title: Effect of holding equine oocytes in meiosis inhibitor-free medium before in vitro maturation and of holding temperature on meiotic suppression and mitochondrial energy/redox potential.
abstract: BACKGROUND
  Evaluation of mitochondrial function offers an alternative to evaluate embryo development for assessment of oocyte viability, but little information is available on the relationship between mitochondrial and chromatin status in equine oocytes. We evaluated these parameters in immature equine oocytes either fixed immediately (IMM) or held overnight in an Earle's/Hank's' M199-based medium in the absence of meiotic inhibitors (EH treatment), and in mature oocytes. We hypothesized that EH holding may affect mitochondrial function and that holding temperature may affect the efficiency of meiotic suppression.
  
  
  METHODS
  Experiment 1 - Equine oocytes processed immediately or held in EH at uncontrolled temperature (22 to 27°C) were evaluated for initial chromatin configuration, in vitro maturation (IVM) rates and mitochondrial energy/redox potential. Experiment 2 - We then investigated the effect of holding temperature (25°C, 30°C, 38°C) on initial chromatin status of held oocytes, and subsequently repeated mitochondrial energy/redox assessment of oocytes held at 25°C vs. immediately-evaluated controls.
  
  
  RESULTS
  EH holding at uncontrolled temperature was associated with advancement of germinal vesicle (GV) chromatin condensation and with meiotic resumption, as well as a lower maturation rate after IVM. Holding did not have a significant effect on mitochondrial distribution within chromatin configurations. Independent of treatment, oocytes having condensed chromatin had a significantly higher proportion of perinuclear/pericortical mitochondrial distribution than did other GV configurations. Holding did not detrimentally affect oocyte energy/redox parameters in viable GV-stage oocytes. There were no significant differences in chromatin configuration between oocytes held at 25°C and controls, whereas holding at higher temperature was associated with meiosis resumption and loss of oocytes having the condensed chromatin GV configuration. Holding at 25°C was not associated with progression of mitochondrial distribution pattern and there were no significant differences in oocyte energy/redox parameters between these oocytes and controls.
  
  
  CONCLUSIONS
  Mitochondrial distribution in equine GV-stage oocytes is correlated with chromatin configuration within the GV. Progression of chromatin configuration and mitochondrial status during holding are dependent on temperature. EH holding at 25°C maintains meiotic arrest, viability and mitochondrial potential of equine oocytes. This is the first report on the effects of EH treatment on oocyte mitochondrial energy/redox potential.
authors.affiliation: Veterinary Clinics and Animal Productions Unit-Dipartimento dell'Emergenza e Trapianti D'Organo (DETO), Università di Bari Aldo Moro, Str, Prov, Casamassima Km 3°, Valenzano 70010, Bari, Italy. nicmartino@libero.it.
authors.forename: Nicola A
authors.initials: NA
authors.lastname: Martino
authors.forename: Maria E
authors.initials: ME
authors.lastname: Dell'Aquila
authors.forename: Manuel
authors.initials: M
authors.lastname: Filioli Uranio
authors.forename: Lucia
authors.initials: L
authors.lastname: Rutigliano
authors.forename: Michele
authors.initials: M
authors.lastname: Nicassio
authors.forename: Giovanni M
authors.initials: GM
authors.lastname: Lacalandra
authors.forename: Katrin
authors.initials: K
authors.lastname: Hinrichs
mesh_terms: D000003:Abattoirs; D000818:Animals; D002470:Cell Survival; D042002:Chromatin Assembly and Disassembly; D003080:Cold Temperature / Q000009:adverse effects; D003470:Culture Media; D004734:Energy Metabolism; D005260:Female; D006736:Horses / Q000502:physiology*; D059471:In Vitro Oocyte Maturation Techniques / Q000662:veterinary; D008540:Meiosis; D018613:Microscopy, Confocal / Q000662:veterinary; D008856:Microscopy, Fluorescence / Q000662:veterinary; D008928:Mitochondria / Q000378:metabolism*; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism; D009866:Oogenesis; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism*
chemical_list: D003470:Culture Media; D017382:Reactive Oxygen Species