Louis, MO, USA: 2009. pp. 47–55.

Yanagimachi R. Mammalian Sperm Acrosome Reaction: Where Does It Begin before Fertilization? Biol. Reprod. 2011;85:4–5. doi: 10.1095/biolreprod.111.092601. PubMed DOI

Bernecic N.C., Donnellan E., O’Callaghan E., Kupisiewicz K., O’Meara C., Weldon K., Lonergan P., Kenny D.A., Fair S. Comprehensive Functional Analysis Reveals That Acrosome Integrity and Viability Are Key Variables Distinguishing Artificial Insemination Bulls of Varying Fertility. J. Dairy Sci. 2021;104:11226–11241. doi: 10.3168/jds.2021-20319. PubMed DOI

Parks J.E., Lynch D.V. Lipid Composition and Thermotropic Phase Behavior of Boar, Bull, Stallion, and Rooster Sperm Membranes. Cryobiology. 1992;29:255–266. doi: 10.1016/0011-2240(92)90024-V. PubMed DOI

De Las Mercedes Carro M., Peñalva D.A., Antollini S.S., Hozbor F.A., Buschiazzo J. Cholesterol and Desmosterol Incorporation into Ram Sperm Membrane before Cryopreservation: Effects on Membrane Biophysical Properties and Sperm Quality. Biochim. Biophys. Acta BBA—Biomembr. 2020;1862:183357. doi: 10.1016/j.bbamem.2020.183357. PubMed DOI