Development of Procedures for Sex-sorting Frozen-Thawed Bovine Spermatozoa
2009
Underwood, SL | Bathgate, R. | Maxwell, WMC | Evans, G.
Dairy bull sperm may be sex-sorted, frozen and used to artificially inseminate heifers with acceptable fertility if the herd is well-managed. One drawback to the technology is that donor bulls must be located within a short distance of the sorting facility in order to collect semen, which limits the number of bulls from which sorted sperm are available. A successful method used to overcome this limitation in sheep is sex-sorting from frozen-thawed semen and refreezing for artificial insemination. This technique is attractive to the dairy industry, and therefore a series of three experiments was designed to investigate the optimal methods to prepare, sex-sort and re-freeze frozen-thawed bovine sperm. Sperm were prepared for sorting by density gradient separation in either PureSperm® or BoviPure[trade mark sign], followed by staining in one of three diluents (Androhep®, Bovine Sheath Fluid + 0.3% BSA or TALP buffer). Sperm were sorted and collected into Test yolk buffer, and frozen in an extender containing 0, 0.25, 0.375 or 0.5% Equex STM Paste. Frozen-thawed sperm were better orientated (p = 0.006) and had fewer damaged membranes (8.7 ± 0.6% vs 19.5 ± 2.4%; p = 0.003) after centrifugation in PureSperm® rather than BoviPure[trade mark sign] gradients. Sperm orientation (p < 0.05) and motility (69.9 ± 3.0 vs 55.6 ± 4.0; p < 0.001) were highest after staining in Androhep® rather than in TALP buffer. Sperm were more motile (58.2 ± 4.7 vs 38.7 ± 3.5; p < 0.001) and had better acrosome integrity (74.3 ± 2.9 vs 66.8 ± 2.0; p < 0.001) after freezing in an extender containing 0.375% Equex STM Paste than in extender without Equex. Hence, a protocol has been developed to allow frozen-thawed bull sperm to be sex-sorted with high resolution between the sexes, then re-frozen and thawed with retention of motility and acrosome integrity.
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