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Survival and fertility of micro-encapsulated ram spermatozoa stored at 5 degrees C



Survival and fertility of micro-encapsulated ram spermatozoa stored at 5 degrees C



Reproduction in Domestic Animals 31(4-5): 665-673



In this study, the viability and fertility of micro-encapsulated ram spermatozoa were examined, and the efficacy of two membrane proteins were evaluated for liquid storage of ram spermatozoa at 5 degree 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 times 10-6 per ml in Tris-glucose-yolk (TGY) diluent (control) or to the same concentration in microcapsules composed of poly L-lysine (polyL) or poly D-lysine (polyD) suspended in TGY. The semen was stored at 5 degree C for 8 days. More spermatozoa were live, motile and had intact acrosomes in the control vs. encapsulated semen (67.9 vs. 20.8% live, 73.0 vs. 36.7% motile, and 60.3 vs. 24.8% acrosome intact, respectively; p lt 0.001). The type of capsule had no effect on the proportion of live or motile spermatozoa, although more cells were acrosome reacted in the polyL than in the polyD capsules or the controls (16.2 vs. 12.7 or 10.0% acrosome reacted, respectively; p lt 0.001). Despite a reduction in the motility of spermatozoa during storage (63.3 vs. 35.6% motile for control and 8-day-old semen, respectively; p lt 0.001), there was no change in the percentages of live/ dead or acrosome intact/reacted cells. In a fertility test, 24 ewes were synchronized in oestrus using progestagen implants and superovulated with a combination of follicle-stimulating hormone and pregnant mares' serum gonadotrophin. The ewes were inseminated in the uterus 39 h after implant removal with control (unencapsulated) or encapsulated semen which had been stored at 5 degree C for 20 h. The inseminate contained 20 times 10-6 total spermatozoa. Control semen was deposited by laparoscopy (n = 12) and encapsulated semen by laparotomy (n = 12 ewes). 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 laparoscopy (38.8 vs. 75.9%, respectively, p lt 0.001), but there were no differences between encapsulated and control spermatozoa in the proportion of eggs fertilized, the proportion of ewes with fertilized eggs, or cell numbers of recovered ova. However, the mean number of accessory spermatozoa observed per ovum was lower for ewes inseminated with polyl-encapsulated than with control semen (5.8 +- 12.4 vs. 8.9 +- 17.1, means +- SD, respectively, p lt 0.05). This is the first report of fertility after insemination of sheep with encapsulated spermatozoa, and confirms that micro-encapsulation techniques for the bovine can be applied to ram spermatozoa. Further research is required to improve the viability of spermatozoa after storage in microcapsules.

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Accession: 002973009

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