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therefore, many strategies (each testing a few factors) have been used to solve this problem. Some of the strategies used include changing the cryodiluents, the cryoprotectants and their concentra- tions, as well as altering the cooling and?or warming rates of the protocol. Other strategies have focused on understanding what di?erences exist between sperm that survive freezing well and those that do not, including seminal plasma composition di?erences, or trying to determine when and where sperm damage occurs and modifying or by-passing that critical point in the protocol. In general, these strategies do not act on the sperm themselves but on the environment surrounding the sperm. However, some strategies have been tested to modify the sperm membranes during their formation (via nutrition) or after ejaculation. This review will focus on changing the cholesterol content of sperm membranes as a strategy to improve the number of sperm that survives cryopreservation as well as the quality of those sperm. Why Cholesterol? Sperm membranes are composed of lipids and proteins with the lipids arranged in a bilayer with the hydrophilic lipid head groups oriented to the membrane exterior and the hydrophobic fatty acyl chains in the membrane interior. The predominant lipids in most membranes are phospholipids and cholesterol. In addition, integral and peripheral proteins are also present. The proteinClipid associations are not random, and the lipid environment around a protein in?uences functional properties of the aggregate unit, so some lipids are preferentially clus- tered around integral membrane proteins (Hammerstedt et al. 1990). Some of the integral proteins function as pores or channels and others as receptors for other molecules. At body temperature, the membrane is in a Reprod Dom Anim

45 (Suppl. 2), 57C66 (2010);

doi: 10.1111/j.1439-0531.2010.01635.x ISSN 0936-6768 ?

2010 Blackwell Verlag GmbH '

?uid'

state in which the phospholipids and proteins can move laterally within the membrane (Amann and Pickett 1987). When membranes are cooled, the phospholipids undergo a phase transition from a liquid state to the crystalline-gel state. Each lipid species undergoes this phase transition at a unique temperature;

therefore, the membrane, being composed of many di?erent lipids, undergoes the phase transition over a relatively wide temperature range. In the crystalline-gel state, the phospholipid fatty acyl chains straighten and lengthen, resulting in a more ordered and packed membrane, where lipid and protein movement is restricted (Amann and Pickett 1987;

Hammerstedt et al. 1990). Moreover, integral proteins are excluded from the crystalline-gel domains within the membrane, so many proteinClipid interactions are lost and proteins cluster into the remaining liquid lipid domains. As a result of the protein aggregation and lipid phase transition, the membrane becomes unstable and functionality is lost (Amann and Pickett 1987). Both the ratio of cholesterol to phospholipids and the amount of polyunsaturated fatty acyl chains composing the phospholipids determine the overall ?uidity of a membrane (Amann and Pickett 1987). Several authors observed that di?erent species produce sperm that can be divided into two groups, related to the resistance of their sperm to '

cold shock'

(Watson 1981). Some species produce sperm that are highly susceptible to cold shock (boar, bull, ram or stallion), others are less sensitive (cat and dog), while others are highly resistant to cold shock (rabbit, human, fowl). When the di?er- ences between the sperm from di?erent species were evaluated at the level of membrane composition, di?erences were detected and, in general, sperm from cold-shock-resistant species exhibited higher levels of polyunsaturated fatty acids and higher cholesterol to phospholipid molar ratios, than the sperm from cold- shock-sensitive species (for an extensive review, see Watson 1981). Cholesterol has multiple e?ects on membranes, including stabilizing the membrane, reducing membrane permeability, facilitating morphological membrane characteristics and enabling cellCcell interactions, in?u- encing the membrane phase transition, providing suit- able microenvironments (chemical and?or physical) for membrane-associated proteins and serving as a mem- brane antioxidant (reviewed by Crockett 1998). The cholesterol to phospholipid ratio is an important determinant of membrane ?uidity and stability at low temperature. Cholesterol modulates the ?uidity of membranes by interacting with the fatty acyl chains of the phospholipids (Watson 1981) and maintains the phospholipids in a random, lamellar arrangement as temperature decreases (Amann and Pickett 1987). In model membranes, increasing the ratio of cholesterol to phospholipid broadens the phase transition, reduces membrane leakage and membrane phase separations (reviewed by Drobnis et al. 1993). Therefore, treating sperm with cholesterol before cryopreservation could reduce the sensitivity of sperm membranes to cooling damage, by eliminating or at least minimizing the lateral phase separation of the lipids (Watson 1981). How Can the Cholesterol be Added? Cholesterol is a hydrophobic molecule and is not soluble in aqueous semen diluents. Attempts have been made to solubilize cholesterol by incorporating it into liposomes and adding these liposomeCcholesterol complexes to sperm. Results have been variable using this technique (from no response to moderate improvement after cooling at 4°C or after cryopreservation), depending on species (Parks et al. 1981;