Proceedings of the 5th International Symposium on EQUINE ...
Proceedings of the 5th International Symposium on EQUINE ...
Proceedings of the 5th International Symposium on EQUINE ...
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Havemeyer Foundati<strong>on</strong> M<strong>on</strong>ograph Series No. 3TABLE 1: Effect <str<strong>on</strong>g>of</str<strong>on</strong>g> cryopreservati<strong>on</strong> <strong>on</strong> embryo grade, size and percent live cellsGrade Diameter (µm) % live cell †Post 20 h Post- 20 hInitial thaw culture S.E. Initial thaw culture S.ETreatmentSlow-cooled 1.4 2.6 2.9 0.2 257 21823811.1 74*Vitrified OPS 1.4 2.9 3.1 0.1 232 184 176 9.9 51Vitrified cryoloop 1.3 2.6 3.3 0.2 211 184 196 11.4 48*Three embryos in <str<strong>on</strong>g>the</str<strong>on</strong>g> slow-cooled treatment group were killed and lost during staining and are not included† sd ± 37liquid nitrogen until thawing in a 4-well dish at37°C. The lid and loop was removed from <str<strong>on</strong>g>the</str<strong>on</strong>g>cryovial under liquid nitrogen. The loopc<strong>on</strong>taining <str<strong>on</strong>g>the</str<strong>on</strong>g> embryo was sequentially placedinto wells, c<strong>on</strong>taining 840 µl H-SOF plus 420 ml<str<strong>on</strong>g>of</str<strong>on</strong>g> H-SOF supplemented with 1 M sucrose for 2.5min, 840 µl H-SOF plus 210 ml <str<strong>on</strong>g>of</str<strong>on</strong>g> H-SOFsupplemented with 1 M sucrose for 3 min, and 840ml H-SOF for 5 min. After thawing, embryos werecultured for 20 h in SOFaa in <str<strong>on</strong>g>the</str<strong>on</strong>g> presence <str<strong>on</strong>g>of</str<strong>on</strong>g> 6%CO 2 , 5% O 2 , 89% N 2 at 38.5°C.Embryos were graded and measured prefreeze,post thaw, and after 20 h <str<strong>on</strong>g>of</str<strong>on</strong>g> culture.Embryos were <str<strong>on</strong>g>the</str<strong>on</strong>g>n stained with propidium iodideand Hoechst 3342 to determine <str<strong>on</strong>g>the</str<strong>on</strong>g> proporti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g>live cells. Embryos were washed in a 100 µldroplet <str<strong>on</strong>g>of</str<strong>on</strong>g> H-SOF under oil and placed in stainingsoluti<strong>on</strong> c<strong>on</strong>taining H-SOF supplemented with 8mg/ml BSA with 10 µg/ml PI and Hoechst 33342.Embryos were placed into a 100 µl droplet <str<strong>on</strong>g>of</str<strong>on</strong>g>staining soluti<strong>on</strong> at 38.5°C for 15 min under foil.After staining embryos were washed twice in H-SOF and mounted <strong>on</strong> a slide in an 11 µl drop <str<strong>on</strong>g>of</str<strong>on</strong>g> H-SOF, covered with a cover-slip, which mounds <strong>on</strong>four drops <str<strong>on</strong>g>of</str<strong>on</strong>g> a paraffin oil/vaseline mixture.Fluorescence was observed with a Nik<strong>on</strong> EclipseE800 narrowband microscope (Filter FITC,TRITC and DAPI) with a 20X/0.75 objective(Nik<strong>on</strong>); nuclei and dead cells <str<strong>on</strong>g>of</str<strong>on</strong>g> embryosfluoresce with bright blue and red colour,respectively, and percent live cells were estimated.It was not possible to count <str<strong>on</strong>g>the</str<strong>on</strong>g> cells, due to <str<strong>on</strong>g>the</str<strong>on</strong>g>large numbers <str<strong>on</strong>g>of</str<strong>on</strong>g> cells per embryo. Three differentpeople independently estimated <str<strong>on</strong>g>the</str<strong>on</strong>g> percent livecells and <str<strong>on</strong>g>the</str<strong>on</strong>g> average <str<strong>on</strong>g>of</str<strong>on</strong>g> <str<strong>on</strong>g>the</str<strong>on</strong>g>se numbers were taken.Results were evaluated by analyses <str<strong>on</strong>g>of</str<strong>on</strong>g>variance. There were no significant differences(P>0.1) in grade or size <str<strong>on</strong>g>of</str<strong>on</strong>g> embryos prior tocryopreservati<strong>on</strong>. Up<strong>on</strong> thawing, an equalproporti<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> embryos fractured in <str<strong>on</strong>g>the</str<strong>on</strong>g> slowcooled,OPS and cryoloop procedure resulting in12.5% (1/8), 18% (2/11) and 12.5% (1/8) fracturedembryos.Because <str<strong>on</strong>g>the</str<strong>on</strong>g> fractured embryos in <str<strong>on</strong>g>the</str<strong>on</strong>g> slowcooledmethod appeared viable <str<strong>on</strong>g>the</str<strong>on</strong>g>y were includedin <str<strong>on</strong>g>the</str<strong>on</strong>g> results. Embryos that survivedcryopreservati<strong>on</strong> were stained, and ranged indiameter from 140–320 µm after 20 h <str<strong>on</strong>g>of</str<strong>on</strong>g> culture.There were no significant differences (P>0.1) inembryo grade or viability post-thaw am<strong>on</strong>gcryopreservati<strong>on</strong> treatments. Embryo grade andviability as assessed by staining were correlatedr =-0.66 (P0.1) as measured bypercent live cells and morphological grades. Inc<strong>on</strong>clusi<strong>on</strong> vitrificati<strong>on</strong> procedures with OPS orcryoloop were similarly effective forcryopreservati<strong>on</strong> <str<strong>on</strong>g>of</str<strong>on</strong>g> small equine embryos. Theseresults are in agreement with those obtained ino<str<strong>on</strong>g>the</str<strong>on</strong>g>r species including bovine (Vajta et al. 1998)and hamsters (Lane et al. 1999). Fur<str<strong>on</strong>g>the</str<strong>on</strong>g>r, bothvitrificati<strong>on</strong> methods produced results which weresimilar to o<str<strong>on</strong>g>the</str<strong>on</strong>g>r studies where morphological gradepost-thaw and subsequent transfers wereperformed. In those studies embryos graded 1 or1.5 prior to cryopreservati<strong>on</strong> were determined tograde 2.4 <strong>on</strong> average and result in 50 to 53%pregnancy rates, respectively (Slade et al. 1985;Squires et al. 1989). It is likely, <str<strong>on</strong>g>the</str<strong>on</strong>g>refore, that<str<strong>on</strong>g>the</str<strong>on</strong>g>se techniques will eventually supercede <str<strong>on</strong>g>the</str<strong>on</strong>g>55