Superfine open pulled straws vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation
C. Cuello A B C , J. Sanchez-Osorio A , C. Almiñana A , M. A. Gil A , I. Parrilla A , J. Roca A , J. M. Vazquez A , E. A. Martinez A and H. Rodriguez-Martinez BA Department of Animal Medicine and Surgery, Veterinary Science, University of Murcia, E-30071 Murcia, Spain.
B Division of Reproduction, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, PO Box 7054, SE-75007 Uppsala, Sweden.
C Corresponding author. Email: ccuello@um.es
Reproduction, Fertility and Development 22(5) 808-817 https://doi.org/10.1071/RD09160
Submitted: 9 July 2009 Accepted: 11 November 2009 Published: 7 April 2010
Abstract
The present study investigated the in vitro development of and cytoskeletal disruption suffered by in vivo-derived porcine blastocysts subjected to superfine open pulled straws (SOPS) vitrification. Blastocysts were either untreated prior to SOPS vitrification or were subjected to one of the following three pretreatment protocols: (1) centrifugation (12 min, 13 000g); (2) 25 min equilibration with 7.5 μg mL–1 cytochalasin B; or (3) equilibration with cytochalasin B followed by centrifugation. After 24 h culture, fresh (n = 32) and vitrified–warmed (n = 188) blastocysts were evaluated by stereomicroscopy, with survival and hatching rates recorded. Some blastocysts were stained with 4′,6′-diamidino-2-phenylindole and processed for cytoskeletal evaluation. Three cytoskeletal patterns were identified: Grade I, intact cytoskeleton; Grade II, gross maintenance of integrity, but with some clumps of actin within the cytoplasm; and Grade III, a highly disrupted cytoskeleton. There were no differences in the survival, hatching and cell death rats, total cell number or cytoskeletal integrity between the different vitrification groups. Cell death was greater for vitrified blastocysts than for fresh blastocysts (3.6 ± 0.4% v. 0.4 ± 0.7%, respectively; P < 0.05) and the percentage of blastocysts with a Grade I cytoskeletal pattern was lower for vitrified compared with fresh blastocysts (60.8% v. 92%, respectively; P < 0.05). The vitrified–warmed blastocysts that hatched during culture exhibited a Grade I cytoskeletal pattern. In conclusion, successful SOPS vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation.
Additional keyword: cytoskeleton.
Acknowledgements
The authors acknowledge the excellent technical support of the late Åsa Jansson (Swedish University of Agricultural Sciences), as well as the assistance of Margareta Wallgren and Fernando Saravia (Swedish University of Agricultural Sciences). This study was supported by the Seneca Foundation of Murcia (01287PD04 and GERM 04543/07), MICINN (AGL2009–12091) and CARM (2I05SU0012). The Spanish Ministry of Education and Science is acknowledged for its grant-based support of JS-O (BES-2005–8894).
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