SURVIVAL AND FERTILITY OF MICRO-ENCAPSULATED RAM SPERMATOZOA STORED AT 5-DEGREES-C

In this study, the viability and fertility of micro-encapsulated ram s permatozoa were examined, and the efficacy of two membrane proteins we re evaluated for liquid storage of ram spermatozoa at 5 degrees C. In the first experiment, the motility, percentage live/dead and acrosome intact/reacted (determined by flow cytometry after staining with SYBR- 14/PI and FITC-PSA/PI, respectively) were assessed after extension of spermatozoa to a concentration of 20 x 10(6) per ml in Tris-glucose-yo lk (TGY) diluent (control) or to the same concentration in microcapsul es composed of poly L-lysine (polyL) or poly D-lysine (polyD) suspende d in TGY. The semen was stored at 5 degrees C for 8 days. More spermat ozoa were live, motile and had intact acrosomes in the control vs. enc apsulated semen (67.9 vs. 20.8% live, 73.0 vs. 36.7% motile, and 60.3 vs. 24.8% acrosome intact, respectively; p < 0.001). The type of capsu le had no effect on the proportion of live or motile spermatozoa, alth ough more cells were acrosome reacted in the polyL than in the polyD c apsules or the controls (16.2 vs. 12.7 or 10.0% acrosome reacted, resp ectively; p < 0.001). Despite a reduction in the motility of spermatoz oa during storage (63.3 vs. 35.6% motile for control and 8-day-old sem en, respectively; p < 0.001), there was no change in the percentages o f live/dead or acrosome intact/reacted cells. In a fertility test, 24 ewes were synchronized in oestrus using progestagen implants and super ovulated with a combination of follicle-stimulating hormone and pregna nt mares' serum gonadotrophin. The ewes were inseminated in the uterus 39 h after implant removal with control (unencapsulated) or encapsula ted semen which had been stored at 5 degrees C for 20 h. The inseminat e contained 20 x 10(6) total spermatozoa. Control semen was deposited by laparoscopy (n = 12) and encapsulated semen by laparotomy (n = 12 e wes). Seventy-seven ova were recovered from 23 of the ewes 48 h after insemination, of which 57 (74%) were fertilized. The recovery rate of ova was lower for those ewes inseminated by laparotomy than by laparos copy (38.8 vs. 75.9%, respectively, p < 0.001), but there were no diff erences between encapsulated and control spermatozoa in the proportion of eggs fertilized, the proportion of ewes with fertilized eggs, or c ell numbers of recovered ova. However, the mean number of accessory sp ermatozoa observed per ovum was lower for ewes inseminated with polyL- encapsulated than with control semen (5.8 +/- 12.4 vs. 8.9 +/- 17.1, m eans +/- SD, respectively, p < 0.05). This is the first report of fert ility after insemination of sheep with encapsulated spermatozoa, and c onfirms that micro-encapsulation techniques for the bovine can be appl ied to ram spermatozoa. Further research is required to improve the vi ability of spermatozoa after storage in microcapsules.