The effectiveness of the stereomicroscopic evaluation of embryo quality in vitrified–warmed porcine blastocysts: An ultrastructural and cell death study C. Cuello a, * , F. Berthelot b , B. Delaleu b , E. Venturi b , L.M. Pastor a , J.M. Vazquez a , J. Roca a , F. Martinat-Botte ´ b , E.A. Martinez a a Department of Animal Medicine and Surgery, Veterinary Science, University of Murcia, Murcia E-30071, Spain b UMR 6175 INRA-CNRS, Haras Nationaux F. Rabelais University, P.R.C. 37380 Nouzilly, France Received 13 March 2006; received in revised form 21 November 2006; accepted 24 November 2006 Abstract The objective of this study was to analyze the validity of the stereomicroscopic evaluation of vitrified–warmed (V–W) porcine blastocysts. Unhatched blastocysts were obtained from Large-white gilts (n = 10). Blastocysts (n = 156) were vitrified using the Open Pulled Straw technology. After warming, V–W blastocysts were cultured for 24 h (V24). Then, their developmental progression was morphologically assessed by stereomicroscopy and classified as: V24 viable re-expanded blastocysts; V24 viable hatched blastocysts or V24 degenerated. Blastocysts which re-expanded or hatched after warming were considered viable. Some fresh blastocysts were not vitrified and were evaluated after 24 h in culture (F24). By stereomicroscopic analysis all the fresh blastocysts were considered viable. Some F24, V24 re-expanded viable, V24 hatched viable and V24 degenerated blastocysts were processed for transmission electron microscopy (n = 13, 19, 9 and 9, respectively) or assessed by TUNEL for cell-death evaluation (n = 16, 21, 11 and 21, respectively). All V24 hatched blastocysts showed similar ultrastructure to fresh blastocysts. However, some V24 re-expanded blastocysts considered viable (6/19) revealed ultrastructural alterations. Degenerated V24 blastocysts showed ultrastructural disintegration. Hatched V24 blastocysts did not differ ( p > 0.05) from F24 hatched blastocysts with regard to the ratio of dead cells (2.8 Æ 0.5% versus 1.9 Æ 0.3%, respectively). However, V24 expanded blastocysts had higher ( p < 0.01) cell death levels (4.3 Æ 3.4%) than those observed in the F24 expanded blastocysts (1.1 Æ 0.3%). The degenerated blastocysts showed the highest cell-death index (19.4 Æ 6.3%). In summary, V–W blastocyst hatching during in vitro culture appears to coincide with good ultrastructure and low cell-death index, suggesting that the hatching rate assessed by stereomicroscopy is more appropriate than embryo re-expansion for an evaluation of V–W blastocyst quality. # 2006 Elsevier Inc. All rights reserved. Keywords: Porcine; Embryo; Vitrification; Ultrastructure; Cell-death and stereomicroscopy 1. Introduction While embryo cryopreservation is routinely used in embryo transfer programs of several mammalian species [1–3], the cryopreservation of pig embryos has been limited by their high sensitivity to chilling injury [4,5]. However, during recent years, important advances in pig embryo cryopreservation have been www.theriojournal.com Theriogenology 67 (2007) 970–982 * Corresponding author at: Departamento Medicina y Cirugı ´a Ani- mal (Reproduccio ´n y Obstetricia). Hospital Clı ´nico Veterinario, Uni- versidad de Murcia, 30071 Murcia, Spain. Tel.: +34968364812; fax: +34968367069. E-mail address: ccuello@um.es (C. Cuello). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.11.011