Influences of environmental conditions, such as changes in media compositions or different
embryo production methods, have great impact on the susceptible stages of the preimplantative
embryo. Various studies have shown that assessed embryo characteristics
(morphology, metabolism, gene expression, survival rate after freezing, transfer to recipient
animal) can be used as sensitive indicators for the embryos quality and developmental
competence, and therefore of the appropriateness of the culture condition used.
Aim of this thesis was to investigate the effects of different culture techniques (purely in vitro,
stepwise in vitro-to-in vivo, or purely in vivo) (i) on the amount of cell organelles (nuclear
material and actin-cytoskeleton), and (ii) on the relative abundance of mRNA expression of
specific genes before and after freezing of bovine blastocysts.
(i) Selected blastocysts were differentially dyed, for nuclear material and for actincytoskeleton,
with fluorescent stains, and a novel computer programme (ColorAnalyzer)
made a differential count of the pixels of two colours. Blastocysts developed purely in vivo
showed significantly more nuclear material than did blastocysts produced by any other
technique. In terms of actin-cytoskeleton quantity, blastocysts derived mainly or purely in
vitro (IVP; Day 27) did not differ significantly from each other, but embryos produced
mainly or purely in vivo (GIFT; MOET) showed a significantly larger amount as compared
with any other group, and differed significantly from each other.
(ii) To assess levels of mRNA expression in bovine blastocysts, RT-qPCR was used. No
significant influence of the culture method on the blastocysts mRNA expression before
freezing was found, the only exception being DSC2. Regardless of the production method
used, the mRNA expression level of all but two (AQP3, and DNMT1A) of the transcripts was
found to be significantly upregulated after freezing.
In conclusion, (i) a novel software was employed, which, in association with a laser scanning
microscope, may be useful for an objective, fast, repeatable assessment of embryo structures
that would allow an evaluation of the culture environment in which the embryo was derived.
(ii) The RT-qPCR findings suggest that after freezing, re-expanded embryos are prepared for
further development. Further studies to evaluate environmental influences on early embryo development are
planned. However, it should be kept in mind that the early embryo can adjust to culture
conditions very rapidly, and any observation represents only a snapshot in the embryos