Improved sperm cryopreservation using cold cryoprotectant
G. N. Clarke A C D , D. Y. Liu A B and H. W. G. Baker A B CA Department of Obstetrics and Gynaecology, University of Melbourne, Victoria 3010, Australia.
B Reproductive Services Division, The Royal Women’s Hospital, Carlton, Victoria 3053, Australia.
C Andrology Unit, Laboratory Services Division, The Royal Women’s Hospital, Carlton, Victoria 3053, Australia.
D To whom correspondence should be addressed. email: gary.clarke@wch.org.au
Reproduction, Fertility and Development 15(7) 377-381 https://doi.org/10.1071/RD03007
Submitted: 3 February 2003 Accepted: 3 December 2003 Published: 3 December 2003
Abstract
It has generally been assumed that very rapid cooling above freezing point would be deleterious to human sperm because it would result in cold shock. Consequently, most routine cryopreservation protocols involve the use of warm (20–30°C) cryoprotectant and slow cooling above the freezing point in order to minimise the risk of cold shock. In order to test this assumption, we added an equal volume of cold (4°C) cryoprotectant in a single aliquot to warm (20, 30 or 37°C) semen to induce rapid cooling. The results of this procedure were compared with those obtained using warm cryoprotectant or with the routine cryopreservation protocol used in this laboratory. The use of cold cryoprotectant resulted in a significant (P = 0.016) improvement (mean 63%, range 42%–79%) in post-thaw motility recovery compared with a standard procedure(mean 47%, range 35%–67%) and a significant (P = 0.016) improvement in post-thaw sperm velocity. A cold glycerol/egg yolk/citrate (GEYC) mixture also gave significantly higher motility recovery than GEYC equilibrated to either room temperature (20°C) or body temperature (37°C). Sperm frozen using the cold cryoprotectant protocol were as efficient at binding to and penetrating the human zona pellucida as sperm frozen with a standard protocol.The modified cryopreservation procedure may lead to improved pregnancy rates in donor insemination and in vitro fertilisation. Further investigation is required to determine how the cold cryoprotectant improves the cryopreservation outcome.
Extra keywords: freezing
Acknowledgments
The authors thank the staff of the Andrology Unit, Laboratory Services Division for technical assistance.
Ackerman, D. R. (1968). The effect of cooling and freezing on the aerobic and anaerobic lactic acid production of human semen. Fertil. Steril. 19, 123–128.
| PubMed |
Ackerman, D. R. (1970). Glycerol metabolism in untreated, cold-shocked and frozen human spermatozoa. Cryobiology 7, 145–147.
| PubMed |
Ackerman, D. R. (1971). Variation due to freezing in the citric acid content of human semen. Fertil. Steril. 22, 58–60.
| PubMed |
Ali, J. I. , Fahim, Z. , Weaver, D. , and Noteboom, W. (1993). The importance of pre-freeze equilibration of glycerol in cryopreservation of human spermatozoa and the biochemical conversion of glycerol. Int. J. Fertil. 38, 180–186.
Berruti, G. (1988). Calpactin-like proteins in human spermatozoa. Exp. Cell Res. 179, 374–384.
| PubMed |
Clarke, G. N. , and Yanagimachi, R. (1978). Actin in mammalian sperm heads. J. Exp. Zool. 205, 125–132.
| PubMed |
Clarke, G. N. , Clarke, F. M. , and Wilson, S. (1982). Actin in human spermatozoa. Biol. Reprod. 26, 319–327.
| PubMed |
Clarke, G. N. , Bourne, H. , Hill, P. , Johnston, W. I. H. , Speirs, A. , McBain, J. C. , and Baker, H. W. G. (1997). Artificial insemination and in vitro fertilization using donor spermatozoa: a report on 15 years of experience. Hum. Reprod. 12, 722–726.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Critser, J. K. , Huse-Benda, A. R. , Aaker, D. V. , Arneson, B. W. , and Ball, G. D. (1988). Cryopreservation of human spermatozoa. III. The effect of cryoprotectants on motility. Fertil. Steril. 50, 314–320.
| PubMed |
Crowe, J. H. , Hoekstra, F. A. , Crowe, L. M. , Anchordoguy, T. J. , and Drobnis, E. (1989). Lipid phase transitions measured in intact cells with Fourier transform infrared spectroscopy. Cryobiology 26, 76–84.
| PubMed |
Darin-Bennett, A. , and White, I. G. (1977). Influence of cholesterol content of mammalian spermatozoa on susceptibility to cold-shock. Cryobiology 14, 466–470.
| PubMed |
David, G. , Czyglik, F. , Mayaux, M. J. , and Schwartz, D. (1980). The success of A.I.D. and semen characteristics: study on 1489 cycles and 192 ejaculates. Int. J. Androl. 3, 613–619.
| PubMed |
de las Heras, M. A. , Valcarcel, A. , Perez, L. J. , and Moses, D. F. (1997). Actin localization in ram spermatozoa: effect of freezing, thawing, capacitation and calcium ionophore-induced acrosomal exocytosis. Tissue Cell 29, 47–53.
| PubMed |
Friberg, J. , and Gemzell, C. (1973). Inseminations of human sperm after freezing in liquid nitrogen vapors with glycerol or glycerol–egg yolk–citrate as protective media. Am. J. Obstet. Gynecol. 116, 330–334.
| PubMed |
Giraud, M. N. , Motta, C. , Boucher, D. , and Grizard, G. (2000). Membrane fluidity predicts the outcome of cryopreservation of human spermatozoa. Hum. Reprod. 15, 2160–2164.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Holt, W. V. , and North, R. D. (1991). Cryopreservation, actin localization and thermotropic phase transitions in ram spermatozoa. J. Reprod. Fertil. 91, 451–461.
| PubMed |
Kann, M. L. , Feinberg, J. , Rainteau, D. , Dadoune, J. P. , Seinman, S. , and Fouquet, J. P. (1991). Localization of calmodulin in perinuclear structures of spermatids and spermatozoa: a comparison of six mammalian species. Anat. Rec. 230, 481–488.
| PubMed |
Kremer, J. , Dejkhuis, J. R. H. , and Jager, S. (1987). A simplified method for freezing and storage of human semen. Fertil. Steril. 47, 838–842.
| PubMed |
Liu, D. Y. , Martic, M. , Clarke, G. N. , Dunlop, M. E. , and Baker, H. W. G. (1999). An important role of actin polymerization in the human zona pellucida-induced acrosome reaction. Mol. Hum. Reprod. 5, 941–949.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Liu, D. Y. , Martic, M. , Clarke, G. N. , Grkovic, I. , Garrett, C. , Dunlop, M. E. , and Baker, H. W. G. (2002). An anti-actin monoclonal antibody inhibits the zona pellucida-induced acrosome reaction and hyperactivated motility of human sperm. Mol. Hum. Reprod. 8, 37–47.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Noiles, E. E. , Thompson, K. A. , and Storey, B. T. (1997). Water permeability, Lp, of the mouse sperm plasma membrane and its activation energy are strongly dependent on interaction of the plasma membrane with the sperm cytoskeleton. Cryobiology 35, 79–92.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Polge, C. , Smith, A. U. , and Parkes, A. S. (1949). Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164, 666.
Vincent, C. , Pruliere, G. , Pajot-Augy, E. , Campion, E. , Garnier, V. , and Reynard, J. P. (1990). Effects of cryoprotectants on actin filaments during the cryopreservation of one-cell rabbit embryos. Cryobiology 27, 9–23.
| PubMed |
Von Bulow, M. , Heid, H. , Hess, H. , and Franke, W. W. (1995). Molecular nature of calicin, a major basic protein of the mammalian sperm head cytoskeleton. Exp. Cell Res. 219, 407–418.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Zeng, W. X. , and Terada, T. (2000). Freezability of boar spermatozoa is improved by exposure to 2-hydroxypropyl-beta-cyclodextrin. Reprod. Fertil. Dev. 12, 223–228.
| PubMed |
