Ultrastructural analysis of early cow embryos after freezing
1998
Pivko, J. | Kubovicova, E. | Grafenau, P. | Oberfranc, M. (Vyskumny Ustav Zivocisnej Vyroby, Nitra (Slovak Republic))
Recovered embryos (blastocysts) from superovulated cows were frozen at a rate of 1 deg C min** (-1) from 22 deg C to -7 deg C and, after crystallization of seeding, at 0.3 deg C min** (-1) up to -35 deg C. After 20 minutes of equilibration, embryos were placed in 0.25 ml straws and plunged into LN2. Embryos were washed in increasing concentrations of glycerine (0.47M, 0.94M, 1.4M), using the medium Dulbecco PBS + 4 mg ml** (-1) BSA +0.2M sucrose. After thawing, glycerine was flushed from embryos at decreasing concentrations, in inverse order, in Dulbecco PBS 1 + 4 mg ml** (-1) BSA +0.2M sucrose. The proportion of damaged thawed embryos assessed by light microscopy and electron microscopy was 14.1 and 26.6%, respectively. Ultrastructure studies revealed damaged organelles of the trophoblast and embryoblast cells, manifested most often by the disintegration of cytoplasmic membranes, their coiling up and the formation of hyalinic rings. Frequent changes of mitochondria consisted in the disintegration of partitions and in an increase of electron optical density of the content of mitochondria looking like enlarged black bodies. The membrane of nucleus formed frequent invaginations into the nucleoplasma. Spread tubulus and patches of endoplasmic reticulum and Golgi complex were found. The destruction of intercell connections of the nexus type appeared frequently. A frequent occurence of multivesicular and residual bodies and of autophagical vacuoles was observed in the trophoblast and embryoblast cells. Based on the electronmicroscopic analysis of frozen cow embryos, it is necessary to expect more than 25% losses of embryos after transfer due to the destruction of cytoplasmic membranes and organelles and to an increase in the occurrence of cytoplasmic lysosomal structures
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