Set up of a serum-free culture system for bovine embryos: Embryo development and quality before and after transient transfer F. George a , C. Daniaux a , G. Genicot a , B. Verhaeghe a , P. Lambert b , I. Donnay a, * a Catholic University of Louvain, Institut des Sciences de la Vie, Unite ´ des Sciences Ve ´te ´rinaires, Place Croix du Sud 5 Box 10, B-1348 Louvain-la-Neuve, Belgium b Catholic University of Louvain, Institut de Statistique, Voie du Roman Pays, 20, B-1348 Louvain-la-Neuve, Belgium Received 20 September 2007; received in revised form 12 November 2007; accepted 12 November 2007 Abstract It is well known that serum in culture medium negatively affects blastocyst quality. The objective of this work was to develop and test a serum-free culture medium which could improve embryo quality, measured by the resistance to freezing, lipid and glutathione content of the resulting blastocysts, as well as the ability of the blastocysts to elongate after transient transfer to recipient cows. In a first experiment we showed that adding a mixture of insulin, transferrin and selenium to serum-free Synthetic Oviduct Fluid medium (SOF–ITS) improved embryo development and quality. In the second experiment, the addition of BSA to SOF–ITS further improved blastocyst development. Moreover, a reduction in lipid content of morulae was observed in SOF–ITS–BSA by comparison with morulae cultured with serum (SOF–FCS). The resistance to freezing measured by hatching rates 24 h post- thawing was also improved for blastocysts with a diameter between 160 and 180 mm cultured in SOF–ITS–BSA by comparison to those produced with serum. In order to evaluate the redox potential of the embryos, reduced glutathione content (GSH) was evaluated both before and after cryopreservation. A significant decrease in glutathione was observed after freezing, whatever the culture medium, but no difference was observed between culture conditions. Transient transfers were performed and elongated D- 13 embryos were recovered. Elongation was more pronounced and the embryonic disk more often visible in embryos cultured in SOF–ITS–BSA than in embryos cultured with FCS. In conclusion, the serum-free system we developed to produce in vitro bovine embryos meets the developmental and qualitative requirements for a large-scale use. # 2007 Elsevier Inc. All rights reserved. Keywords: Embryo development; Embryo quality; Assisted reproductive technology; Early development; Cryopreservation 1. Introduction Successful cryopreservation of in vitro produced (IVP) bovine embryos is essential for their widespread use in the embryo transfer industry. However, these embryos are more sensitive to cryopreservation than their in vivo counterparts [1]. The accumulation of intracellular lipids may be at least partly responsible for this greater sensitivity to freezing [2–5] and addition of serum to culture medium increases such lipid accumulation in bovine embryos [4,6,7]. More- over, the partial delipidation of IVP bovine embryos improves their post-thawing survival [3,8] and removal of serum from the culture medium has been shown to improve the resistance of blastocysts to cryopreservation [9]. www.theriojournal.com Available online at www.sciencedirect.com Theriogenology 69 (2008) 612–623 * Corresponding author. Tel.: +32 10 478748; fax: +32 10 473717. E-mail address: donnay@vete.ucl.ac.be (I. Donnay). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.11.008