title: Improving the effect of incubation and oxidative stress on thawed spermatozoa from red deer by using different antioxidant treatments.
abstract: Antioxidants could improve sperm media, extending the viability of spermatozoa and protecting their DNA. The protective ability of lipoic acid, melatonin, Trolox and crocin was tested on red deer spermatozoa incubated at 37 degrees C. Cryopreserved spermatozoa were thawed and incubated with 1 mM or 0.1 mM of each antioxidant, with or without oxidative stress (100 muM Fe(2+)). Motility (CASA), viability, mitochondrial membrane potential and acrosomal status were assessed. Lipoperoxidation (malondialdehyde production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were checked at 4 h. Incubation alone increased ROS and decreased motility. Oxidative stress intensified these effects, increasing lipoperoxidation and DNA damage. Lipoic acid had little protective effect, whereas 1 mM melatonin showed limited protection. Trolox lowered ROS and lipoperoxidation both in oxidised and non-oxidised samples. In oxidised samples, Trolox prevented DNA and acrosomal damage, and ameliorated motility. Crocin at 1 mM showed similar results to Trolox, but noticeably stimulated motility and had no effect on lipoperoxidation. In a second experiment, a broader range of crocin and melatonin concentrations were tested, confirming the effects of crocin (positive effects noticeable at 0.5-0.75 mM), but showing an increase in lipoperoxidation at 2 mM. Melatonin was increasingly effective at 2.5 and 5 mM (ROS, lipoperoxidation and DNA status). Crocin seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied, especially the consequences of motility stimulation and confirming its effect on lipoperoxidation. Melatonin might be useful at relatively high concentrations, compared to Trolox.
authors.affiliation: Biology of Reproduction Group, National Wildlife Research Institute (IREC) (UCLM-CSIC-JCCM), 02071 Albacete, Spain.
authors.forename: Alvaro E
authors.initials: AE
authors.lastname: Domínguez-Rebolledo
authors.forename: María R
authors.initials: MR
authors.lastname: Fernández-Santos
authors.forename: Alfonso
authors.initials: A
authors.lastname: Bisbal
authors.forename: José Luis
authors.initials: JL
authors.lastname: Ros-Santaella
authors.forename: Manuel
authors.initials: M
authors.lastname: Ramón
authors.forename: Manuel
authors.initials: M
authors.lastname: Carmona
authors.forename: Felipe
authors.initials: F
authors.lastname: Martínez-Pastor
authors.forename: J Julián
authors.initials: JJ
authors.lastname: Garde
mesh_terms: D000177:Acrosome / Q000648:ultrastructure; D000818:Animals; D000975:Antioxidants / Q000008:administration & dosage*; D002338:Carotenoids / Q000008:administration & dosage; D002839:Chromans / Q000008:administration & dosage; D015925:Cryopreservation / Q000662:veterinary*; D004247:DNA / Q000032:analysis; D004249:DNA Damage / Q000187:drug effects; D003670:Deer; D006358:Hot Temperature; D020287:In Situ Nick-End Labeling; D015227:Lipid Peroxidation / Q000187:drug effects; D008297:Male; D008550:Melatonin / Q000008:administration & dosage; D053078:Membrane Potential, Mitochondrial; D018384:Oxidative Stress / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000032:analysis; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000737:chemistry / Q000502:physiology* / Q000648:ultrastructure; D008063:Thioctic Acid / Q000008:administration & dosage
chemical_list: D000975:Antioxidants; D002839:Chromans; D017382:Reactive Oxygen Species; D002338:Carotenoids; D008063:Thioctic Acid; C029036:crocin; D004247:DNA; D008550:Melatonin; C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid