Table 716 continued f TCA 325 extender

Component Quantity

TRIS 2.487 g

Citric acid 1.39 g

Dextrose 0.838 g pH 6.85

Osmotic pressure (mOsm kg-1) 325.0

(g) TCA 390 extender

Component Quantity

TRIS 2.9 g

Dextrose 1.0 g pH 6.85

Osmotic pressure (mOsm kg-1) 390.0

aTRIS = TRIS(hydroxymethyl)-aminomethane optimum effect of glycerol was obtained with an equilibration time of 3-5 h. However, it has also been reported that exposure of spermatozoa to glycerol for only a few seconds still resulted in cryopreservation when glycerol was included in a glucose-skimmed milk extender or a glucose-egg yolk extender and the sample was stored in ampoules (Rajamannan et al., 1968) -hence the popularity and success of many of the extenders listed. Further work supported this finding that exposure of spermatozoa to glycerol under the correct circumstances for just a few seconds could be as beneficial (as regards cryopreservation) as exposure over 2-3 min or over 4 h (Berndtson and Foote, 1969; Nishikawa et al., 1972a,b). Nishikawa (1975) found that 5-10% final glycerol concentration in an HF-20 extender with an equilibration time of 30 s ensured good fertility. Conception rates of up to 60% were reported after storage in 1 ml straws. Again this work supported the significant variability found in the freezing capacity of spermatozoa from different stallions.

As indicated, Nishikawa (1975) obtained good results with 10% glycerol diluent. Other work suggested that glycerol levels could just as successfully be lowered to 5% but inclusion rates of 0, 1 and 3% were less effective in maintaining motility (Nishikawa et al., 1968; Nishikawa, 1975). Other workers have come up with similar results. Piao and Wang (1988) suggested that 6% glycerol was the most beneficial. Pace and Sullivan (1975) demonstrated that reducing glycerol from 7% to 2% was advantageous, obtaining conception rates of 12% and 46%, respectively. Barsel (1994) suggested that 5% glycerol was superior to both 3% and 7% in a lactose-EDTA-egg yolk extender. Cochran et al. (1984), investigating the effect of extender, freezing rate and thawing temperature on motility, demonstrated that an inclusion rate of 4% in an extender containing 20% egg yolk was superior to 2% or 6% glycerol as regards progressive motility. This superiority of 4% glycerol was further confirmed by Christanelli et al. (1985). Burns and Reasner (1995) concluded that a sucrose-glucose-dried skimmed milk freezing extender with 2% glycerol was most beneficial as regards motility and progressive motility, plus lateral head displacement, but an inclusion rate of 1% glycerol was best for velocity and progressive velocity. Unfortunately, no inseminations were carried out. It has also been suggested that glycerol accelerates the rate at which maximal post-thaw motility is reached (Nishikawa, 1975). In addition to altering the inclusion rates, it has been suggested that the slow addition of glycerol, or addition at 4°C, may alleviate some of the adverse effects (Pickett and Amann, 1993), but this has not been fully investigated.

Clearly it is impossible to recommend either an absolute inclusion rate for glycerol or an optimum equilibration time. Both are highly dependent upon other factors - for example, other components of the extender, individual stallions, or freezing and thawing rate. It was once thought that an adverse reaction may occur between glycerol and egg yolk, though some more recent research casts doubt on this assumption (Loomis et al., 1983; Cochran et al., 1984).

In an attempt to provide additional protection for acrosome membranes and help to preserve motility post freezing and thawing, concanavalin A has been added to the freezing diluent. Concanavalin A has the ability to coat and thus protect membranes. Betaine, included at 0-3%, has also been used as a possible cryoprotectant, with some limited success (Koskinen et al., 1989). More recently the inclusion of liposomes, which have proved successful in bulls, has been tried with some success with equine semen (Heitland et al., 1995). Phenolic antioxidants have proved successful in rams (Erokhin et al., 1996).

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