1. Introduction
Freezing mammalian spermatozoa offers many advantages to the livestock industry, particularly in conjunction with genetic evaluation and selection programmes, such as in
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sire reference schemes Maxwell, 1984 . However, the biggest obstacle to the exploita- tion of frozen semen is that freezing and thawing of spermatozoa of any species
generally leads to a decrease in the percentage of motile cells post-thawing as a result of Ž
. damage to membrane structures Quinn et al., 1969; Nath, 1972 . As a consequence,
fertility following artificial insemination is poorer than with fresh semen in most species and can only be partially compensated for by using greater numbers of spermatozoa in
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the insemination dose Watson, 1995 . Damage to sperm membranes primarily occurs during the freezing and thawing
process over the temperature range y158C to y608C and not during storage in liquid Ž
. nitrogen Mazur, 1965 . In the case of ram spermatozoa, most damage occurs between
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y108C and y258C; the region of ice crystallisation Salamon and Maxwell, 1995 . The process of cell dehydration that accompanies slow freezing is potentially beneficial for
cell survival, whereas rapid freezing rates are considered more likely to cause cell death Ž
. Watson, 1995 .
There are some reports in the literature dealing with the effects of freezing rate on Ž
post-thaw motility and acrosome integrity Watson and Martin, 1975; Fiser and Fairfull, .
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Ž 1986 . O’Neill 1998 observed that semen frozen rapidly from 58C to y258C at
. y58Crmin
had significantly better viability, mitochondrial activity and acrosome Ž
. integrity than after a slower y0.58Crmin freezing rate over the same interval. In these
studies, there was no direct evaluation of the fertilising ability of the sperm. Therefore, the objective of this study was to re-examine the effects of freezing rate, described by
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O’Neill 1998 , in the context of testing in vitro procedures for evaluating the fertilising capacity of frozen-thawed ram spermatozoa. The overall aim was to provide a method
for evaluating fertilizing ability of ram spermatozoa without the need to resort to in vivo methods. The procedure employed the techniques of in vitro maturation, fertilisation and
culture to assess the fertilisation rate and developmental competence of ovine oocytes. The procedure was validated by parallel in vivo studies using adult ewes.
2. Materials and methods
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