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Survival Rate and Ultrastructure of Vitrified Bovine in vitro and in vivo Developed Embryos

Överlevnadsprocent och ultrastruktur hos vitrifierade in vitro och in vivo embryon från nötkreatur

Acta Veterinaria Scandinavica volume 35, pages 417–426 (1994)Cite this article

Abstract

The capacity of different vitrification media and methods was tested onto in vivo and in vitro produced bovine morula/blastocysts and their ultrastructure and survival studied post-thawing. Two vitrification solutions were finally selected, named 40 ES (40% ethylene glycol in PBS containing 0.5 M sucrose) and 35 EFS (composed of 35% (v/v) ethylene glycol in PBS containing 0.5 M/l sucrose and 30% (w/v) Ficoll 70). The straws were either precooled or not precooled in nitrogen vapour, plunged and stored in LN2 for 10–25 days, and then thawed in a 20° C waterbath. The content of the straws was rediluted in 1M sucrose solution in PBS and later cocultured with BOEC for 48 h. The overall survival rates for in vitro and in vivo embryos were 36% (12 of 33) and 20% (3 of 15) after 24 h and 21% (7 of 33) and 33% (5 of 15 ) after 48 h. The survival rates for precooled embryos were significantly higher than for not precooled (48% vs 13% after 24 h and 44% vs 4% after 48 h) when tested across vitrification media. The in vitro-produced embryos presented an ultrastructure similar to the pre-freeze state, irrespective of the vitrification media used. The in vivo developed embryos showed a rather modified post-thaw ultrastructure, with clear signs of osmotic changes at both the trophoblastic and embryonic cells. The results indicated that in vitro and in vivo developed bovine embryos can survive vitrification using ethylene glycol as a cryoprotectant.

Sammanfattning

Olika mediers förmåga till vitrifiering testades på in vivo och in vitro producerde embryon och ultrastruktur och överlevnadsförmåga studerades efter tining. Baserat på inledande försök valdes 2 vitrifieringsmedier för fortsatta studier; 40 ES (40% v/v ethylene glycol i PBS mad 0.5 M sucrose) och 35 EFS (35% v/v ethylene glycol i PBS med 0.5 M sucrose och 30% (w/v) Ficoll 70). Embryon equilibrerades i respektive medium och aspirerades i strån vilka kyldes eller ej kyldes under en minut i kväveånga innan de fördes ned i flytande kväve. Stråna tinades i vattenbad (+20°) och vitrifieringsmediet späddes bort i PBS med 1M sackaros. Embryona odlades in vitro med BOEC varefter utvecklingsgraden utvärderades efter 24 och 48 timmar. Både in vivo och in vitro embryon överlevde vitrifiering i huvudsak i samma omfattning. Överlevnadgraden hos embryon som kyldes över kväveånga innan de fördes ned i flytande kväve var högre än hos embryon som direkt fördes ned i kväve (48% jämfört med 13% efter 24 timmar och 44% jämfört med 4% efter 48 timmar) oavsett medium. Efter frysning och tining hade in vitro producerade embryon en bättre ultrastruktur än in vivo embryon. De senare uppvisade klara tecken på osmotiska förändringar hos både trofoblast- och embryonalceller. Resultaten visar klart att in vitro embryon kan överleva vitrifiering med ethylenglykol som vitrifieringsmedium.

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Acknowledgments

We thank Ms A. Coloring for her assistance in the IVF laboratory and Mrs M. Ekwall and Mr H. Ekwall for their expert assistance with the electron microscopy. The present study was supported by the Swedish Council for Forestry and Agricultural Research.

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Authors and Affiliations

  1. Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, Uppsala

    U. Darvelid, M. Shamsuddin, B. Larsson & H. Rodriguez Martinez

  2. Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences, P. O. Box 7039, S-75007, Uppsala, Sweden

    H. Gustafsson

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  1. U. Darvelid
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  2. H. Gustafsson
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  3. M. Shamsuddin
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  4. B. Larsson
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  5. H. Rodriguez Martinez
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Darvelid, U., Gustafsson, H., Shamsuddin, M. et al. Survival Rate and Ultrastructure of Vitrified Bovine in vitro and in vivo Developed Embryos. Acta Vet Scand 35, 417–426 (1994). https://doi.org/10.1186/BF03548317

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Acta Veterinaria Scandinavica

ISSN: 1751-0147