title: The influence of antioxidant, cholesterol and seminal plasma on the in vitro quality of sorted and non-sorted ram spermatozoa.
abstract: In an effort to improve the number of functional spermatozoa following sex-sorting and cryopreservation, the effects on in vitro sperm characteristics of the additives: (i) catalase (pre-sorting); (ii) cholesterol-loaded cyclodextrins (CLCs; pre-sorting); and (iii) seminal plasma (post-thawing) were investigated. For all experiments, spermatozoa (three males, n=3 ejaculates/male) were processed using a high speed flow cytometer before cryopreservation, thawing and incubation for 6h. Catalase had no effect (P>0.05) on post-thaw motility characteristics (as measured by CASA) of sex-sorted ram spermatozoa, but pre-sort addition of CLCs reduced (P<0.05) sperm quality after post-thaw incubation for 0 h (motility), 3h (motility, average path velocity, viability and acrosome integrity) and 6h (motility, average path and curvilinear velocity, straightness, linearity, viability and acrosome integrity). Seminal plasma had a differential effect (P<0.001) on sex-sorted and non-sorted spermatozoa. Post-thaw supplementation of increasing levels of seminal plasma caused all motility characteristics of sex-sorted, frozen-thawed spermatozoa to decline (P<0.05); conversely, non-sorted, frozen-thawed spermatozoa exhibited improvements (P<0.05) in motility, viability, acrosome integrity and mitochondrial respiration. In summary, incorporation of catalase, CLCs and seminal plasma into the sorting protocol failed to improve post-thaw sperm quality and, consequently efficiency of sex-sorting of ram spermatozoa. The paradoxical effect of seminal plasma supplementation on the in vitro characteristics of ram spermatozoa provides further evidence that sex-sorting by flow cytometry produces a selected population of cells with different functions compared with non-sorted spermatozoa.
authors.affiliation: Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, The University of Sydney, Sydney, NSW 2006, Australia. simong@vetsci.usyd.edu.au
authors.forename: S P
authors.initials: SP
authors.lastname: de Graaf
authors.forename: G
authors.initials: G
authors.lastname: Evans
authors.forename: L
authors.initials: L
authors.lastname: Gillan
authors.forename: M M P
authors.initials: MM
authors.lastname: Guerra
authors.forename: W M C
authors.initials: WM
authors.lastname: Maxwell
authors.forename: J K
authors.initials: JK
authors.lastname: O'Brien
mesh_terms: D000177:Acrosome / Q000187:drug effects / Q000502:physiology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D002374:Catalase / Q000494:pharmacology; D002784:Cholesterol / Q000494:pharmacology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary; D005434:Flow Cytometry / Q000379:methods / Q000662:veterinary; D008297:Male; D012661:Semen / Q000502:physiology*; D012662:Semen Preservation / Q000662:veterinary; D012743:Sex Preselection / Q000379:methods / Q000662:veterinary*; D012756:Sheep / Q000502:physiology*; D013081:Sperm Motility / Q000187:drug effects / Q000502:physiology; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology; D013997:Time Factors
chemical_list: D000975:Antioxidants; D002784:Cholesterol; D002374:Catalase