title: Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.
abstract: Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates.
authors.affiliation: Department of Veterinary Medicine and Animal Production, Federico II University of Naples-Via F., Naples, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Serafini
authors.forename: V
authors.initials: V
authors.lastname: Longobardi
authors.forename: M
authors.initials: M
authors.lastname: Spadetta
authors.forename: D
authors.initials: D
authors.lastname: Neri
authors.forename: B
authors.initials: B
authors.lastname: Ariota
authors.forename: B
authors.initials: B
authors.lastname: Gasparrini
authors.forename: R
authors.initials: R
authors.lastname: Di Palo
mesh_terms: D000177:Acrosome / Q000648:ultrastructure; D020101:Acrosome Reaction; D000818:Animals; D001399:Azure Stains; D001947:Breeding; D002462:Cell Membrane / Q000648:ultrastructure*; D005260:Female; D005298:Fertility; D006736:Horses; D007315:Insemination, Artificial / Q000662:veterinary; D008297:Male; D011247:Pregnancy; D018873:Pregnancy Rate; D012372:ROC Curve; D012680:Sensitivity and Specificity; D013076:Sperm Count / Q000662:veterinary; D013081:Sperm Motility; D013094:Spermatozoa / Q000502:physiology / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods / Q000662:veterinary*; D014343:Trypan Blue
chemical_list: D001399:Azure Stains; D014343:Trypan Blue