Ž .
Ž freezing Pursel and Johnson, 1975 , contains the TEST combination Graham et al.,
. 1972 . The comparative merits of buffer systems were discussed in some detail by
Ž .
Ž .
Watson 1990 and Salamon and Maxwell 1995a .
4. Practical aspects of semen cryopreservation
The practical details of semen processing for cryopreservation have been described Ž
. previously Pursel and Park, 1985; Salamon and Maxwell, 1995a; Watson, 1990 .
Ž .
Semen is either packaged in straws 0.25 and 0.5 ml for freezing and storage, or frozen as pellets on shallow depressions in dry ice. Straws are either frozen in the vapour phase
above liquid nitrogen or in a controlled-rate freezing machine. The practical require- ments of artificial insemination techniques have considerable influence over the choice
of technique. Bull spermatozoa are routinely packaged in 0.25-ml straws; these contain a
Ž .
known number of live spermatozoa typically 10–15 million which can be inseminated directly from the straw after thawing. Boar semen, on the other hand, has to be frozen in
much larger quantities and pellets of approximately 200 ml volume are frequently preferred. These can be stored frozen in 10–15 ml tubes, each tube providing sufficient
Ž .
spermatozoa for one insemination. Larger straws Weitze et al., 1991 and flattened Ž
. plastic bags Bwanga et al., 1991 have been introduced for coping with the higher
volume requirements. Laparoscopic insemination pipettes for sheep, deer and exotic ruminants have been
developed with the 0.25 ml plastic straw in mind, especially as lower sperm numbers are needed for this mode of insemination than for the transcervical route. Typically, the
straw is fitted into the insemination pipette immediately after thawing, and insemination can be performed within seconds. The same principle applies to the pipettes used for
transcervical insemination in cattle, where either of the two standard straw sizes can be used. A disadvantage of this approach is that there is no easy way to thaw the samples
while simultaneously reducing the cryoprotectant concentration. This is where the pellet technique offers a clear advantage because, where desired, thawing can be rapidly
performed by pouring the pellets into a solution specifically formulated for the purpose; ‘‘wet thawing’’. Thawing of straws is usually performed by immersion in a warm
waterbath; this has its own advantages in that standardized temperature and time combinations can be used. Some experimentation with thawing at relatively high
Ž .
temperatures 60–708C
has been undertaken. The comparative merits of different Ž
. thawing techniques have been discussed in detail by Salamon and Maxwell 1995a and
Ž .
Pursel and Park 1985 . It is generally considered that fast rewarming rates are required for best sperm recovery. This has been attributed to the possibility that small intra-
cellular ice crystals formed in some cells during freezing might grow during a slow rewarming process.
Systematic studies of sperm plasma membrane properties in relation to cryopreserva- tion have provided some new insights into the factors which determine cell survival
during the freeze–thaw process. Particularly interesting from a practical viewpoint is the realization that if cryoprotectant addition and removal are carried out in a number of
steps, the excessive cell volume excursions which cause membrane rupture can be
Ž .
avoided Gao et al., 1993, 1995 .
Commercially produced semen freezing machines have been available for a number of years. In principle, these should be of considerable help for both handling large
numbers of samples by a standardized method, and should also permit accurate cooling of the samples at the desired rates. While these systems may meet the first objective,
they have so far proved inadequate to meet the second. This is because during the
Ž .
freezing process, the samples release sufficient heat latent heat of fusion to cause a sharp increase in temperature; no successful method of dissipating this heat rapidly has
so far been developed, and sample temperature consequently does not decrease in tandem with the fall in chamber temperature. In fact, the sample temperature can remain
static for 2–3 min before cooling is resumed. Several investigators have shown that this period between freezing and resumption of cooling, the freezing point plateau, is
Ž .
detrimental to sperm survival. Parkinson and Whitfield 1987 showed that reduction of this plateau improved the fertility of bull spermatozoa, and similar results have been
Ž .
obtained with boar semen Pursel and Park, 1985; Bwanga et al., 1991 . The introduc- tion of larger volume freezing methods such as the maxi-straw or plastic bag is likely to
exacerbate this problem and there is clearly a need for the development of more effective temperature control systems.
5. Disease control issues
