THE EFFECT OF EXTENDING RAM SPERM BEFORE AND AFTER CRYOPRESERVATION ON THEIR VIABILITY AND VELOCITY
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The current study aimed to assess the effect of adding cryopreserving extender (soy-bean lecithin-SBLE), reduced glutathione (GSH), and seminal plasma (SP) before and after thawing on viability and velocity of cryopreserved ram sperm in liquid nitrogen. Fresh ejaculates (Ovchepolska pramenka rams, n=10) were collected and pooled. One portion was extended up to 50 million/ml with SBLE (control C-a), SBLE and GSH 5 mM (E1-a), SBLE and SP 20 vol% (E2-a), and SBLE, GSH 5 mM and SP 20 vol% (E3-a), respectively. The second portion was extended with SBLE to 100 million/ml. Both portions were cryopreserved in liquid nitrogen. Following thawing, the second portion was extended in the same manner to 50 million/ml and was separated into C-b, E1-b, E2-b, and E3-b, respectively. Each group was sampled in ten replicates immediately following thawing. Thawed samples were analyzed for viability (Hancock-2 stain), and velocity (Hamilton Thorne, USA). Each sample included at least 200 cells and the results were expressed in percent values (mean±SEM). Normality (Kolmogorov) and variance comparison (factorial-ANOVA) were performed in Statistica 8 with a significance level p<0.05. E2-a (57.58% ±2.40) and E3-a (56.94% ±1.85) yielded significantly higher viability compared to the C-a (40.73 ±1.53). There were no significant differences between C-b (50.00% ±2.33), E1-b (43.61% ±1.37), E2-b (49.16% ±1.50), and E3-b (48.50% ±1.85). In conclusion, the addition of SBLE, GSH, and SP prior vs after cryopreservation has a significant effect on thawed ram sperm viability and velocity.
Barbas, J. P., & Mascarenhas, R. D. (2008). Cryopreservation of domestic animal sperm cells. Cell and Tissue Banking, 10(1), 49–62. doi:10.1007/s10561-008-9081-4
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-Shock Damage on Ram Sperm Membrane1. Biology of Reproduction, 63(5), 1531–1537. doi:10.1095/biolreprod63.5.1531
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
Camara DR, Mello-Pinto MMC, Pinto LC, Brasil OO, Nunes JF, Guerra MMP (2011) Effects of reduced glutathione and catalase on the kinematics and membrane functionality of sperm during liquid storage of ram semen. Small Rumin Res 100:44–49
de Graaf SP, Evans G, Gillan L, Guerra MMP, Maxwell WMC & O’Brien JK 2007 The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa. Theriogenology 67 217–227
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
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.
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
Leahy, T., & Gadella, B. M. (2011). Sperm surface changes and physiological consequences induced by sperm handling and storage. Reproduction, 142(6), 759.
Leahy T & de Graaf SP 2012 Seminal plasma and its effect on ruminant spermatozoa during processing. Reproduction in Domestic Animals 47, 207–213.
Leahy, T., Rickard, J. P., Bernecic, N. C., Druart, X., & de Graaf, S. P. (2019). Ram seminal plasma and its functional proteomic assessment. Reproduction, 157(6), R243-R256.
Marti, E., Mara, L., Marti, J. I., Muiño-Blanco, T., & Cebrián-Pérez, J. A. (2007). Seasonal variations in antioxidant enzyme activity in ram seminal plasma. Theriogenology, 67(9), 1446-1454.
Morrier, A., Castonguay, F., & Bailey, J. L. (2003). Conservation of fresh ram spermatozoa at 5 C in the presence of seminal plasma. Canadian journal of animal science, 83(2), 221-227.
Muiño‐Blanco, T., Pérez‐Pé, R., & Cebrián‐Pérez, J. A. (2008). Seminal plasma proteins and sperm resistance to stress. Reproduction in domestic animals, 43, 18-31.
Nikolovski, M., Dovenska, M., Ilievska, K., Adamov, N., Atanasov, B., Radeski, M., Kirovski, D., Petkov, V., & Dovenski, T. (2019). Homologous seminal plasma and glutathione promote pre-capacitation motility and structural stability of cryopreserved ram spermatozoa. Mac Vet Rev 42(2), 169-179
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.
Pini, T., Leahy, T., & de Graaf, S. P. (2018). Sublethal sperm freezing damage: Manifestations and solutions. Theriogenology, 118, 172–181. doi:10.1016/j.theriogenology.2018
Silva S, Soares A, Batista A, Almeida F, Nunes J, Peixoto C, Guerra M (2011) 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 46:874–881
Tseng, H. C., Lee, R. K. K., Hwu, Y. M., Lu, C. H., Lin, M. H., & Li, S. H. (2013). Mechanisms underlying the inhibition of murine sperm capacitation by the seminal protein, SPINKL. Journal of Cellular Biochemistry, 114(4), 888-898.
World Health Organization (2010). WHO Laboratory Manual for the Examination of Human Semen. In: Cooper, T.G. (Ed.). Standard procedures (pp. 37). WHO Press
Zeitoun, M. M., & Al-Damegh, M. A. (2014). Effect of nonenzymatic antioxidants on sperm motility and survival relative to free radicals and antioxidant enzymes of chilled-stored ram semen. Open Journal of Animal Sciences, 5(01), 50.