title: Supplementing culture and vitrification-warming media with l-ascorbic acid enhances survival rates and redox status of IVP porcine blastocysts via induction of GPX1 and SOD1 expression.
abstract: The present study sought to determine the effect of adding l-ascorbic acid (AC) to (1) in vitro culture medium and (2) vitrification and warming solutions on redox status and developmental ability and quality of IVP porcine embryos. In both experiments, embryo quality was analysed in terms of total cell number (TCN), DNA fragmentation, intracellular peroxide levels and expression of three oxidative stress-related genes: glutathione peroxidase 1 (GPX1), superoxide dismutase 1 (SOD1) and 2 (SOD2). In the first experiment, fresh blastocysts were found to upregulate SOD1 expression when cultured with medium supplemented 100 μM AC. No differences were found between culture groups in the other analysed parameters. In the second experiment, blastocysts cultured with or without AC were divided into two groups: vitrified and warmed with solutions containing 0 or 100 μM AC. Addition of AC during culture and vitrification-warming upregulated the expression of GPX1 and SOD1 genes, enhanced survival rates and decreased peroxide levels at 24h post-warming. In addition, peroxide levels were negatively correlated with relative GPX1- and SOD1-transcript abundances, whereas GPX1 was positively correlated with embryo survival at 24h post-warming. No effects of AC-supplementation were seen for TCN, DNA fragmentation or relative SOD2-transcript abundance in vitrified blastocysts. In conclusion, the addition of AC to culture and vitrification-warming media increases gene expression of antioxidant enzymes SOD1 and GPX1. This appears to improve redox balance and is suggested to ultimately enhance embryo cryosurvival.
authors.affiliation: Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, E-17071 Girona, Spain. Electronic address: miriam.castillo@udg.edu.
authors.forename: Miriam
authors.initials: M
authors.lastname: Castillo-Martín
authors.affiliation: Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, E-17071 Girona, Spain.
authors.forename: Sergi
authors.initials: S
authors.lastname: Bonet
authors.affiliation: Biotechnology of Animal and Human Reproduction (TechnoSperm), Department of Biology, Institute of Food and Agricultural Technology, University of Girona, E-17071 Girona, Spain.
authors.forename: Roser
authors.initials: R
authors.lastname: Morató
authors.affiliation: Unit of Animal Reproduction, Department of Animal Medicine and Surgery, Faculty of Veterinary Medicine, Autonomous University of Barcelona, E-08193 Bellaterra (Barcelona), Spain.
authors.forename: Marc
authors.initials: M
authors.lastname: Yeste
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D001205:Ascorbic Acid / Q000494:pharmacology*; D001755:Blastocyst / Q000187:drug effects* / Q000502:physiology; D015925:Cryopreservation / Q000662:veterinary; D003470:Culture Media / Q000494:pharmacology; D005307:Fertilization in Vitro; D005979:Glutathione Peroxidase / Q000235:genetics*; D010084:Oxidation-Reduction / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000032:analysis / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics*; D000072105:Superoxide Dismutase-1; D013552:Swine / Q000196:embryology*; D015854:Up-Regulation / Q000187:drug effects; D058989:Vitrification; D000094765:Glutathione Peroxidase GPX1
chemical_list: D000975:Antioxidants; D003470:Culture Media; D017382:Reactive Oxygen Species; C000606290:SOD1 protein, human; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D001205:Ascorbic Acid; D000094765:Glutathione Peroxidase GPX1
keywords: Blastocysts; Cryosurvival; Cryotop; GPX1; In vitro produced; ROS levels; SOD1; SOD2