AN OVERVIEW OF CRYOPRESERVATION AND CAUSES OF SPERM DAMAGE
resuspension in freezing extender. Extender can be added conserva- tively until the desired concentration is reached, after which straws are filled. This is done manually or with the assistance of a semi- automated system. During filling, one must ensure that the cotton plug is completely saturated with semen, and a 0.5–1.0 cm air bubble is introduced to the straw to allow room for expansion during thaw- ing. After filling, each straw is sealed using a heat sealer, glass ball or sealing powder to close the open end of the straw. Using a flicking motion or a quick single shake, the air bubble should be moved to the centre of the straw, and then straws are arranged on a rack evenly spaced in horizontal orientation for cooling and freezing. When a given stallion ' s semen is being frozen for the first time, a
test freeze may be useful to determine the optimal freezing extender for a particular individual. Semen is divided into aliquots (typically 3–4) and a different freezing extender is used for each aliquot. Post- thaw semen analysis allows for the determination of the most appro- priate freezing extender for that stallion.
SEMEN COOLING AND FREEZING
A single standardised stallion semen cryopreservation protocol does not exist, and the variable pregnancy rates achieved with thawed frozen semen may be in part due to differences in freezing proto- cols. Cooling and freezing can be performed either with a program- mable freezer or using a manual freezing protocol in a Styrofoam box over liquid nitrogen vapour (Clulow et al., 2008 ). As sperm cells cool from room temperature to ultimately −196°C, the ideal rate is slow enough to allow cellular dehydration yet fast enough to pre- vent prolonged sperm exposure to hyperconcentrated conditions as water freezes. Freezing happens over two different stages: the first step allows semen to cool from room temperature to 5°C at a rate of 3–5°C/min, with the ideal time depending on the specific extender. The second stage cools semen from 5 to −196°C and occurs at a faster rate than the first step (20–50°C/min) (Alvarenga et al., 2016 ). If a programmable freezer is available, it can be programmed to set the desired freezing curve(s). Using a manual process, the rack of straws is placed in a refrigerator for 20 min, then rests at 3 cm above liquid nitrogen vapour for 20 min, and then is plunged into the liq- uid nitrogen. Cryopreserved semen may be stored indefinitely at −196°C provided the liquid nitrogen supply is maintained.
SOURCES OF CRYODAMAGE
Expected per cycle pregnancy rates when breeding with cryopreserved- thawed semen are lower than those for cooled or fresh semen (Barbacini et al., 1999 ; Loomis & Graham, 2008 ; Metcalf, 2007 ; Vidament et al., 1997 ). To some extent, the success of semen cryopreservation is dependent on the innate stallion- dependent ability to freeze and thaw well
(Salazar et al., 2011 ).
Although semen quality is typically optimised in stallions who are neither young (immature spermatogenesis) nor aged (testicular
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degeneration) (Dowsett & Knott, 1996 ), there is a ‘stallion factor’ which results in a normal distribution of cryosurvival among stal- lions, that is, the ability to withstand the freeze–thaw cycle and still produce pregnancies. Multiple studies have suggested that between 20% and 50% of stallions are ‘poor’ freezers (i.e., a normal distribu- tion), meaning their semen is not amenable to freezing using con- ventional methods (Loomis & Graham, 2008 ; Salazar et al., 2011 ). By investigating specific sources of sperm damage, one can iden- tify ways to manipulate the process in an attempt to improve cryosurvival. Spermatozoa undergo a barrage of insults during the cryopres-
which contains
ervation process and the resulting sperm population is compro- mised (Neild et al., 2003 ). There are substantial stressors applied to spermatozoa during freezing, specifically as a result of temperature changes, osmotic stresses, and ice formation. A brief examination of sperm structure may help clarify the pathogenesis of freezing- related damage (Varner & Johnson, 2011 ). The mature spermatozoa consists of a head and flagellum (tail). The head contains the nucleus and the acrosome, while the flagellum is responsible for sperm movement. The entirety of the spermatozoa is surrounded by a plasma mem- brane as the outermost component. The plasma membrane consists of a lipid bilayer, a phospholipid- water interface, and a glycocalyx. The polar phospholipids are arranged with the hydrophobic regions directed internally, and the hydrophilic regions towards the exter- nal polar solvent (water); proteins are intermingled throughout the bilayer. The specific ratio of cholesterol to phospholipids varies by species and plays a role in freezing success. At a normal temperature under normal conditions, the cholesterol and protein arrangement within the lipid bilayer maintains a liquid state. During cooling, a phase change occurs as lipids move into a gel or crystalline state and as membrane proteins become aggregated, the membrane becomes unstable. In this way, temperature changes or cold shock, may result in compromised barrier function which can be irreversible (Loomis & Graham, 2008 ; Watson, 2000 ). Another cause of stress is the addition of a freezing extender, cryoprotectants,
to the concentrated semen
(Loomis & Graham, 2008 ; Moore et al., 2006 ; Watson, 2000 ). Cryoprotectants are necessary but also damaging and can be di- vided into two broad categories—penetrating and non- penetrating. Non- penetrating cryoprotectant agents are unable to cross the sperm plasma membrane due to their relatively high molecu- lar weight. These substances create a hypertonic environment outside of the cell which encourages water to leave the cell; this results in dehydration of the sperm cell, but also decreases the likelihood of intracellular ice crystals formation. Examples of non- penetrating cryoprotectants include sucrose, egg yolk, and other sugars (Alvarenga et al., 2016 ). Penetrating cryoprotectants, on the contrary, exert their effects intracellularly by permeating the sperm plasma membrane; examples include ethylene glycol, propyl- ene glycol, glycerol, and DMSO. Penetrating cryoprotectants cross the plasma membrane and enter the sperm cell but do so more slowly than water. Thus, even though the sperm cell attempts to reach equilibrium, dehydration and undesirable osmotic effects still
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