title: Caspase 3 Activity and Lipoperoxidative Status in Raw Semen Predict the Outcome of Cryopreservation of Stallion Spermatozoa.
abstract: Stallion-to-stallion variability in the quality of cryopreserved ejaculates postthaw affects the commercial acceptability of frozen semen and thus is a major constraint for the equine industry. In recent years, the molecular mechanisms associated with sperm damage during cryopreservation have become better understood. Identification of the freezability of the ejaculates before the freezing process is initiated will have a major impact on the equine industry. We studied three markers of oxidative stress in sperm, including 8-iso-PGF2alpha, 8-OH guanosine, and 4-hydroxynonenal (4-HNE); the presence of active caspase 3; and their changes after sperm cryopreservation. Although 4-HNE levels increased after cryopreservation (from 7% to 33%, P < 0.001), 8OH-guanosine and 8-ISO-PGF2alpha levels decreased after cryopreservation (from 130 to 35 arbitrary fluorescence units, P < 0.01, and from 1280 to 1233, P < 0.01, respectively). Postthaw sperm quality was classified as poor, average, or good using the 25th and 75th percentiles of all assays of sperm quality studied (motility, velocity, membrane functionality, and thiol content) as thresholds. Using these values, a sperm postthaw quality index was proposed. Receiver operating characteristic curves and the Youden J statistic were used to investigate the value of the measured parameters in fresh sperm as predictors of potential freezability. Using these techniques, we identified markers of bad freezers (percentages of caspase 3-positive dead sperm [area under the curve (AUC) = 0.820, P < 0.05] and percentages of caspase 3- and 4-HNE-positive sperm [AUC = 0.872, P < 0.05]) and good freezers (percentages of caspase 3-negative live sperm [AUC = 0.815, P < 0.05], percentages of live sperm with high thiol content [AUC = 0.907, P < 0.01], and percentages of 8-ISO-PGF2alpha-positive sperm [AUC = 0.900, P < 0.01]. Moreover, we described for the first time the presence of 8-ISO-PGF2alpha in stallion spermatozoa and revealed the importance of considering different markers of oxidative stress.
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Patricia Martín
authors.initials: PM
authors.lastname: Muñoz
authors.affiliation: Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, León, Spain.
authors.forename: Cristina Ortega
authors.initials: CO
authors.lastname: Ferrusola
authors.affiliation: Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, León, Spain.
authors.forename: Luis Anel
authors.initials: LA
authors.lastname: Lopez
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain.
authors.forename: Chiara
authors.initials: C
authors.lastname: Del Petre
authors.affiliation: Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, León, Spain.
authors.forename: Mercedes Alvarez
authors.initials: MA
authors.lastname: Garcia
authors.affiliation: Department of Molecular Biology, University of León, León, Spain.
authors.forename: Paulino
authors.initials: P
authors.lastname: de Paz Cabello
authors.affiliation: Reproduction and Obstetrics Department of Animal Medicine and Surgery, University of León, León, Spain.
authors.forename: Luis
authors.initials: L
authors.lastname: Anel
authors.affiliation: Laboratory of Equine Reproduction and Equine Spermatology, Veterinary Teaching Hospital, University of Extremadura, Cáceres, Spain fjuanpvega@unex.es.
authors.forename: Fernando J
authors.initials: FJ
authors.lastname: Peña
mesh_terms: D000447:Aldehydes / Q000378:metabolism; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism*; D015925:Cryopreservation / Q000379:methods*; D015237:Dinoprost / Q000031:analogs & derivatives / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D006736:Horses; D008297:Male; D018384:Oxidative Stress / Q000502:physiology; D012661:Semen / Q000378:metabolism*; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D006151:Guanosine; C075750:8-epi-prostaglandin F2alpha; C046215:8-hydroxyguanosine; D015237:Dinoprost; D053148:Caspase 3; C027576:4-hydroxy-2-nonenal
keywords: 4-HNE; 8-ISO-PGF2α; 8-OH guanosine; ROS; cryopreservation; equids; flow cytometry; sperm; stallion