Vitrification of early-stage bovine and equine embryos L.F. Campos-Chillo `n, T.K. Suh, M. Barcelo-Fimbres, G.E. Seidel Jr., E.M. Carnevale * Animal Reproduction and Biotechnology Laboratory, Foothills Campus, Colorado State University, Fort Collins, CO 80523-1683, USA Received 22 February 2008; received in revised form 23 July 2008; accepted 4 August 2008 Abstract The objectives of this study were to: (1) determine an optimal method and stage of development for vitrification of bovine zygotes or early embryos; and (2) use the optimal procedure for bovine embryos to establish equine pregnancies after vitrification and warming of early embryos. Initially, bovine embryos produced by in-vitro fertilization (IVF) were frozen and vitrified in 0.25 mL straws with minimal success. A subsequent experiment was done using two vitrification methods and super open pulled straws (OPS) with 1- or 8-cell bovine embryos. In Method 1 (EG-O), embryos were exposed to 1.5 M ethylene glycol (EG) for 5 min, 7 M ethylene glycol and 0.6 M galactose for 30 s, loaded in an OPS, and plunged into liquid nitrogen. In Method 2 (EG- DMSO), embryos were exposed to 1.1 M ethylene glycol and 1.1 M dimethyl sulfoxide (DMSO) for 3 min, 2.5 M ethylene glycol, 2.5 M DMSO and 0.5 M galactose for 30 s, and loaded and plunged as for EG-O. Cryoprotectants were removed after warming in three steps. One- and eight-cell bovine embryos were cultured for 7 and 4.5 d, respectively, after warming, and control embryos were cultured without vitrification. Cleavage rates of 1-cell embryos were similar (P > 0.05) for vitrified and control embryos, although the blastocyst rates for EG-O and control embryos were similar and higher (P < 0.05) than for EG-DMSO. The blastocyst rate of 8-cell embryos was higher (P < 0.05) for EG-O than EG-DMSO. Therefore, EG-O was used to cryopreserve equine embryos. Equine oocytes were obtained from preovulatory follicles. After ICSI, injected oocytes were cultured for 1–3 d. Two- to eight-cell embryos were vitrified, warmed and transferred into recipient’s oviducts. The pregnancy rate on Day 20 was 62% (5/8) for equine embryos after vitrification and warming. In summary, a successful method was established for vitrification of early-stage bovine embryos, and this method was used to establish equine pregnancies after vitrification and warming of 2- to 8-cell embryos produced by ICSI. # 2009 Elsevier Inc. All rights reserved. Keywords: Vitrification; Bovine; Equine; Embryos; ICSI 1. Introduction Vitrification is an alternative to freezing [1]. This technique involves rapid cooling and warming rates, small volumes, high viscosity and the use of high concentrations of cryoprotectant solutions to bring about a glass physical state in which crystalline ice does not form [2]. Vitrification is rapid, inexpensive, and has been used to cryopreserve embryos (from several mammals) at various stages of development [2]. Valuable genetics can be preserved through the cryopreservation of sperm, oocytes, or embryos. Meth- ods to cryopreserve sperm and embryos have been successful [3]. However, despite advances in cryobiol- ogy, cryopreservation of oocytes from most species is inefficient, and results are inconsistent [4]. In humans, cryopreservation of zygotes offers a theoretical advan- tage, because the center of the lipid phase transition curve (T m ) is lower in zygotes than in oocytes [5]. However, www.theriojournal.com Available online at www.sciencedirect.com Theriogenology 71 (2009) 349–354 * Corresponding author. Tel.: +1 970 491 8626; fax: +1 970 491 7005. E-mail address: emc@colostate.edu (E.M. Carnevale). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2008.08.001