ORIGINAL_ARTICLE Reduced glutathione (GSH) and homologous ram seminal plasma (HSP), used as additives in cryopreserving (CP) media prior to freezing, showed conflicting results in retaining structural integrity and progressive motility in post-thawed ram spermatozoa. The aims of this research were: (1) to assess the effect of GSH and/or HSP supplementation via soybean-lecithin CP extender on cryopreserved ram spermatozoa viability, morphology and motility pattern; and (2) to assess the effect of incubation in the context of the previous aim. Quantitatively and qualitatively, homogenized and pooled ram ejaculates (N=10) were extended with one of the following extenders: control (C) – tris-based, GSH and HSP-free, experimental-1 (E1) – C + GSH 5 mM, experimental-2 (E2) – C + HSP 20 % and experimental-3 (E3) - GSH 5 mM + HSP 20 %. Following thawing, samples were taken at 0- and 3-hours from each group (n=10) and were assessed for spermatozoa viability, morphology, and motility pattern. C-0h samples yielded a spermatozoa population with low viability, altered head morphology and highly deviated motility pattern. E3-3h samples yielded spermatozoa with unaffected viability, head morphology and high progressive motility. In conclusion, E3 extender added to cryopreserved-thawed ram spermatozoa is most efficient in obtaining high viability, unaltered head morphology, and progressive motility. https://macvetrev.mk/Files/Article/2019/10.2478/macvetrev-2019-0022/macvetrev-2019-0022.pdf 2020-10-15T09:00:00 169 179 10.2478/macvetrev-2019-0022 ram ejaculates cryopreservation homologous seminal plasma reduced glutathione Martin Nikolovski nikolovski_m@fvm.ukim.edu.mk false 1 Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, 1000 Skopje, North Macedonia LEAD_AUTHOR Monika Dovenska false 2 Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, 1000 Skopje, North Macedonia AUTHOR Ksenija Ilievska false 3 Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, 1000 Skopje, North Macedonia AUTHOR Nikola Adamov false 4 Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, 1000 Skopje, North Macedonia AUTHOR Branko Atanasov false 5 Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, 1000 Skopje, North Macedonia AUTHOR Miroslav Radeski false 6 Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, 1000 Skopje, North Macedonia AUTHOR Daniela Kirovski false 7 Faculty of Veterinary Medicine – Belgrade, University of Belgrade, Serbia AUTHOR Vladimir Petkov false 8 Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, 1000 Skopje, North Macedonia AUTHOR Toni Dovenski false 9 Faculty of Veterinary Medicine - Skopje, Ss. Cyril and Methodius University in Skopje, 1000 Skopje, North Macedonia AUTHOR Salamon, S., Maxwell, W.M. (2000). Storage of ram semen. Animal Reproduction Science 62(1-3): 77-111. 1 10.1016/S0378-4320(00)00155-X De Paz, P., Mata-Campuzano, M., Tizado, E.J., Álvarez, M., Álvarez-Rodríguez, M., Herraez, P., Anel, L. (2011). The relationship between ram sperm head morphometry and fertility depends on the procedures of acquisition and analysis used. Theriogenology 76(7): 1313-1325. PMid:21798583 2 10.1016/j.theriogenology.2011.05.038 Maroto-Morales, A., Ramón, M., García-Álvarez, O., Montoro, V., Soler, A.J., Fernández-Santos, M.R., Garde, J.J. (2015). Sperm head phenotype and male fertility in ram semen. Theriogenology 84(9): 1536-1541. PMid:26318229 3 10.1016/j.theriogenology.2015.07.038 Robayo, I., Montenegro, V., Valdés, C., Cox, J. (2008). CASA assessment of kinematic parameters of ram spermatozoa and their relationship to migration efficiency in ruminant cervical mucus. Reproduction in Domestic Animals 43(4): 393-399. PMid:18282216 4 10.1111/j.1439-0531.2007.00920.x Drevet, J.R. (2006). The antioxidant glutathione peroxidasefamilyandspermatozoa: Acomplexstory. Molecular and Cellular Endocrinology 250(1-2): 70-79. PMid:16427183 5 10.1016/j.mce.2005.12.027 Barrios, B., Pérez-Pé, R., Gallego, M., Tato, A., Osada, J., Muiño-Blanco, T., Cebrián-Pérez, J.A. (2000). Seminal plasma proteins revert the cold- chock damage on ram sperm membrane1. Biology of Reproduction 63(5): 1531- 1537. PMid:11058562 6 10.1095/biolreprod63.5.1531 Marti, E., Marti, J.I., Muino-Blanco, T., Cebrian- Perez, J.A. (2008). Effect of the cryopreservation process on the activity and immunolocalization of antioxidant enzymes in ram spermatozoa. Journal of Andrology 29(4): 459- 467. PMid:18296478 7 10.2164/jandrol.107.003459 Pérez-Pé, R., Cebrián-Pérez, J.A., Muiño-Blanco, T. (2001). Semen plasma proteins prevent cold- shock membrane damage to ram spermatozoa. Theriogenology 56(3): 425-434. 8 10.1016/S0093-691X(01)00574-X Pérez-Pé, R., Grasa, P., Fernández-Juan, M., Peleato, M.L., Cebrián-Pérez, J.Á., Muiño-Blanco, T. (2002). Seminal plasma proteins reduce protein tyrosine phosphorylation in the plasma membrane of cold- shocked ram spermatozoa. Molecular Reproduction and Development 61(2): 226-233. PMid:11803559 9 10.1002/mrd.1152 Bucak, M.N., Ateşşahin, A., Yüce, A. (2008). Effect of anti-oxidants and oxidative stress parameters on ram semen after the freeze-thawing process. Small Ruminant Research 75(2-3): 128-134. 10 10.1016/j.smallrumres.2007.09.002 Budai, C., Egerszegi, I., Olah, J., Javor, A., Kovacs, A. (2014). The protective effect of antioxidants on liquid and frozen stored ram semen-review. J Anim Sci Biotechnol. 47, 46-52. 11 Bucak, M.N., Ateşşahin, A., Varışlı, Ö., Yüce, A., Tekin, N., Akçay, A. (2007). The influence of trehalose, taurine, cysteamine and hyaluronan on ram semen. Theriogenology 67(5): 1060-1067. PMid:17280711 12 10.1016/j.theriogenology.2006.12.004 Ghaoui, R.E.H., Gillan, L., Thomson, P.C., Evans, G., Maxwell, W.M.C. (2006). Effect of Seminal plasma fractions from entire and vasectomized rams on the motility characteristics, membrane status, and in vitro fertility of ram spermatozoa. Journal of Andrology 28(1): 109-122. PMid:16928891 13 10.2164/jandrol.106.001099 Juyena, N.S., Stelletta, C. (2011). Seminal plasma: an essential attribute to spermatozoa. Journal of Andrology 33(4): 536-551. PMid:22016346 14 10.2164/jandrol.110.012583 Maxwell, W.M., Stojanov, T. (1996). Liquid storage of ram semen in the absence or presence of some antioxidants. Reproduction, Fertility and Development 8(6): 1013. 15 10.1071/RD9961013 Uysal, O., Bucak, M.N. (2007). Effects of oxidized glutathione, bovine serum albumin, cysteine and lycopene on the quality of frozen-thawed ram semen. Acta Veterinaria Brno 76(3): 383-390. 16 10.2754/avb200776030383 Rovegno, M., Feitosa, W.B., Rocha, A.M., Mendes, C.M., Visintin, J.A., D‘Avila Assumpção, M.E.O. (2012). Assessment of post-thawed ram sperm viability after incubation with seminal plasma. Cell and Tissue Banking 14(2): 333-339. PMid:22661118 17 10.1007/s10561-012-9317-1 El-Hajj Ghaoui, R., Thomson, P., Leahy, T., Evans, G., Maxwell, W. (2007). Autologous whole ram seminal plasma and its vesicle-free fraction improve motility characteristics and membrane status but not in vivo fertility of frozen- thawed ram spermatozoa. Reproduction in Domestic Animals 42(5): 541-549. PMid:17845611 18 10.1111/j.1439-0531.2006.00819.x Mataveia, G.A., Terblanche, S.J., Nöthling, J.O., Gerber, D. (2010). Effect of heterologous seminal plasma and semen extenders on motility of frozen-thawed ram spermatozoa. J.S.Afr.Vet.Ass. 81(3): 139-142. PMid:21247037 19 10.4102/jsava.v81i3.135 García-Álvarez, O., Maroto-Morales, A., Martínez-Pastor, F., Garde, J.J., Ramón, M., Fernández- Santos, M.R., Soler, A. J. (2009). Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection. Theriogenology 72(2): 160-168. PMid:19349069 20 10.1016/j.theriogenology.2009.02.002 Maxwell, W.M.C., de Graaf, S.P., Ghaoui, R. E.H., Evans, G. (2007). Seminal plasma effects on sperm handling and female fertility. Soc Reprod Fertil Suppl. 64, 13-38. PMid:17491139 21 10.5661/RDR-VI-13 De Graaf, S.P., Evans, G., Gillan, L., Guerra, M.M.P., Maxwell, W.M.C., O‘Brien, J.K. (2007). The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa. Theriogenology 67(2): 217-227. PMid:16934321 22 10.1016/j.theriogenology.2006.07.008 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(3): 386-392. PMid:25684063 23 10.1111/rda.12500 Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes Text with EEA relevance OJ L 276, 20.10.2010, p. 33-79. 24 Hancock, J.L. (1951). A Staining Technique for the Study of Temperature-Shock in Semen. Nature 167(4243): 323-324. PMid:14806490 25 10.1038/167323b0 World Health Organization (2010). WHO laboratory manual for the examination of human semen. In: T.G. Cooper, (Ed.). Standard procedures (pp. 37). WHO Press. 26 Mortimer, S.T., Maxwell, W.M.C. (1999). Kinematic definition of ram sperm hyperactivation. Reproduction, Fertility and Development 11(1): 5. 27 10.1071/RD99019 Beasley, T.M., Schumacker, R.E. (1995). Multiple regression approach to analyzing contingency tables: post hoc and planned comparison procedures. The Journal of Experimental Education 64(1): 79-93. 28 10.1080/00220973.1995.9943797 Maxwell, W.M.C., De Graaf, S.P., Ghaoui, R. E., Evans, G. (2006). Seminal plasma effects on sperm handling and female fertility. Society of Reproduction and Fertility supplement 64, 13. 29 10.5661/RDR-VI-13 Ashworth, P.J., Harrison, R.A., Miller, N.G., Plummer, J.M., Watson, P.F. (1994). Survival of ram spermatozoa at high dilution: protective effect of simple constituents of culture media as compared with seminal plasma. Reproduction, Fertility and development 6(2): 173-180. 30 10.1071/RD9940173 Mann, T. (1954). The biochemistry of semen. In: Mann, T. (Ed.). Biochemistry of semen (pp. 12). London: Methuen. 31 10.5962/bhl.title.6940 Bergeron, A., Villemure, M., Lazure, C., Manjunath, P. (2005). Isolation and characterization of the major proteins of ram seminal plasma. Mol Reprod Dev. 71(4): 461-470. PMid:15892046 32 10.1002/mrd.20310 Cardozo, J., Fernández-Juan, M., Cebrián-Pérez, J., Muiño-Blanco, T. (2007). Identification of RSVP14 and RSVP20 components by two-dimensional electrophoresis and western-blotting. Reproduction in Domestic Animals 43, 15-21. PMid:18199253 33 10.1111/j.1439-0531.2006.00845.x Colás, C., Junquera, C., Pérez-Pé, R., Cebrián-Pérez, J. A., Muiño-Blanco, T. (2009). Ultrastructural study of the ability of seminal plasma proteins to protect ram spermatozoa against cold-shock. Microscopy Research and Technique 72(8): 566-572. PMid:19322897 34 10.1002/jemt.20710 Malo, A.F., Gomendio, M., Garde, J., Lang-Lenton, B., Soler, A.J., Roldan, E.R. (2006). Sperm design and sperm function. Biology Letters 2(2): 246-249. PMid:17148374 PMCid:PMC1618917 35 10.1098/rsbl.2006.0449 Salamon, S., Maxwell, W.M. (2000). Storage of ram semen. Animal Reproduction Science 62(1-3): 77-111. 36 10.1016/S0378-4320(00)00155-X Mortimer, S. (1997). A critical review of the physiological importance and analysis of sperm movement in mammals. Human Reproduction Update 3(5): 403-439. PMid:9528908 37 10.1093/humupd/3.5.403