Proceedings of the 5th International Symposium on EQUINE ...

Havemeyer Foundation Monograph Series No. 3METABOLIC ACTIVITY OF EQUINE BLASTOCYSTSCRYOPRESERVED IN ETHYLENE GLYCOL ANDGALACTOSEM. K. O’Donovan, N. Oberstein, J. E. Bruemmer, M. Lane*, G. E. Seidel, Jr.,D. K. Gardner* and E. L. SquiresAnimal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado80523, USA; * Colorado Center for Reproductive Medicine, Englewood, Colorado 80110, USAINTRODUCTIONThe inability <strong>of</strong> expanded equine blastocysts tosurvive standard cryopreservation protocols is welldocumented (Yamamoto et al. 1982; Squires et al.1989; Young et al. 1997). The objective <strong>of</strong> thisstudy was to compare <strong>the</strong> efficacy <strong>of</strong> 3 protocolsfor cryopreserving equine embryos 300–600 Fm indiameter. It was hypo<strong>the</strong>sised that exposingexpanded equine blastocysts to galactose andethylene glycol at various concentrations for 40min would allow proper equilibration to occurprior to cryopreservation, thus improving postthaw survival.MATERIALS AND METHODSMorphological Grade 1 or 2 embryos recovered onDay 7 post ovulation, between 300–600 Fm indiameter were randomly assigned to one <strong>of</strong> 3cryoprotectant treatments: 1) (n=8) 0.3 Mgalactose (10 min), 2.0 M ethylene glycol (EG) +0.3 M galactose (40 min); 2) (n=5) 0.3 M galactose(10 min), 2.0 M EG + 0.3 M galactose (15 min),and 4.5 M EG + 0.3 M galactose (25 min); or 3)(n=8) 0.3 M galactose (10 min), 2.0 M EG + 0.3 Mgalactose (10 min), 4.5 M EG + 0.3 M galactose(15 min), 2.0 M EG + 0.8 M galactose (10 min).All embryos were loaded into 0.25 ml straws andcooled to -6°C at 4°C/min, held 5 min, seeded,cooled to -35°C at 0.5°C/min and plunged intoliquid nitrogen. Embryos were thawed in air (15 s)followed by a 37°C water bath (15 s). Uponthawing, cryoprotectants were removed in 3 or 4steps based on <strong>the</strong> specific treatment. Viability wasassessed following <strong>the</strong> freeze/thaw procedure bygrading <strong>the</strong> embryos and recording changes indiameter over time. Additionally, metabolicactivity <strong>of</strong> <strong>the</strong> embryos pre-freeze and post thawwas compared. Measurements <strong>of</strong> glucose andpyruvate uptake and production <strong>of</strong> lactate wereanalysed using micr<strong>of</strong>luorescence assays (Gardneret al. 1996). Embryos were stained after <strong>the</strong> lastmetabolic assay using Hoechst 33342 (10 µg/ml)and propidium iodide (10 µg/ml) to determinepercent live cells.RESULTSTwenty-one embryos were randomly assigned toone <strong>of</strong> 3 treatments, n 1 =8, n 2 =5, n 3 =8. Meandiameters <strong>of</strong> embryos were not different amongtreatments (P>0.1) but varied within treatmentover time (P