This experiment was carried out to develop a new extender for long term preservation of equine sperm at 5°C. Semen was collected from 4 riding horse stallions by means of an artificial vagina and filtered through gauze. Gel-free semen was diluted 2 to 3 times with a pre-extender and then centrifuged (450 × g, 10 min). Precipitated spermatozoa were suspended in the extender for preservation to give a final sperm concentration of 1 × 10 ⁸/ml and stored at 5°C for 72 hr. Sperm motility was examined every day. Preserved equine spermatozoa in Modena extender (developed for boar semen preservation at 15°C) containing egg yolk maintained more superior motility than those in some other boar semen extenders during the storage at 5°C (p<0.05). As additives to equine semen extender, Casein was a very beneficial protein as compared with BSA, Ovalbumin and Livetin (p<0.05). Two %Casein extender which was made based on the Modena was superior to Martin, Kenney and CGH-27 extender for maintaining the viability of equine sperm, and was further improved by the addition of 5%egg yolk. The conception rates for semen stored in Casein-egg yolk extender at 5°C were 63.6% (14/22) within 0-72 hr and 75% (3/4) within 96-120 hr of storage, respectively. The total conception rate for semen preserved for up to 120 hr at 5°C was 65.4% (17/26). We have named this new casein-yolk extender (see in Table 5) ‘Iwate extender’.

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In this study, we examined the relationship between the osmotic fragility and K-Cl cotransport (KCC) activity in the density-separated erythrocytes from thoroughbred horses. Erythrocytes from four healthy thoroughbreds were collected, and separated into five fractions according to their density. The KCC activities of the cells were determined as the K⁺ congener Rb⁺ influx in the cells suspended in isotonic medium in which the main salts were constructed with N-methyl-D-glucamine (NMDG)/NO₃ or NMDG/Cl and the medium pH were varied from 7.0 to 7.8. The decrease in medium pH was associated with an increase in the KCC activities, particularly the KCC activity at pH 7.0 was 4- and 50-fold higher than that at pH 7.4 and 7.8, respectively. The KCC activity in the dense cells was also significantly higher than that in the light cells. The decrease in medium pH and/or the density of erythrocytes was associated with a progressive increase in the osmotic fragility. The osmotic fragility measured in the NMDG/NO₃ medium always showed a tendency to be higher than that incubated in the NMDG/Cl medium in each medium pH and/or cell fraction. These results suggest that 1) the increase in the KCC activity may cause shrinkage of the cell volume and decrease the fragility; and 2) the KCC has a functional interrelation to the change in the erythrocyte fragility with the fall in pH. The KCC of equine erythrocytes therefore plays an important role in the prevention of fragilocytes and protection of haemolysis in blood vessels with exercise.

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