Animal Reproduction (AR)
Animal Reproduction (AR)

Reduction of seminal plasma concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa

Reza Rajabi-Toustani; Mohammad Roostaei-Ali Mehr; Rasool Motamedi-Mojdehi

Downloads: 1
Views: 266


  This study was conducted to investigate the effect of different levels of seminal plasma (SP) and cold-shock on ram spermatozoa during 36 h storage at 5°C. In both ejaculated spermatozoa coated with egg yolk (second ejaculate; coated spermatozoa) and epididymal spermatozoa, samples were treated with 0, 50 and 100% seminal plasma. Different levels of seminal plasma were added on the basis of ram spermatocrit (32%). Then half of aliquots were suddenly put on ice water (cold-shock) and other half were gradually (0.25°C/min) chilled (non- cold shock). Sperm motility, viability and functional membrane integrity were determined in both aliquots at 0, 12, 24 and 36 h storage at 5°C. Under non- cold shock and cold-shock conditions, coated spermatozoa treated with 0% SP showed the highest motility compared to ejaculated spermatozoa (first ejaculate; uncoated spermatozoa) after 12, 24 and 36 h of storage at 5°C (P<0.05). Under non- cold shock and cold-shock conditions, viability and functional membrane integrity was higher in the coated spermatozoa treated with 0% SP than in the uncoated spermatozoa during 36 h storage (P<0.05). There was no significant difference between coated spermatozoa treated with 0 and 50% SP in the percentage of motility and viability after 24 and 36 h of storage (P>0.05). Under non- cold shock and cold-shock conditions, the percentage of motility of epididymal spermatozoa treated with 0% SP was significantly (P<0.05) higher than those treated with 100% SP after 36 h of storage at 5°C. In conclusion, removal of seminal plasma and/or reduction (up to 50%) of its concentration can decrease detrimental effects of seminal plasma on chilled ram spermatozoa.


coated spermatozoa, cold-shock, ram spermatozoa, seminal plasma


Aboagla EME, Terada T. Trehalose-enhanced fluidity of the goat sperm membrane and its protection during freezing. Biol Reprod. 2003;69(4):1245-50. PMid:12801983.

Bathgate R, Maxwell W, 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. Reprod Domest Anim. 2006;41(1):68-73. PMid:16420332.

Bergeron A, Crête MH, Brindle Y, Manjunath P. Low-density lipoprotein fraction from hen’s egg yolk decreases the binding of the major proteins of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. Biol Reprod. 2004;70(3):708-17. PMid:14613896.

Bispo CAS, Pugliesi G, Galvão P, Rodrigues MT, Ker PG, Filgueiras B, Carvalho GR. Effect of low and high egg yolk concentrations in the semen extender for goat semen cryopreservation. Small Rumin Res. 2011;100(1):54-8.

Björndahl L, Söderlund I, Kvist U. Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment. Hum Reprod. 2003;18(4):813-6. PMid:12660276.

Bogart R, Mayer DT. The effects of egg yolk on the various physical and chemical factors detrimental to spermatozoan viability. J Anim Sci. 1950;9(2):143-52. PMid:15415361.

Calvete JJ, Raida M, Gentzel M, Urbanke C, Sanz L, Töpfer-Petersen E. Isolation and characterization of heparin- and phosphorylcholine-binding proteins of boar and stallion seminal plasma: primary structure of porcine pB1. FEBS Lett. 1997;407(2):201-6. PMid:9166899.

Cebrián J, Muiño-Blanco T, Pérez-Pé RCA. Manejo y conservación del semen. In: Abecia Martínez A, Forcada Miranda F, editors. Manejo reproductivo en ganado ovino. Zaragoza, Spain: Editorial Servet; 2010.

Darin-Bennett A, White IG. Influence of the cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology. 1977;14(4):466-70. PMid:560945.

de Menezes EB, van Tilburg M, Plante G, de Oliveira RV, Moura AA, Manjunath P. Milk proteins interact with goat Binder of SPerm (BSP) proteins and decrease their binding to sperm. Cell Tissue Res. 2016;366(2):427-42. PMid:27432314.

De Pauw IM, Van Soom A, Maes D, Verberckmoes S, de Kruif A. Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa. Theriogenology. 2003;59(5-6):1109-22. PMid:12527060.

Desnoyers L, Manjunath P. Major proteins of bovine seminal plasma exhibit novel interactions with phospholipid. J Biol Chem. 1992;267(14):10149-55. PMid:1577785.

Dott HM, Harrison RA, Foster GC. The maintenance of motility and the surface properties of epididymal spermatozoa from bull, rabbit and ram in homologous seminal and epididymal plasma. J Reprod Fertil. 1979;55(1):113-24. PMid:34036.

Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, Crowe JH. Cold shock damage is due to lipid phase transitions in cell membranes: A demonstration using sperm as a model. J Exp Zool. 1993;265(4):432-7. PMid:8463792.

Evans G, Maxwell WMC, Salamon S. Salamon’s artificial insemination of sheep and goats. London: Butterworths; 1987.

Foulkes JA, Sweasey D, Goodey RG. Fertility of bull spermatozoa in egg-yolk diluents of varied lipid fatty acid composition. J Reprod Fertil. 1980;60(1):165-9. PMid:7431317.

Garcia MA, Graham EF. Effects of low-molecular-weight fractions (LMWF) from milk, egg yolk, and seminal plasma on freezability of bovine spermatozoa. Cryobiology. 1987;24(5):429-36. PMid:3652723.

García W, Tabarez A, Palomo MJ. Effect of the type of egg yolk, removal of seminal plasma and donor age on ram sperm cryopreservation. Anim Reprod. 2017;14(4):1124-32.

García-Artiga C. Test de endósmosisen ovino. In: Proceedings of the 7th International Meeting on Animal Reproduction; 1994; Murcia, Spain. Murcia: Consejería de Agricultura Ganadería y Pesca; 1994. p. 77-81.

Gilmore JA, Liu J, Peter AT, Critser JK. Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol Reprod. 1998;58(1):28-36. PMid:9472919.

Graham JK, Foote RH. Effect of several lipids, fatty acyl chain length, and degree of unsaturation on the motility of bull spermatozoa after cold shock and freezing. Cryobiology. 1987;24(1):42-52. PMid:3816287.

Graham JK. Effect of seminal plasma on the motility of epididymal and ejaculated spermatozoa of the ram and bull during the cryopreservation process. Theriogenology. 1994;41(5):1151-62. PMid:16727467.

Iwamoto T, Tanaka H, Osada T, Shinagawa T, Osamura Y, Gagnon C. Origin of a sperm motility inhibitor from boar seminal plasma. Mol Reprod Dev. 1993;36(4):475-81. PMid:8305210.

Jeyendran RS, Van der Ven HH, Zaneveld LJ. The hypoosmotic swelling test: an update. Arch Androl. 1992;29(2):105-16. PMid:1456832.

Kaabi M, Paz P, Alvarez M, Anel E, Boixo JC, Rouissi H, Herraez P, Anel L. Effect of epididymis handling conditions on the quality of ram spermatozoa recovered post-mortem. Theriogenology. 2003;60(7):1249-59. PMid:14511779.

Kawano N, Shimada M, Terada T. Motility and penetration competence of frozen–thawed miniature pig spermatozoa are substantially altered by exposure to seminal plasma before freezing. Theriogenology. 2004;61(2-3):351-64. PMid:14662134.

Killen ID, Caffery GJ. Uterine insemination of ewes with the aid of a laparoscope. Aust Vet J. 1982;59(3):95. PMid:6218800.

Lamirande E, Belles-Isles M, Gagnon C. Characteristics of a seminal plasma inhibitor of sperm motility. Ann N Y Acad Sci. 1984;438:125-31. PMid:6242010.

Lasley JF, Mayer DT. A variable physiological factor necessary for the survival of bull spermatozoa. J Anim Sci. 1944;3(2):129-35.

Lusignan MF, Bergeron A, Crête MH, Lazure C, Manjunath P. Induction of epididymal boar sperm capacitation by pB1 and BSP-A1/-A2 proteins, members of the BSP protein family. Biol Reprod. 2007;76(3):424-32. PMid:17123943.

Manjunath P, Nauc V, Bergeron A, Ménard M. Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen’s egg yolk. Biol Reprod. 2002;67(4):1250-8. PMid:12297543.

Manjunath P, Thérien I. Role of seminal plasma phospholipid-binding proteins in sperm membrane lipid modification that occurs during capacitation. J Reprod Immunol. 2002;53(1-2):109-19. PMid:11730909.

Martin JC, Klug E, Günzel AR. Centrifugation of stallion semen and its storage in large volume straws. J Reprod Fertil Suppl. 1979;(27):47-51. PMid:289825.

Martins JAM, Souza CEA, Silva FDA, Cadavid VG, Nogueira FC, Domont GB, Oliveira JTA, Moura AA. Major heparin-binding proteins of the seminal plasma from Morada Nova rams. Small Rumin Res. 2013;113(1):115-27.

Martinus RD, Molan PC, Shannon P. Deleterious effect of seminal plasma in the cryo-preservation of bovine spermatozoa. N Z J Agric Res. 1991;34(3):281-5.

Maxwell WM, Salamon S. Liquid storage of ram semen: a review. Reprod Fertil Dev. 1993;5(6):613-38. PMid:9627724.

Maxwell WMC, Mendoza G, White IG. Post-thawing survival of motile ram sperm after isolation by layering on protein columns. Theriogenology. 1984;21(4):601-6. PMid:16725910.

Maxwell WMC, Watson PF. Recent progress in the preservation of ram semen. Anim Reprod Sci. 1996;42(1-4):55-65.

Mehr MR-A, Mousavi M, Ghadamyari M. Effect of seminal plasma proteins on membrane cholesterol efflux of ram epididymal spermatozoa. Small Rumin Res. 2015;129:88-91.

Moore AI, Squires EL, Graham JK. Effect of seminal plasma on the cryopreservation of equine spermatozoa. Theriogenology. 2005;63(9):2372-81. PMid:15910920.

Muiño-Blanco T, Pérez-Pé R, Cebrián-Pérez J. Seminal Plasma Proteins and Sperm Resistance to Stress. Reprod Domest Anim. 2008;43(Suppl 4):18-31. PMid:18803753.

Nass SJ, Miller DJ, Winer MA, Ax RL. Male accessory sex glands produce heparin-binding proteins that bind to cauda epididymal spermatozoa and are testosterone dependent. Mol Reprod Dev. 1990;25(3):237-46. PMid:2331373.

National Research Council. Nutrient requirements of sheep. 6th ed. Washington: National Academies Press; 1985.

Phillips PH, Lardy HA. A yolk-buffer pabulum for the preservation of bull semen. J Dairy Sci. 1940;23(5):399-404.

Poiani A. Complexity of seminal fluid: a review. Behav Ecol Sociobiol. 2006;60(3):289-310.

Quinn PJ, Chow PY, White IG. Evidence that phospholipid protects ram spermatozoa from cold shock at a plasma membrane site. J Reprod Fertil. 1980;60(2):403-7. PMid:7431346.

Rajabi-Toustani R, Roostaei-Ali Mehr M, Motamedi-Mojdehi R. Effect of different levels of egg yolk on ram sperm coating and preserving at 5°C. Iran J Vet Res. 2014;15(2):168-71.

Ritar AJ, Salamon S. Effects of month of collection, method of processing, concentration of egg yolk and duration of frozen storage on viability of Angora goat spermatozoa. Small Rumin Res. 1991;4(1):29-37.

Roostaei Ali-Mehr M, Rajabi-Toustani R, Motamedi-Mojdehi R. Leptin receptor in ram epididymal spermatozoa. Engineering. 2012;4(20B):121-3.

Roostaei-Ali Mehr M, Sharafi F. The effect of seminal plasma on the quality of coated ram frozen-thawed spermatozoa. Iran J Vet Res. 2013;14:305-12.

SAS Institute Inc. SAS® user’s guide. Statistics. Cary, NC; 2002.

Schmehl MK, Anderson SP, Vazquez IA, Graham EF. The effect of dialysis of extended ram semen prior to freezing on post-thaw survival and fertility. Cryobiology. 1986;23(5):406-14. PMid:3769516.

Thérien I, Bleau G, Manjunath P. Phosphatidylcholine-binding proteins of bovine seminal plasma modulate capacitation of spermatozoa by heparin. Biol Reprod. 1995;52(6):1372-9. PMid:7632845.

Thérien I, Moreau R, Manjunath P. Major proteins of bovine seminal plasma and high-density lipoprotein induce cholesterol efflux from epididymal sperm. Biol Reprod. 1998;59(4):768-76. PMid:9746724.

van Tilburg M, Sousa S, Lobo MDP, Monteiro-Azevedo ACOM, Azevedo RA, Araújo AA, Moura AA. Mapping the major proteome of reproductive fluids and sperm membranes of rams: from the cauda epididymis to ejaculation. Theriogenology. 2021;159:98-107. PMid:33126182.

Vishwanath R, Shannon P, Curson B. Cationic extracts of egg yolk and their effects on motility, survival and fertilising ability of bull sperm. Anim Reprod Sci. 1992;29(3-4):185-94.

Watson P. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci. 2000;60-61:481-92. PMid:10844218.

Way AL, Killian GJ. Capacitation and induction of the acrosome reaction in bull spermatozoa with norepinephrine. J Androl. 2002;23(3):352-7. PMid:12002437.

Submitted date:

Accepted date:

60ae9216a9539550e824c502 animreprod Articles
Links & Downloads

Anim Reprod

Share this page
Page Sections