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ADDENDUM I
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Slow cooling injury can be caused by solute toxicity (mechanisms not yet elucidated) or by
shrinkage of the cells resulting from the hypertonic extracellular solution. Intracellular ice formation
(rapid cooling injury) occurs when a cell is not able to maintain osmotic equilibrium with the external
environment (Fig. 2). Due to the sudden exaggerated extracellular ice formation, the increase in
solutes is so dramatically that the cell cannot respond to it with exosmosis. The cytoplasm will
consequently become increasingly supercooled, as such increasing the likelihood of intracellular ice
formation (Muldrew et al., 2004). There are multiple hypotheses that attempt to explain how
extracellular ice interacts with the plasma membrane in the initiation of intracellular ice formation,
but they are all topic of debate. The nature of cellular injury caused by freezing and thawing is very
complex.
Amann and Pickett (1987) assumed that for stallion spermatozoa, in an extender of given
composition, there should be a cooling rate that maximizes sperm survival. Figure 3 represents such
a theoretical cooling curve, depicting the sperm survival as a function of the cooling rate. Slow
cooling (pathway A to B) will cause sperm damage due to excessive dehydration (solution effect),
while rapid cooling (pathway C to D) will cause sperm damage because of intracellular ice formation.
The optimal cooling rate is between B and C, and so far this optimal cooling rate can only be
determined empirically (Amann and Pickett, 1987). A theoretical approach for determining the
optimal cooling rate for the cryopreservation of bull semen has been proposed based on the
different compositions in the extender used (Woelders and Chaveiro, 2004). So far, no such work has
been presented for stallion spermatozoa.
A very remarkable observation concerning cryo-injury was the demonstration that
membrane integrity of ram sperm during cryopreservation clearly retained throughout the freeze-
thaw procedure, and that the permeabilization occurred not only after the samples had been thawed,
but once they reached threshold temperatures (Holt et al., 2005). These findings are in contrast with
the concept that the lethality is situated in the intermediate zone of temperature (between freezing
point and -60°C) that the cells must traverse twice during the freeze and thaw cycle (Mazur, 1963).