Blastomere Content of Cultured/Frozen Bovine Demiembryos

Abstract

The purpose of the study was to evaluate whether a period of co-culture with bovine oviduct epithelial cells (BOEC) could improve the tolerance of bisected bovine embryos to freezing and thawing. Day 6 embryos were bisected and the resulting demiembryos were stained with Hoechst 33342 and cell counts were made by counting intact blastomere nuclei. Of these, 11 were stained as freshly manufactured demiembryos, 25 after co-culture for 24 h with BOEC and 37 stained after 24 h co-culture and freezing and thawing. The staining revealed, that there was no significant difference in cell count of demiembryos that were stained immediately after bisection, compared to those, that were co-cultured for a 24 h period. Also, the co-cultured/frozen/thawed demiembryos had a significant decrease in cell numbers compared to the non-frozen demiembryos. We conclude, that a 24 h period of co-culture with BOEC does not result in appreciable cellular proliferation in demiembryos and therefore instead of improving the survival of frozen/thawed demiembryos by giving them opportunity to multiply their cell number and thus make them more resistant to cell damage, rather compromised the viability of cryopreserved demiembryos.

Sammendrag

Tidligere forsøg har vist, at dyrkning med BOEC ikke forbedrer frysbarheden af halve kvægembryoner, demiembryoner. Hensigten med denne dyrkningsmetode skulle være, at give demiembryonerne tid til at restituere sig efter mikromanipulationsprocessen og give dem lejlighed til yderligere at forøge celleantallet, således at de bliver mere velegnede til at overleve frysningen. Formålet med nærværende forsøg var at få et indtryk af celleantallet i dyrkede og frosne demiembryoner ved at farve med Hoecht 33342 (Sigma). Af 73 farvede og bedømte demiembryoner var de 11 friske, de 25 var dyrkede i 24 timer med bovine Ovidukt epithel celler (BOEC), og de 37 var dyrkede, frosne og optøede.

Resultaterne var, at der efter en 24 t dyrkningsperiode skete et mindre, ikke signifikant fald i antallet af demiembryoner med normalt antal blastomerer, men efter frysning og optøning viste der sig at være significant færre demiembryoner end før frysningen med det normale antal celler. Det fremgår således, at det ikke var muligt at forøge antallet af blastomerer i demiembryoner ved at dyrke med BOEC, og at fryseprocessen reducerede celle-antallet yderligere.