title: Development of procedures for sex-sorting frozen-thawed bovine spermatozoa.
abstract: 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, 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 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.
authors.affiliation: Centre for Advanced Technologies in Animal Genetics and Reproduction (ReproGen), Faculty of Veterinary Science, The University of Sydney, Sydney, NSW 2006, Australia. shelleyu@vetsci.usyd.edu.au
authors.forename: S L
authors.initials: SL
authors.lastname: Underwood
authors.forename: R
authors.initials: R
authors.lastname: Bathgate
authors.forename: W M C
authors.initials: WM
authors.lastname: Maxwell
authors.forename: G
authors.initials: G
authors.lastname: Evans
mesh_terms: D000177:Acrosome / Q000502:physiology; D000818:Animals; D001562:Benzimidazoles; D002417:Cattle; D002469:Cell Separation / Q000379:methods / Q000662:veterinary*; D002499:Centrifugation, Density Gradient / Q000379:methods / Q000662:veterinary; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D005260:Female; D005456:Fluorescent Dyes; D006358:Hot Temperature; D007202:Indicators and Reagents; D008297:Male; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D012732:Sex Determination Analysis / Q000662:veterinary*; D012996:Solutions; D013081:Sperm Motility; D013094:Spermatozoa / Q000166:cytology* / Q000502:physiology
chemical_list: D001562:Benzimidazoles; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D012996:Solutions; C017807:bisbenzimide ethoxide trihydrochloride