Infertility pp 101-114 | Cite as

Sperm Preservation: Fundamental Cryobiology and Practical Implications

  • P. F. Watson
  • J. K. Critser
  • P. Mazur


Human spermatozoa were first frozen successfully almost 40 years ago [1]. Although developments in dilution media, packaging and storing have occurred in the intervening period, little is known specifically about the effects of freezing and thawing on spermatozoa. This is largely due to the fact that a sufficient number of cells survive the challenge to give a reasonable expectation of fertilisation; but the shift to the exclusive use of cryopreserved semen for donor insemination, occasioned by the awareness of the risks particularly of AIDS transmission [2–4], has resulted in a renewed critical interest in the process of cryopreservation. This review will cover the more significant contributions over the past few years, and develop an argument for a sustained fundamental approach to sperm cryobiology.


Acquire Immune Deficiency Syndrome Artificial Insemination Human Spermatozoon Acrosome Reaction Fresh Spermatozoon 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bunge RG, Keettel W, Sherman J. Clinical use of frozen semen, report of 4 cases. Fert Steril 1954; 5: 520–9.Google Scholar
  2. 2.
    Stewart G, Tyler JPP, Cunningham AL, Barr JA, Driscoll GL, Gold J, Lamont BJ. Transmission of human T-cell lymphotropic virus type III (HTLV-III) by artificial insemination by donor. Lancet 1985; ii:581–4.Google Scholar
  3. 3.
    DHSS. Acquired immune deficiency syndrome (AIDS) and artificial insemination — guidance for doctors and AI clinics. London: Department of Health and Social Security, 1986.Google Scholar
  4. 4.
    American Fertility Society. New guidelines for the use of semen donor insemination: 1990. Fert Steril 1990; 53 (Suppl 1): 1S - 13S.Google Scholar
  5. 5.
    Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 1984; 247: C125 — C142.PubMedGoogle Scholar
  6. 6.
    Critser JK, Kleinhans FW, Mazur P. Cryopreservation of human sperm. Cryobiology 1991; 28: 525–6.Google Scholar
  7. 7.
    Duncan AE, Watson PF. Predictive water loss curves for ram spermatozoa during cryopreservation: comparison with experimental observations. Cryobiology 1992; 29: 95–105.PubMedCrossRefGoogle Scholar
  8. 8.
    Watson PF, Kunze E, Cramer P, Hammerstedt RH. A comparison of critical osmolality, hydraulic conductivity and its activation energy in fowl and bull spermatozoa. J Androl 1992; (in press).Google Scholar
  9. 9.
    Nodes EE, Mazur P, Boldt HD, Kleinhans FW, Critser JK. Hydraulic conductivity (Le) and its activation energy (E a ) in human sperm. J Androl 1992 (Suppl): P-30.Google Scholar
  10. 10.
    Dick DAT. Cell water. Washington DC: Butterworths, 1966.Google Scholar
  11. 11.
    Nodes EE, Ruffing NA, Kleinhans FW et al. Critical tonicity determination of sperm using dual fluorescent staining and flow cytometry. In: Johnson LA, Rath D, eds. Boar semen preservation II. Berlin, Hamburg: Paul Parey Scientific Publishers, 1991; 359–64.Google Scholar
  12. 12.
    Rall WF, Mazur P, McGrath JJ. Depression of the ice-nucleation temperature of rapidly cooled mouse embryos by glycerol and dimethyl sulfoxide. Biophys J 1983; 41: 1–12.PubMedCrossRefGoogle Scholar
  13. 13.
    Ford WCL, Harrison A. D-[1–14C]mannitol and [U-14C]sucrose as extracellular space markers for human spermatozoa and the uptake of 2-deoxyglucose. J Reprod Fertil 1983; 69: 479–87.PubMedCrossRefGoogle Scholar
  14. 14.
    Laufer N, Segal S, Yaffe H, Svartz H, Grover NB. Volume and shape of normal human spermatozoa. Fertil Steril 1977; 28: 456–8.PubMedGoogle Scholar
  15. 15.
    Jeyendran RS, Karuhn RF, Van der Ven HH, Perez-Pelaez M. Volumetric analysis of human spermatozoa. Andrologia 1987; 19: 54–7.PubMedCrossRefGoogle Scholar
  16. 16.
    Weidel L, Prins GS. Cryosurvival of human spermatozoa frozen in eight different buffer systems. J Androl 1987; 8: 41–7.PubMedGoogle Scholar
  17. 17.
    Mahadevan M, Trounson AO. Effects of cryoprotective media and dilution methods on the preservation of human spermatozoa. Andrologia 1983; 15: 355–66.PubMedCrossRefGoogle Scholar
  18. 18.
    Watson PF. Artificial insemination and the preservation of semen. In: Lamming GE, ed. Marshall’s physiology of reproduction. Edinburgh: Churchill Livingstone, 1990; 747–869.Google Scholar
  19. 19.
    Sherman JK. Current status of clinical cryobanking of human semen. In: Paulson JD, Negro-Vilar A, Lucena E, Martini L, eds. Andrology: male fertility and sterility. Orlando: Academic Press, 1986; 517–47.Google Scholar
  20. 20.
    Watson PF. The preservation of semen in mammals. In: Finn CA, ed. Oxford reviews of reproductive biology. Oxford: Oxford University Press, 1979; 283–350.Google Scholar
  21. 21.
    Sherman JK. Questionable protection by intracellular glycerol during freezing and thawing. J Cell Comp Physiol 1963; 61: 67–83.PubMedCrossRefGoogle Scholar
  22. 22.
    Critser JK, Huse-Benda AR, Aaker DV, Arneson BW, Ball GD. Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility. Fertil Steril 1988; 50: 314–20.PubMedGoogle Scholar
  23. 23.
    Jeyendran RS, Van der Ven HH, Kennedy W, Perez-Pelaez M, Zaneveld LJD. Comparison of glycerol and a zwitter ion buffer system as cryoprotective media for human spermatozoa. J Androl 1984; 5: 1–7.PubMedGoogle Scholar
  24. 24.
    Mazur P, Miller RH. Permeability of the human erythrocyte to glycerol in 1 and 2M solutions at 0 and 20°C. Cryobiology 1976; 13: 507–22.PubMedCrossRefGoogle Scholar
  25. 25.
    Mazur P. Cryobiology: the freezing of biological systems. Science 1970; 168: 939–49.PubMedCrossRefGoogle Scholar
  26. 26.
    Fiser PS, Fairfull RW. The effect of glycerol concentration and cooling velocity on cryosurvival of ram spermatozoa frozen in straws. Cryobiology 1984; 21: 542–51.PubMedCrossRefGoogle Scholar
  27. 27.
    Watson PF, Duncan AE. Effect of salt concentration and unfrozen water fraction on the viability of slowly frozen ram spermatozoa. Cryobiology 1988; 25: 131–42.PubMedCrossRefGoogle Scholar
  28. 28.
    Kincade RS, Colvin KE, Kleinhans FW, Critser ES, Mazur P, Critser JK. The effects of cooling rate and repeated freezing on human sperm cryosurvival. J Androl 1989 (Suppl):48P.Google Scholar
  29. 29.
    Freund M, Wiederman J. Factors affecting the dilution, freezing and storage of human semen. J Reprod Fertil 1966; 11: 1–17.CrossRefGoogle Scholar
  30. 30.
    Trelford JD, Mueller F. Observations and studies on the storage of human sperm. Can Med Assoc J 1969; 100: 62–5.PubMedGoogle Scholar
  31. 31.
    Watson PF. Recent advances in sperm freezing. In: Thompson W, Joyce DN, Newton JR, eds. In vitro fertilization and donor insemination. London: Royal College of Obstetricians and Gynaecologists, 1985; 261–7.Google Scholar
  32. 32.
    Serafini P, Marrs RP. Computerised staged-freezing technique improves sperm survival and preserves penetration of zona-free hamster ova. Fertil Steril 1986; 45: 854–8.PubMedGoogle Scholar
  33. 33.
    Critser JK, Huse-Benda AR, Aaker DV, Arneson BW, Ball GD. Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction. Fertil Steril 1987; 47: 656–63.PubMedGoogle Scholar
  34. 34.
    McLaughlin EA, Ford WCL, Hull MGR. A comparison of the freezing of human semen in the uncirculated vapour above liquid nitrogen and in a commercial semiprogrammable freezer. Hum Reprod 1990; 5: 724–8.PubMedGoogle Scholar
  35. 35.
    Fiser PS, Hansen C, Underhill KL, Shrestha JNB. The effect of induced ice nucleation (seeding) on the post-thaw motility and acrosomal integrity of boar spermatozoa. Anim Reprod Sci 1991; 24: 293–304.CrossRefGoogle Scholar
  36. 36.
    Mahadevan MM. Cryobiological and biochemical studies of human serum. PhD Thesis, Monash University, Melbourne, 1981.Google Scholar
  37. 37.
    Rodriguez OL, Berndtson WE, Ennen BD, Pickett BW. Effect of rates of freezing, thawing and level of glycerol on the survival of bovine spermatozoa in straws. J Dairy Sci 1975; 41: 129–36.Google Scholar
  38. 38.
    Senger PL, Becker WC, Hilliers JK. Effect of thawing rate and post-thaw temperature on motility and acrosomal maintenance in bovine semen frozen in plastic straws. J Anim Sci 1976; 42: 932–6.PubMedGoogle Scholar
  39. 39.
    Fiser PS, Ainsworth L, Lanford G. Effect of osmolality of skim-milk diluents and thawing rate on cryosurvival of ram spermatozoa. Cryobiology 1981; 18: 399–403.PubMedCrossRefGoogle Scholar
  40. 40.
    Cochran JD, Amann RP, Froman DP, Pickett BW. Effects of centrifugation, glycerol level, cooling to 5°C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Theriogenology 1984; 22: 25–38.PubMedCrossRefGoogle Scholar
  41. 41.
    Pickett BW, Berndtson WE. Preservation of bovine spermatozoa by freezing in straws: a review. J Dairy Sci 1974; 57: 1287–301.PubMedCrossRefGoogle Scholar
  42. 42.
    Polge C. Fertilization in the pig and horse. J Reprod Fertil 1978; 54: 561–70.Google Scholar
  43. 43.
    Wilmut I, Polge C. The low temperature preservation of boar semen. III. The fertilizing capacity of frozen and thawed boar semen. Cryobiology 1977; 14: 483–91.PubMedCrossRefGoogle Scholar
  44. 44.
    Barwin BN. Artificial insemination. In: Paulson JD, Negro-Vilar A, Lucena E, Martini L, eds. Andrology: male fertility and sterility. Orlando: Academic Press, 1986; 461–74.Google Scholar
  45. 45.
    Steinberger E, Rodriguez-Rigau LI, Smith KD. Comparison of results of AID with fresh and frozen semen. In: David G, Price WS, eds. Human artificial insemination and semen preservation. New York: Plenum Press, 1980; 283–94.Google Scholar
  46. 46.
    Richardson DW. Factors influencing the fertility of frozen semen. In: Richardson DW, Joyce D, Symonds EM, eds. Frozen human semen. London: Royal College of Obstetricians and Gynaecologists, 1979; 33–58.Google Scholar
  47. 47.
    Cramer DW, Walker AM, Schiff I. Statistical methods in evaluating the outcome of infertility therapy. Fertil Steril 1979; 32: 80–6.PubMedGoogle Scholar
  48. 48.
    Smith KD, Rodriguez-Rigau LJ, Steinberger E. The influence of ovulatory dysfunction and timing of insemination on the success of artificial insemination donor (AID) with fresh or frozen semen. Fertil Steril 1981; 36: 496–502.PubMedGoogle Scholar
  49. 49.
    Richter MA, Haning RV, Shapiro SS. Artificial donor insemination: fresh versus frozen semen; the patient as her own control. Fertil Steril 1984; 41: 277–80.PubMedGoogle Scholar
  50. 50.
    Hammond MG, Jordan S, Sloan CS. Factors affecting pregnancy rates in a donor insemination program using frozen semen. Am J Obstet Gynecol 1986; 155: 480–5.PubMedGoogle Scholar
  51. 51.
    Bordson BL, Ricci E, Dickey RP, Dunaway H, Taylor SN, Curole DN. Comparison of fecundability with fresh and frozen semen in therapeutic donor insemination. Fertil Steril 1986; 46: 466–9.PubMedGoogle Scholar
  52. 52.
    Brown CA, Boone WR, Shapiro SS. Improved cryopreserved semen fecundability in an alternating fresh-frozen artificial insemination program. Fertil Steril 1988; 50: 825–7.PubMedGoogle Scholar
  53. 53.
    Leridon H. The efficacy of natural insemination: a comparative standard for AID. In: David G, Price WS, eds. Human artificial insemination and semen preservation. New York: Plenum Press, 1980; 191–6.Google Scholar
  54. 54.
    Mazur P. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology 1977; 14: 251–72.PubMedCrossRefGoogle Scholar
  55. 55.
    Muldrew K, McGann LE. Mechanisms of intracellular ice formation. Biophys J 1990; 57: 525–32.PubMedCrossRefGoogle Scholar
  56. 56.
    Yanagimachi R. Mammalian fertilization. In Knobil E, Neill JD, eds. The physiology of reproduction. New York: Raven Press, 1988; 135–85.Google Scholar
  57. 57.
    Hammerstedt RH, Graham JK, Nolan JP. Cryopreservation of mammalian sperm: what we ask them to survive. J Androl 1990; 11: 73–88.PubMedGoogle Scholar
  58. 58.
    Slavik T. Effect of glycerol on the penetrating ability of fresh ram spermatozoa with zona-free hamster eggs. J Reprod Fertil 1987; 79: 99–103.PubMedCrossRefGoogle Scholar
  59. 59.
    Karagiannidis A. The distribution of calcium in bovine spermatozoa and seminal plasma in relation to cold shock. J Reprod Fertil 1976; 46: 83–90.PubMedCrossRefGoogle Scholar
  60. 60.
    Robertson L, Watson PF. Calcium transport in diluted or cooled ram semen. J Reprod Fertil 1986; 77: 177–85.PubMedCrossRefGoogle Scholar
  61. 61.
    Simpson AM, White IG. Effect of cold shock and cooling rate on calcium uptake of ram spermatozoa. Anim Reprod Sci 1986; 12: 131–43.CrossRefGoogle Scholar
  62. 62.
    Robertson L, Watson PF. The effect of egg yolk on the control of intracellular calcium in ram spermatozoa cooled and stored at 5°C. Anim Reprod Sci 1987; 15: 177–87.CrossRefGoogle Scholar
  63. 63.
    Parrish JJ, Foote RH. Fertility of cooled and frozen rabbit sperm measured by competitive fertilization. Biol Reprod 1986; 35: 253–7.PubMedCrossRefGoogle Scholar
  64. 64.
    Chan SYW, Li SQ, Wang C. TEST-egg yolk buffer storage increases the capacity of human sperm to penetrate hamster eggs in vitro. Int J Androl 1987; 10: 517–24.PubMedCrossRefGoogle Scholar
  65. 65.
    Critser JK, Arneson BW, Aaker DV, Huse-Benda AR, Ball GD. Cryopreservation of human spermatozoa. II. Post-thaw chronology of motility and of zona-free hamster ova penetration. Fertil Steril 1987; 47: 980–4.PubMedGoogle Scholar
  66. 66.
    Mattner P, Entwistle KW, Martin ICA. Passage, survival and fertility of deep-frozen ram semen in the genital tract of the ewe. Aust J Biol Sci 1969; 22: 181–7.Google Scholar
  67. 67.
    Keel BA, Black JB. Reduced motility longevity in thawed human spermatozoa. Arch Androl 1980; 4: 213–15.PubMedCrossRefGoogle Scholar
  68. 68.
    Maxwell WMC. Artificial insemination of ewes with frozen—thawed semen at a synchronized oestrus. I. Effect of time of onset of oestrus, ovulation and insemination on fertility. Anim Reprod Sci 1986; 10: 301–8.CrossRefGoogle Scholar
  69. 69.
    Maxwell WMC. Artificial insemination of ewes with frozen—thawed semen at a synchronized oestrus. II. Effect of dose of spermatozoa and site of intrauterine insemination on fertility. Anim Reprod Sci 1986; 10: 309–16.CrossRefGoogle Scholar

Copyright information

© Royal College of Obstetricians and Gynaecologists 1992

Authors and Affiliations

  • P. F. Watson
  • J. K. Critser
  • P. Mazur

There are no affiliations available

Personalised recommendations