Ž .
Animal Reproduction Science 62 2000 3–22 www.elsevier.comrlocateranireprosci
Basic aspects of frozen storage of semen
W.V. Holt
Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, UK
Abstract
Basic concepts of cryopreservation and the causes of cryoinjury are reviewed. The possible roles of cryoprotectants and additives are considered in the context of their putative interactions
with the sperm plasma membrane. Modern approaches to the laboratory assessment of spermato- zoa after freeze-thawing are also briefly discussed. q 2000 Elsevier Science B.V. All rights
reserved.
Keywords: Cryobiology; Spermatozoa; Cryoprotectant; Functional assessment
1. Introduction
The discovery of glycerol as a cryoprotectant marked a quantum advance in semen cryopreservation, but subsequent research has only made relatively small improvements
to the basic techniques established in the early 1950s. Nevertheless, the influence of this technology upon the animal breeding industry, especially dairying, has been profound.
The remarkable success with bull semen has not been matched in other mammals such as pigs, sheep and exotic species. Such species differences result from at least two
Ž . sources of variability: i the physiology and biochemistry of the spermatozoa them-
Ž . selves, and ii variations in the anatomy and physiology of sperm transport in the
Ž .
female reproductive tract. Surprisingly few viable bull spermatozoa tens of thousands are sufficient to achieve fertilization in the cow, while conversely the pig requires many
millions. This quantitative difference between species is an important determinant of the fertility of cryopreserved semen and means that development of successful freezing
Tel.: q44-171-449-6630; fax: q44-171-586-2870. Ž
. E-mail address: bill.holtioz.ac.uk W.V. Holt .
0378-4320r00r - see front matter q 2000 Elsevier Science B.V. All rights reserved. Ž
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procedures necessarily involves more than the identification or application of novel cryoprotectants and additives. If large numbers of spermatozoa are required for concep-
tion there will be less tolerance of poor sperm survival during cryopreservation. It could also be argued that species bearing large litters must generate a minimum number of
fertilized eggs otherwise they fail to produce sufficient concentrations of pregnancy-re- cognition factors.
Having mentioned that the quirks of individual species can have major influences upon the success of artificial insemination procedures with cryopreserved semen, it has
been left to the authors of accompanying reviews to deal with species-specific require- ments. This review will mainly treat the spermatozoon as a generic cell type which
undergoes various stresses during cryopreservation leading to survival, cell death or functional impairment. The reader should be aware that because of space limitations the
literature has not been exhaustively cited. The principles and practice of semen
Ž cryopreservation have been reviewed several times Bwanga, 1991; Hammerstedt and
Graham, 1992; Hammerstedt et al., 1990; Hofmo and Almlid, 1991; Salamon and .
Maxwell, 1995a,b; Watson, 1979, 1990, 1995; Wolf and Patton, 1989 , and these authors refer to most of the original literature up to the early 1990s. The reviews by
Salamon and Maxwell are notable for their inclusion of research performed in the former Soviet Union.
2. Basic principles of semen cryopreservation and cryoinjury
