THE USE OF L-4 MEMBRANE-FILTER IN FILTRAT ING EQUINE SEMEN

In this study the feasability of filtrating equine semen to select mot ile and morphological intact spermatozoa with the leucocyte adsorption membrane filter Leucosorb L4 was tested by 101 ejaculates from 11 war mblood stallions. The results were compared with these of centrifugati on. After dilution the ejaculates were filtrated through the L4 membra ne filter and centrifuged in the split-sample procedure. They were sto red at +5 degrees Celsius. The motility was evaluated before, immediat ly after, as well as 24, 48 and 72 hours after the preparation. Slides for evaluation of the morphology were dual-stained before processing and after 8 and 30 hours. Before and after filtration as well as centr ifugation samples were stained with Carboxyfluorescindiacetate (CFDA) and Propidiumiodid (PI). The membrane integrity was evaluated by fluor escensmicroscopy. The influence of the different selection methods on fertility was assessed in an insemination trial. Within these trial ce ntrifugated semen (400 Mio. progressive motile spermatozoa/portion) wa s used for 57 mares and filtrated semen for 44 mares (200 Mio. progres sive motile spermatozoa/portion). Pregnancy rates per oestrus and seas onal fertility were analyzed. The following results were obtained: 1. There was a significant increase in viable sperm with stained acrosome and in progressive motility immeditly after L4 membrane filtration an d centrifugation. Additionally total motility improved and there were less spermatozoa with damaged heads or other morphological alterations (p<0,009) after filtration. 2. Altogether filtration resulted at all times in better values for motility, viability and morphology than cen trifugation (p=0,0218). Progressive motility, total motility and amoun t of living spermatozoa was higher. Damaged heads and other defects an d also the amount of dead spermatozoa were lower after filtration than centrifugation. During centrifugation the amount of spermatozoa with membrane defects increased (p=0,0041). Compared with the results of di luted sperm after filtration the number of membrane defects decreased (p=0,004). The exact filtration mechanism is unknown. 3. After centrif ugation the recovery rate of motile and total sperm was significantly higher (p = 0,0016) than after filtration. A possible reason for this fact might be that the filter was not rinsed subsequently. 4. Pregnanc y rates of the mares inseminated with the filtrated semen were 69,2% ( stallion A) and 80,6% (stallion B). Using the centrifugated semen preg nancy rates of 47,6% (A) and 66,6% (B) were obtained. It ought to be e mphasized that the insemination dose of progessively motile spermatozo a was twice as high in comparision to the filtrated semen. Altogether the leucocyte Leucosorb(R) membrane filter L4 filtration resulted at a ll times in better values for motility, morphology, viability and fert ility. This method could be applicated in the preparation of fresh sem en for artificial insemination in horses.