Cryobiology of equine spermatozoa: principles, factors affecting on semen quality and

Since the first foal was born after AI with cryopreserved equine semen, many aspects of semen cryopreservation in the horse have still remained empirical and relatively little information is available on the basic cryobiologic and biophysical stresses imposed during freezing and thawing.

There is considerable variation in the lipid composition of the sperm plasma membrane in different mammalian species. The plasma membrane of stallion sperm differs primarily with regard to its relatively high cholesterol content (37% of total lipids, in boar sperm this ratio is 24%) (Yanagimachi 1994, cited: Gadella et al. 2001).

Semen lipids play a major role in motion characteristics, sensitivity to cold shock and fertilizing capacity of sperm. It is important to note that the distribution of long chain polyunsaturated fatty acids in stallion sperm is more similar to boars than that of the bulls. Bulls produce sperm that are more resistant to cold shock and freeze well, whereas sperm from boars and stallions have very low tolerance to cold shock and in general, freeze poorly. Sperm of bulls have higher levels of 22:6 fatty acids, whereas sperm from stallions and boars have much higher levels of 22:5 fatty acids (Parks and Lynch 1992). The variation on membrane fluidity could be an explanation for the variability on sperm freezability observed between individual stallions. The major variable is the amount of cholesterol in the sperm plasma membrane between different males within a species and even between different ejaculates from a single male.

Furthermore, the cholesterol content seems to be related to the rate of capacitation possibly because cholesterol must be depleted from the plasma membrane during this process (Yanagimachi 1994, cited: Gadella et al. 2001). The sperm plasma membrane serves as the main physical barrier to the outside environment and is a primary site of freeze-thaw damage. Such damage includes membrane destabilization due to lateral lipid rearrangement, loss of lipids from the membrane, and peroxidation of membrane lipids as a result of formation of reactive oxygen species (De Leeuw et al. 1990).

Lipid-based cryoprotectants such as egg phosphatidylcholine or soy phosphatidylcholine may provide a physical barrier to freeze-thaw damage and prevent membrane phase separation by influencing lipid packing at the membrane surface. These exogenous lipids from the freezing extender does not incorporate into the sperm membrane but strongly associates with the membrane surface (Ricker et al.

2006).

Cryopreservation requires exposure of spermatozoa to extreme variations in temperature and osmolality. Post-thaw survival of cryopreserved spermatozoa exhibits a maximum at a presumptive optimum cooling rate, and the optimum cooling rate is also dependent on the warming rate, the optimum rates presumably being due to sperm permeability properties. The optimum cooling and warming rates may also be significantly dependent on the specific cryoprotective additive and buffer solution in which the spermatozoa are cryopreserved (Leibo 2006). During freezing, the most critical time for sperm damage is the period of extracellular ice crystal formation. The solution at this stage is cooled down to between -6 and -15 °C (Pickett and Amann 1993, Caiza de la Cueva et al. 1997). Both too slow and too rapid freezing were found to be associated with lethal cryoinjury. If freezing progresses at very slow rates, the dehydration will take place over a longer time period resulting in high degree of shrinking associated with fatal cellular disruption. (Mazur et al. 1972). However, the cooling rate must be slow enough to allow water to leave the cells by osmosis in sufficient quantity. The cellular damage that spermatozoa encounter at rapid rates of cooling has often been attributed to the formation of intracellular ice. However, no direct evidence of intracellular ice has been presented. In the study of Morris et al.

(2007) they concluded that cell damage to horse spermatozoa, at cooling rates of up to 3000 °C/min, is not caused by intracellular ice formation rather the cells are subjected to an osmotic shock when they are thawed. The observed differences in the viability and motility measurements suggest that different mechanisms of cellular injury may be occurring at ‘‘slow’’ and ‘‘rapid’’ rates of cooling (Morris et al. 2007). Sperm cells are generally frozen at quite rapid rates in the range of 15–60 °C/min, which have been empirically determined as giving the best survival rates (Watson 2000). Optimal cooling rates for stallion sperm are about 29 °C/min in the absence of cryoprotective agents and about 60 °C/min in their presence, as calculated at subzero temperatures (Devireddy et al. 2002). Stallion spermatozoa are extremely sensitive to chilling injury also when cooled from 37 °C to approximately 8 °C at rates > 0.3 °C/min. The cold shock effect includes abnormal patterns of swimming (circular or backwards), rapid loss of motility, acrosomal damage, plasma membrane damage, reduced metabolism and loss of intracellular components (Moran et al. 1992).

The capacity for spermatozoa to respond with cell volume adjusment is determined by several factors including membrane phospholipid composition, water permability (Lp), lipid phase transition temperature, Na+/K+ ATPase activity, ion channels, and cytoskeletal elements (Pommer et al. 2002). Cells with a higher Lp will reach equilibrium faster (Devireddy et al. 2002). Spermatozoa subjected to hyperosmolal

environment (up to 450 mosmol/kg) appear to have intact membranes (viable cells) and are capable of preserving their mitochondria, as demonstrated by a high mitochondrial membrane potential (MMP). However when sperm were subjected to hypotonic solutions, MMP and viability markedly decresed. Thus the thawing process may be more detrimental to spermatozoa than the freezing process (Ball and Vo 2001, Pommer et al. 2002).

Oxidative stress is defined as the imbalance between pro-oxidative and antioxidative molecules in a biological system. This imbalance can lead to damage to the structure of cells and macromolecules such as plasma membrane components, proteins, and DNA (Aitken et al. 1999). Because of the high content of polyunsaturated fatty acids (PUFA) in the plasma membrane, mammalian sperm are sensitive to oxidative stress (Parks and Lynch 1992, Aitken 1995). In several experiments the effect of sperm freezing/thawing and storage (Kankofer et al. 2005) on production of ROS and effectivness of various antioxidants (Aurich et al. 1997; Ball et al. 2000, 2001;

Baumber et al. 2000, 2003a; Sarlós et al. 2002; Gadea et al. 2005) added to the semen were evaluated. While the uncontrolled generation of reactive oxygen species (ROS) by defective spermatozoa can have detrimental effects on sperm function, controlled production of ROS plays physiologically relevant roles in signalling events controlling sperm capacitation, the acrosome reaction, hyperactivation and sperm–oocyte fusion (Baumber et al. 2000). In stallion semen, ROS are generated mainly by immature, damaged and abnormal spermatozoa and by contaminating leukocytes. Although lipid peroxidation is well characterized for mammalian sperm, equine spermatozoa appear relatively more resistant to membrane peroxidation than sperm of other domestic animals (Baumber et al., 2000; Neild et al., 2005). Cryopreservation of equine sperm, however, increased lipid peroxidation particularly over the region of the sperm midpiece (Neild et al. 2005, Ball 2009).

Factors affecting on semen quality and freezability

Differences in sperm membrane composition, biochemistry and metabolism between both species and individuals within a species may be responsible for differences in membrane permeability to water and cryoprotectants. Glycerol toxicity could be also one reason for the variation on stallion sperm freezability. Hammerstedt and Graham (1992) reviewed cellular effects caused by glycerol that included changes in cytoplasmic events due to increased viscosity by intracellular glycerol, altered polymerization of tubulin, alteration of microtubule association, effects on bioenergetic balances and direct alteration of the plasma membrane and glycocalyx.

Increasing cryoprotectant permeability, either by altering membrane composition or by using alternative cryoprotectants may improve cryosurvival rates of sperm that normally survive freezing poorly. Indeed, increasing the cholesterol content of stallion (Moore 2005b) increases their osmotic tolerance. In addition, for stallion sperm that do not survive cryopreservation well using standard procedures, changing the cryoprotectant to a smaller more permeable cryoprotectant such as formamide or dimethyl formamide can improve cryosurvival (Squires et al. 2004; Alvarenga et al.

2005). Fertility trials have also been performed that showed a significant improvement on fertility of stallion semen frozen with dimethyl-formamide (DMF) when compared with glycerol (Medeiros et al. 2002, Moffet et al. 2003, Medeiros 2003).

Due to the great variation in semen quality between and within stallions, factors affecting quality should carefully be controlled. Routinely used medicines like Eqvalan or Quadrisol (Janett et al. (2001, 2005) , stress situations as training or competition (Dinger et al. 1986, Lange et al. 1997, Janett et al. 2006), seasonality (Janett et al. 2003) or nutrition like feeding a nutriceutical rich in docosahexaenoic acid /DHA/ (Brinsko et al. (2005b) can influence the fresh and frozen sperm quality.

Current freezing protocols for stallion semen involve a two-step dilution procedure in which semen is first diluted with a primary extender, centrifuged and then diluted a second time prior to freezing in an extender containing cryoprotectants. The first dilution employs either saline/sugar extenders or skim milk extenders with or without egg yolk used to dilute fresh semen. The dilution rate is either 1:1 or the semen is diluted to a concentration of ~50 million spermatozoa/ml. The success of centrifugation depends on duration (10–15 min) and centrifugation force (350–

700×g). Despite the development of an ever increasing range of freezing extenders, each claiming some improvement or benefit over the other (Martin et al. 1979, Loomis et al. 1984, Heitland et al. 1996, Ecot et al. 2000, Allen 2005). In spite of the elevated research on the alternative cryoprotectans, glycerol at a concentration of 3–

5% has been the major penetrating cryoprotectant routinely used to freeze stallion semen. The yolk of fresh chicken or duck eggs at a concentration of 10–20% v:v has remained the preferred source of protein in the freezing mixture. Sugars (usually combination of fructose and glucose, alternatively raffinose or trehalose) are often added to media which act as non-penetrating cryoprotectants (Squires et al. 2004). The most commonly used freezing containers are 0.5-ml straws. The centrifuged, extended semen is usually cooled to 4°C before freezing which takes place in liquid nitrogen vapour by suspending the rack of pre-filled straws a few centimetres above the liquid nitrogen in a specially adapted freezing bath (Boyle 1999), or in a computer-controlled automated freezing machine (Allen 2005). Using computer-controlled rate freezers

different freezing curves can be set, for example the recommendation of Digitcool devise (IMV CryoBio-System, L’Aigle, France) for equine sperm cryopreservation is the following: from 4 to -10 C at 10 C/min, from -10 to -100 C at 20 C/min, from -100 to -140 C at 60 °C/min. The samples were then plunged into liquid nitrogen.

Some alternative methods such as unique freezing technique /UFT/ (Vartorella 2003, Goolsby et al. 2004), ultra-low temperature freezers (Álamo et al. 2005), 'Multi-Thermal-Gradient' (MTG) technology (Zirkler et al. 2005, Saragusty et al. 2007) also have been utilised and showed comparable results than conventional liquid nitrogen methodology. These techniques may be suitable to replace the tradional method. A sublethal environmental stress, through the application of a high hydrostatic pressure (HHP) impulse (30 MPa pressure for 90 min) before cryopreservation significantly improved the post-thaw motility, viability and fertility of frozen bull sperm (Pribenszky et al. 2007, Kútvölgyi et al. 2008). However the preliminary study showed that the response for different pressure/time combinations is more individual in stallion sperm and it was not found substantial improvement in most of the examined attributes, only the VCL parameter of CASA after using 5MPa pressure for 60 minutes, increased after freezing/thawing (Horváth et al. 2007).