title: Insulin concentrations used in in vitro embryo production systems: a pilot study on insulin stability with an emphasis on concentrations measured in vivo.
abstract: BACKGROUND
  Insulin has been used as a stimulatory factor for in vitro cell culture since many years. Even for routine in vitro embryo production (IVP), insulin is added to the media during different steps. There is a strong difference in concentrations used in vitro compared to what is measured in vivo in follicular fluid or serum. We performed a pilot study on insulin stability to explain possible reasons for that variation.
  
  
  RESULTS
  We measured insulin concentrations before and after bovine oocyte maturation in an experiment by using a quantitative ELISA (Mercodia bovine insulin ELISA immunoassay) and found that concentrations were stable up to 22 h of incubation. We compared our results with eleven in vivo studies measuring insulin in either serum or follicular fluid and nine IVP-protocols using insulin. In all studies, in vitro concentrations were much higher compared with those found physiologically in vivo. Limited knowledge is available concerning the different activity and stability of insulin in vitro versus in vivo.
  
  
  CONCLUSIONS
  The concentrations of insulin used in vitro are quite high in comparison to physiological concentrations found in serum or follicular fluid. One explanation may be a different stability or activity of insulin in vitro even if we could measure stable concentrations of insulin in our pilot study. More precise dose-effect studies have to be performed to draw clear conclusions about the consequences of the use of such high doses as they might have negative consequences for the developing embryo. Insulin has direct effects on the regulation of the metabolism and could even influence the epigenetic programming of the metabolism with unknown consequences for the offspring later in life.
authors: Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden. denise.laskowski@slu.se.
authors: Denise
authors: D
authors: Laskowski
authors: Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors: Ylva
authors: Y
authors: Sjunnesson
authors: Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors: Hans
authors: H
authors: Gustafsson
authors: Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors: Patrice
authors: P
authors: Humblot
authors: Department of Animal Breeding and Genetics, Section of Molecular Animal Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors: Göran
authors: G
authors: Andersson
authors: Division of Reproduction, Department of Clinical Sciences, Swedish University of Agricultural Sciences, Uppsala, Sweden.
authors: Renée
authors: R
authors: Båge
mesh_terms: D000818:Animals; D002417:Cattle; D003470:Culture Media / Q000592:standards*; D004355:Drug Stability; D046149:Embryo Culture Techniques / Q000592:standards / Q000662:veterinary*; D066298:In Vitro Techniques; D007328:Insulin / Q000032:analysis / Q000378:metabolism*; D010865:Pilot Projects
chemical_list: D003470:Culture Media; D007328:Insulin
keywords: Blastocyst; Bovine; Embryo development; In vitro model; Insulin; Oocyte maturation