Adding hormones sequentially could be an effective approach for IVM of dog oocytes M. Evecen a ,U ¨ . Cirit b, *, K. Demir a , A.I ˙ . Hamzaog ˘lu a , G. Bakırer a , S. Pabuccuog ˘lu a , S. Birler a a Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Istanbul University, 34320, Avcilar, Istanbul, Turkey b Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine, Dicle University, 21280, Diyarbakir, Turkey Received 13 May 2010; received in revised form 26 October 2010; accepted 4 January 2011 Abstract There have not been successful and repeatable methods for in vitro embryo production in the dog. Up to date, only one blactocyst has been achieved on in vitro culture. Since reproductive physiology of the dog is different from that of other mammalian species, it seems that a suitable method for in vitro production of canine embryos is still far from being designed and routinely applied, and an effective protocol is needed. Therefore, the aim of the present study was to examine the effects of adding hormones sequentially, for mimicking the dog’s in vivo endocrine milieu, on maturation of immature dog oocytes in vitro. At the end of the 96 h IVM period, nuclear maturation rates were evaluated by the aceto-orcein staining method. In comparison relating IVM rates, the sequential hormone addition was more beneficial on IVM rates (MI + MII) than the traditional hormone addition and control groups (48.1%, 38.9% and 23.0% respectively; P  0.0001). As a result, hormone addition sequentially may be an effective approach for the IVM of the immature dog oocytes. We suggest that attempts to define the adequate conditions for IVM in the dog should extend towards this new perspective. © 2011 Elsevier Inc. All rights reserved. Keywords: Dog; Oocyte; IVM; Hormone 1. Introduction The potential benefits of assisted reproduction tech- niques, such as in vitro maturation (IVM) and in vitro fertilization (IVF) in canids, are linked to protection and saving of species threatened by extinction due to worldwide habitat destruction and pollution [1]. How- ever, the efficiency of these technologies is still very limited. Until now, no offspring have been produced in the dog by transferring embryos derived from in vitro matured oocytes. It seems that an adequate protocol for in vitro production of canine embryos is still far from being designed and routinely applied, and an effective method is needed. In several mammalian species, stud- ies have attempted to optimize oocyte maturation in vitro by modelling culture systems upon physiological conditions [2]. Canine oocytes seem to be quite refrac- tory to the different treatments that have been tested and their maturation and fertilization rates still remain low [3]. The reproductive physiology of the domestic dog is unique from that of other mammalian species. The dog’s reproductive cycle is distinguished by a prolonged proestrus and estrus. Irrespective of whether pregnancy occurs, estrus is followed by diestrus, a * Corresponding author. Tel.: +90 412 248 80 20 (8667); fax: +90 412 248 80 21. E-mail address: ucirit@dicle.edu.tr (U ¨ . Cirit). Available online at www.sciencedirect.com Theriogenology 75 (2011) 1647–1651 www.theriojournal.com 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.01.004