title: Comparison of methods for assessing integrity of equine sperm membranes.
abstract: Sperm membrane integrity (SMI) is thought to be an important measure of stallion sperm quality. The objective was to compare three methods for evaluating SMI: flow cytometry using SYBR-14/propidium iodide (PI) stain; an automated cell counting device using PI stain; and eosin-nigrosin stain. Raw equine semen was subjected to various treatments containing 20 to 80% seminal plasma in extender, with differing sperm concentrations, to simulate spontaneous loss of SMI. The SMI was assessed immediately, and after 1 and 2 d of cooled storage. Agreement between methods was determined according to Bland-Altman methodology. Eosin-nigrosin staining yielded higher (2%) overall mean values for SMI than did flow cytometry. Flow cytometry yielded higher (6%) overall mean values for SMI than did the automated cell counter. As percentage of membrane-damaged sperm increased, agreement of SMI measurement between methods decreased. When semen contained 50-79% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -26.9 to 24.3%; i.e., a 51.2% span) than for SMI determined by flow cytometry and the automated cell counter (range = -3.1 to 17.0%; 20.1% span). When sperm populations contained <50% membrane-intact sperm, the 95% limits of agreement between SMI determined by flow cytometry and eosin-nigrosin staining were greater (range = -35.9 to 19.0%; 54.9% span) than for SMI determined by flow cytometry and the automated cell counter (range = -11.6 to 28.7%; 40.3% span). We concluded that eosin-nigrosin staining assessments of percent membrane-intact sperm agreed less with flow cytometry when <80% of sperm had intact membranes, whereas automated cell counter assessments of percent membrane-intact sperm agreed less with flow cytometry when <30% of sperm had intact membranes.
authors.affiliation: Department of Large Animal Clinical Sciences, Texas A&M University, College Station, TX, USA.
authors.forename: M L
authors.initials: ML
authors.lastname: Foster
authors.forename: C C
authors.initials: CC
authors.lastname: Love
authors.forename: D D
authors.initials: DD
authors.lastname: Varner
authors.forename: S P
authors.initials: SP
authors.lastname: Brinsko
authors.forename: K
authors.initials: K
authors.lastname: Hinrichs
authors.forename: S
authors.initials: S
authors.lastname: Teague
authors.forename: K
authors.initials: K
authors.lastname: Lacaze
authors.forename: T L
authors.initials: TL
authors.lastname: Blanchard
mesh_terms: D000814:Aniline Compounds; D000818:Animals; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure*; D004396:Coloring Agents; D004801:Eosine Yellowish-(YS); D005434:Flow Cytometry / Q000662:veterinary; D005456:Fluorescent Dyes; D006736:Horses; D008297:Male; D009930:Organic Chemicals; D011419:Propidium; D013076:Sperm Count; D013094:Spermatozoa / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000662:veterinary
chemical_list: D000814:Aniline Compounds; D004396:Coloring Agents; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C093207:SYBR-14; D011419:Propidium; C002712:nigrosin; D004801:Eosine Yellowish-(YS)