title: Cholesterol-loaded-cyclodextrins improve the post-thaw quality of stallion sperm.
abstract: An unacceptable proportion of stallion sperm do not survive the freeze-thaw process. The hypothesis of this study was that adding cholesterol to a stallion semen extender would stabilise the sperm membrane, resulting in an improved post-thaw semen quality in terms of increased sperm viability, membrane integrity and fluidity, and reduced oxidative stress. Semen was collected from three stallions and diluted in four extenders: TALP; TALP+0.75mg methyl-β-cyclodextrin-cholesterol (MβCD)/mL (MβCD0.75); TALP+1.5mg MβCD-cholesterol/mL (MβCD1.5); and Equipro. Following 15min incubation, samples were centrifuged and diluted to 100×10(6)sperm/mL, frozen in 0.5mL straws and stored in liquid nitrogen. Sperm from each treatment was assessed for progressive linear motility (PLM) and acceptable membrane integrity under hypotonic conditions on a phase contrast microscope at 1000× while viability, membrane fluidity and superoxide generation were assessed by flow cytometry. The MβCD1.5 and MβCD0.75 treatments had a greater proportion of viable sperm than the TALP treatment (P<0.01). There was no effect of treatment on PLM or membrane integrity. The MβCD1.5 treatment had a greater proportion of viable sperm positive for membrane fluidity than the TALP treatment (P<0.05). The MβCD1.5 and MβCD0.75 treatments had a lesser proportion of viable sperm positive for superoxide generation than the TALP treatment (P<0.001). This study has demonstrated that adding cholesterol to stallion sperm prior to cryopreservation increases post-thaw viability, with these viable sperm being of better quality in terms of increased membrane fluidity and reduced superoxide generation.
authors.affiliation: Department of Life Sciences, Faculty of Science and Engineering, University of Limerick, Limerick, Ireland.
authors.forename: C
authors.initials: C
authors.lastname: Murphy
authors.affiliation: Department of Life Sciences, Faculty of Science and Engineering, University of Limerick, Limerick, Ireland.
authors.forename: A M
authors.initials: AM
authors.lastname: English
authors.affiliation: Department of Life Sciences, Faculty of Science and Engineering, University of Limerick, Limerick, Ireland.
authors.forename: S A
authors.initials: SA
authors.lastname: Holden
authors.affiliation: Department of Life Sciences, Faculty of Science and Engineering, University of Limerick, Limerick, Ireland. Electronic address: sean.fair@ul.ie.
authors.forename: S
authors.initials: S
authors.lastname: Fair
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000737:chemistry / Q000494:pharmacology*; D003451:Cryoprotective Agents / Q000737:chemistry / Q000494:pharmacology*; D003505:Cyclodextrins / Q000737:chemistry / Q000494:pharmacology*; D006736:Horses / Q000502:physiology*; D008297:Male; D055101:Semen Analysis; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000187:drug effects
chemical_list: D003451:Cryoprotective Agents; D003505:Cyclodextrins; D002784:Cholesterol
keywords: Cholesterol; Equine; Membrane fluidity; Reactive oxygen species; Sperm; Viability