Research-Article| Volume 72, ISSUE 10, P2683-2690, October 1989

Induction of Bovine Sperm Capacitation by TEST-Yolk Semen Extender1

  • A. Ijaz
    Departments of Animal Science, and Large Animal Clinical Sciences, University of Minnesota, St. Paul 55108
    Search for articles by this author
  • A.G. Hunter
    Departments of Animal Science, and Large Animal Clinical Sciences, University of Minnesota, St. Paul 55108
    Search for articles by this author
  • Author Footnotes
    1 Published as Paper Number 16,388 of the Scientific Journal Series of the Minnesota Agricultural Experiment Station on research conducted under Minnesota Agricultural Experiment Station Project Number 72 supported by Hatch funds.
      This paper is only available as a PDF. To read, Please Download here.


      Ejaculated bull semen was diluted 1:10 in the TEST-yolk buffer, cooled slowly to 4°C, and stored for up to 48 h. Aliquots were taken at 0, 4, 8, 16, 24, and 48 h and washed once or three times in bovine serum albumin-saline and the sperm pellets resuspended in this saline. Fertilization of zona-free hamster oocytes was used to assess sperm capacitation. Motility differed between samples washed once or three times (53.7 vs. 21.7%). Motility was highest at 4 h storage but did not differ between 16, 24, or 48 h of storage. More sperm without intact acrosomes were found at 4 h than at 0 h, but the percentage did not change further until after 24 h. Penetration of oocytes was not different between sperm washed once or three times (28.5 vs. 26.9%). No penetration occurred at 0 h, and highest penetration rates occurred at 4 and 8 h of storage (32.1 and 33.4%). Penetration rates at 16, 24, and 48 h were not different (25.3, 25.2, 22.5%). In conclusion, storage of bull sperm in TEST-yolk buffer for 4 to 48 h resulted in capacitation. Even though capacitation was induced by 4 h, at least 71% of the sperm population had not undergone an acrosome reaction by 48 h of storage. This may explain why penetrability was maintained over this period.


        • Austin C.R.
        Observations on the penetration of the sperm into the mammalian egg.
        Aust. J. Sci. Res. 1951; B4: 581
        • Bedford J.M.
        Significance of the need for sperm capacitation before fertilization in eutherian mammals.
        Biol. Reprod. 1983; 28: 108
        • Berger T.
        • Marrs R.P.
        • Saito H.
        • Kletzky O.A.
        Factors affecting human sperm penetration of zona-free hamster ova.
        Am. J. Obstet. Gynecol. 1983; 145: 397
        • Blackshaw A.W.
        The motility of ram and bull spermatozoa in dilute suspension.
        J. Gen. Physiol. 1953; 36: 444
        • Bolanos J.R.
        • Overstreet J.W.
        • Katz D.F.
        Human sperm penetration of zona-free hamster eggs after storage of the semen for 48 hours at 2°C to 5°C.
        Fertil. Steril. 1983; 39: 536
        • Bousquet D.
        • Brackett B.G.
        Penetration of zona-free hamster ova as a test to assess fertilizing ability of bull sperm after frozen storage.
        Theriogenology. 1982; 17: 199
        • Bousquet D.
        • Brackett B.G.
        • Dressel M.A.
        • Allen C.H.
        Efforts to correlate laboratory with field observations on bull sperm fertility.
        Theriogenology. 1983; 20: 601
        • Brackett B.G.
        • Oh Y.K.
        • Evans J.F.
        • Donawick W.J.
        Fertilization and early development of cow ova.
        Biol. Reprod. 1980; 23: 189
        • Bryan J.H.D.
        • Akruk S.R.
        A naphthol yellow S and erythrosin B staining procedure for use in studies of the acrosome reaction of rabbit spermatozoa.
        Stain Technol. 1977; 52: 47
        • Byrd W.
        In vitro capacitation and chemically induced acrosome reaction in bovine spermatozoa.
        J. Exp. Zool. 1981; 215: 35
        • Chan S.Y.W.
        • Li S.Q.
        • Wang C.
        TEST-egg yolk buffer storage increases the capacity of human sperm to penetrate hamster eggs in vitro.
        Int. J. Androl. 1987; 10: 517
        • Chang M.C.
        The fertilizing capacity of spermatozoa deposited into the Fallopian tubes.
        Nature. 1951; 168: 697
        • Chang M.C.
        A detrimental effect of seminal plasma on the fertilizing capacity of sperm.
        Nature. 1957; 179: 258
        • Dott H.M.
        • Walton A.
        Effects of dilution and washing on ram spermatozoa studied by the flow dialysis technique.
        J. Reprod. Fertil. 1960; 1: 350
        • Dukelow W.R.
        • Chemoff H.N.
        • Williams W.L.
        Properties of decapacitation factor and presence in various species.
        J. Reprod. Fertil. 1967; 14: 393
        • Duncan D.B.
        Multiple range test and multiple F test.
        Biometrics. 1955; 11: 1
        • Graham E.F.
        • Crabo B.G.
        • Brown K.I.
        Effect of some Zwitter ion buffers on the freezing and storage of spermatozoa.
        I. Bull. J. Dairy Sci. 1972; 55: 372
        • Graham J.K.
        • Foote R.H.
        Dilauroylphos phatidylcholine liposome effects on acrosome reaction and in vitro penetration of zona-free hamster eggs by bull sperm: 1. A fertility assay for fresh semen.
        Gamete Res. 1987; 16: 133
        • Graham J.K.
        • Foote R.H.
        • Parrish J.J.
        Effect of dilauroylphosphatidylcholine on the acrosome reaction and subsequent penetration of bull spermatozoa into zona-free hamster eggs.
        Biol. Reprod. 1986; 35: 413
        • Hensleigh H.C.
        • Hunter A.G.
        In vitro maturation of bovine cumulus enclosed primary oocytes and their subsequent in vitro fertilization and cleavage.
        J. Dairy Sci. 1985; 68: 1456
        • Hunter A.G.
        • Nornes H.O.
        Characterization and isolation of a sperm-coating antigen from rabbit seminal plasma with capacity to block fertilization.
        J. Reprod. Fertil. 1969; 20: 419
        • Ijaz A.
        • Hunter A.G.
        Effect of buffers containing TES and/or tris on bovine sperm motility and their ability to penetrate zona-free hamster oocytes.
        Biol. Rcprod. 1988; 38 (Abstr.): 97
        • Iritani A.
        • Niwa K.
        Capacitation of bull sperm and fertilization in vitro of cattle follicular oocytcs matured in culture.
        J. Reprod. Fertil. 1977; 50: 119
        • Jaskey D.G.
        • Cohen M.R.
        Twenty-four to ninety-six hour storage of human spermatozoa in TEST-yolk buffer.
        Fertil. Steril. 1981; 35: 205
        • Johnson W.L.
        • Hunter A.G.
        Seminal antigens’: their alterations in the genital tract of female rabbits and during partial in vitro capacitation with beta amylase and beta glucuronidase.
        Biol. Reprod. 1972; 7: 332
        • Johnson A.R.
        • Syms A.J.
        • Lipshultz L.I.
        • Smith R.G.
        Conditions influencing human sperm capacitation and penetration of zona-free hamster ova.
        Fertil. Steril. 1984; 41: 603
        • Jones R.C.
        • Holt W.V.
        The effect of washing on the ultrastructure and cytochemistry of ram spermatozoa.
        J. Reprod. Fertil. 1974; 41: 159
        • Langlais J.
        • Roberts K.D.
        A molecular membrane model of sperm capacitation and the acro some reaction of mammalian spermatozoa.
        Gamete Res. 1985; 12: 183
        • Lenz R.W.
        • Ball G.D.
        • Lohse J.K.
        • First N.L.
        • Ax R.L.
        Chondroitin sulphate facilitates an acrosome reaction in bovine spermatozoa as evidenced by light microscopy, electron microscopy and in vitro fertilization.
        Biol. Reprod. 1983; 28: 683
        • Lorton S.P.
        • First N.L.
        Hyaluronidase does not disperse the cumulus oophorus surrounding bovine ova.
        Biol. Reprod. 1979; 21: 301
        • Maker A.
        • Jakobi P.
        Effects of shaking and cenrifugation on human sperm motility.
        Arch. Androl. 1981; 7: 21
        • Oliphant G.
        • Singhas C.A.
        Iodination of rabbit sperm plasma membrane: relationship of specific surface proteins to epididymal function and sperm capacitation.
        Biol. Reprod. 1979; 21: 937
        • Pickett B.W.
        • Sullivan J.J.
        • Byers W.W.
        • Pace M.M.
        • Remmenga E.E.
        Effect of centrifugation and seminal plasma on motility and fertility of stallion and bull spermatozoa.
        Fertil. Steril. 1975; 26: 167
        • Reddy J.M.
        • Stark R.A.
        • Zaneveld L.J.D.
        A high molecular weight antifertility factor from human seminal plasma.
        J. Reprod. Fertil. 1979; 57: 437
      1. SAS Institute, Inc. 1982. SAS User's guide:statistics. 1982 ed. Cary, NC.

        • Singer R.
        • Bonnet M.
        • Allabonf P.
        • Chomers T.
        Sensitivity of human spermatozoa to various isolation procedures: differences in relation to sperm count.
        Int. J. Fertil. 1979; 24: 33
        • Talbot P.
        • Chacon R.
        Detection of modifications on the tail of capacitated guinea pig sperm using lectins.
        J. Exp. Zool. 1981; 216: 435
        • Talbot P.
        • Chacon R.S.
        Ultrastructural observations on binding and membrane fusion between human sperm and zona pellucida-free hamster oocytes.
        Fertil. Steril. 1983; 37: 240
        • Tarlatzis B.C.
        • Murillo O.
        • Laufer N.
        • Makler A.
        • Naftolin F.
        • Dechemey A.H.
        The effects of different methods of centrifugation on sperm motility, morphology and acrosome integrity.
        J. Androl. 1984; 5 (Abstr.): 14
        • Thompson J.G.E.
        • Cummins J.M.
        The effect of washing and protein supplementation on the acrosome reaction of ram spermatozoa in vitro.
        Anim. Reprod. Sci. 1985; 9: 75
        • Wheeler M.B.
        • Seidel Jr., G.E.
        Zona-pellucida penetration assay for capacitation of bovine sperm.
        Gamete Res. 1987; 18: 237
        • Yanagimachi R.
        Penetration of guinea pig spermatozoa into hamster eggs in vitro.
        J. Reprod. Fertil. 1972; 28: 477
        • Yanagimachi R.
        Zona-free hamster eggs: their use in assessing fertilizing capacity and examining chromosomes of human spermatozoa.
        Gamete Res. 1984; 10: 187
        • Yanagimachi R.
        • Yanagimachi H.
        • Rogers B.J.
        The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa.
        Biol. Reprod. 1976; 15: 471
        • Young L.G.
        • Goodman S.A.
        Analysis of lipid and protein components of ejaculated bull sperm surface and seminal plasma.
        Gamete Res. 1982; 6: 281
        • Zavos P.M.
        • Goodpasture J.C.
        • Zaneveld L.J.D.
        • Cohen M.R.
        Motility and enzyme activity of human spermatozoa stored for 24 hours at +5°C and −196°C.
        Fertil. Steril. 1980; 34: 607