title: High pre-freezing dilution improves post-thaw function of ram spermatozoa.
abstract: Despite considerable cryobiology research there is no industry standard for the concentration to which ram spermatozoa should be diluted before freezing. Ram semen is highly concentrated and often frozen at a high sperm concentration, necessitating the use of small laparoscopic insemination doses. The aim of this paper was to ascertain the effect of dilution on the integrity of frozen-thawed ram spermatozoa. In the first experiment, spermatozoa were extended with a Tris-buffered diluent before freezing or after thawing to yield a final sperm concentration of 20 x 10(6)/ml, or were not diluted. Motility characteristics, viability and acrosome integrity of spermatozoa were analysed over a 6h incubation period at 37 degrees C. In the second experiment, spermatozoa were either diluted before freezing, subjected to sex-sorting or not diluted before freezing. Thawed spermatozoa were separated into sub-populations using centrifugal counter-current distribution (CCCD) and the profile of partition and functional integrity (viability, chlortetracycline status and Annexin-V binding) in the sub-populations assessed. Dilution before freezing significantly improved post-thaw viability, acrosome integrity and total motility whereas dilution post-thaw decreased viability and motility of spermatozoa. Sperm heterogeneity, as assessed by CCCD profile, was not different for control, diluted and sex-sorted spermatozoa. Analysis of CCCD sub-populations showed the proportion of functional cells (displaying the F-Pattern or no PS translocation) was similar for all sperm types. The results show that ram spermatozoa retain normal function at higher pre-freeze dilution rates than are commonly used in the sheep industry. The application of these findings would result in more practicable and functional artificial insemination doses.
authors.affiliation: Faculty of Veterinary Science, University of Sydney, NSW 2006, Sydney, Australia. tamaral@vetsci.usyd.edu.au
authors.forename: T
authors.initials: T
authors.lastname: Leahy
authors.forename: J I
authors.initials: JI
authors.lastname: Marti
authors.forename: N
authors.initials: N
authors.lastname: Mendoza
authors.forename: R
authors.initials: R
authors.lastname: Pérez-Pé
authors.forename: T
authors.initials: T
authors.lastname: Muiño-Blanco
authors.forename: J A
authors.initials: JA
authors.lastname: Cebrián-Pérez
authors.forename: G
authors.initials: G
authors.lastname: Evans
authors.forename: W M C
authors.initials: WM
authors.lastname: Maxwell
mesh_terms: D000818:Animals; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D005615:Freezing; D007201:Indicator Dilution Techniques; D008297:Male; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods* / Q000662:veterinary; D012756:Sheep; D013075:Sperm Capacitation / Q000502:physiology; D013076:Sperm Count / Q000662:veterinary; D013094:Spermatozoa / Q000166:cytology* / Q000502:physiology*