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Deliverables
One of the results of this project was that we were able to induce large offspring (Lazzari et al., 2002) that often are less vital, with a much higher birth weight and with anomalies and malformations (Kruip and Den Daas, 1997). These deviations are called the large offspring syndrome (LOS) (Young et al, 1998) and have something in common with the Wiedemann-Beckman syndrome (gigantism) in human. We were able to induce LOS by adding one particular serum to the culture medium or by changing the culture conditions. The latter was even more important than the former (Wrenzycki, 2001). However, both demonstrate that an inferior environment of the developing embryo plays a crucial role in the induction of LOS. These results support furthermore the hypothesis that animal health and welfare can have an embryonic origin. The here used technologies, IVP and NT, can be used succesfully for a more general study of the origin of other diseases than the ones by infections.Due to technical problems we still do not know whether the oocyte quality, related to the quality of the follicle where it comes from, matters but the hypothesis is still valuable. More research is needed to develop a valuable test for the developmental potency of the oocyte. This is not only important for the broad scale application of IVP but also for the application of NT.
The culture conditions for IVP and NT have consequences for the morphological outgrowth of embryos and fetus and for the expression of genes. In most cases the expression of some genes was enhanced. These changes in gene expression in early embryos are responsible for the LOS.
The results of this project, although referred to small groups, suggest that there is a correlation between cell number at day 7, embryo size at day 12 and average weight of the calves at birth within the groups. This result is very important for the progress of the study on the large calves syndrome in cattle because it indicates the possibility of screening the effect of different culture systems as early as day 7 and day 12. The importance of this result is not only scientific but it is also practical because it offers an early and relatively cheap screening system of different culture procedures that could contribute substantially to the LOS. Like these morphological parameters also the deviated expression of some genes can be used as screening method on the culture systems.
In the first major study the effects of two basic culture systems (Tissue Culture Medium (TCM) with 5% CO2 in air or Synthetic Oviduct Fluid (SOF) with 7% O2, 88% N2, 5% CO2) and various protein supplements [serum, bovine serum albumin (BSA) or polyvinyl alcohol (PVA) = defined medium)] on the relative abundance of a panel of developmentally important gene transcripts in bovine morulae and blastocysts were analyzed and compared with that obtained for their in vivo derived counterparts.
The specific transcripts analyzed in this study included gap junction protein connexin43 (Cx43), desmosomal protein plakophilin (Plako), desmosomal glycoproteins desmocollin II and III (Dc II, III), cellular adhesion protein E-cadherin (E-cad), tight junction protein Zonula occludens (ZO-1), glucose transporter-1 (Glut-1), PolyA-Polymerase (PolyA), heat shock protein 70.1 (Hsp 70.1) and Interferon tau (IF-tau). The predominant observation of this study was that the basic culture system had profound effects on the relative amount of specific transcripts in bovine embryos whereas the protein source had only weak effects. Significantly more differences in the relative abundance of the analyzed gene transcripts were detected between in vivo and in vitro derived embryos at the morula stage than at the blastocyst stage.
More differences were found between embryos produced in the TCM-system and in vivo derived embryos than between SOF-generated embryos and their in vivo counterparts. Interestingly, no differences in gene transcription patterns were found in the relative abundance of gene transcripts in embryos generated under chemically defined conditions in two different laboratories. This provides a major step forward towards a standardization of in vitro production systems for bovine embryos (Wrenzycki et al., 2001).
In a second study the effects of two diverging culture systems (TCM supplemented with serum versus SOF supplemented with PVA) on the relative abundance of transcripts for Insulin-like growth factor I and II ligands and their receptors (IGF I and II, IGF Ir, IGF IIr) were investigated in bovine pre-implantation embryos starting with the immature oocyte up to the hatched blastocyst. IGF I transcripts were not detected at any stage of bovine development including the hatched blastocyst. In both culture systems IGF Ir, II and IGF IIr were expressed throughout pre-implantation development in a varying pattern. Significant differences between the two culture systems were found at the expanded blastocyst stage at which significantly higher amounts of IGF II were observed in the TCM- than in the SOF-system. These data clarify that IGFI most likely predominantly has a paracrine mode of action in the early bovine embryo whereas IGF II, IGF IIr and IGF Ir follow an autocrine mechanism. Expression of these genes is modulated by culture conditions (Yaseen et al., 2001).
The relative abundance of a panel of genes (Hsp 70.1, Cu/Zn-SOD (Copper-Zinc superoxide dismutase), Glut-1, -3, -4 (Glucose-transporter-1, -3, -4); IGF-Ir, IGF IIr, bFGF (basic fibroblastic growth factor), H4 (Histone 4) has also been analyzed for bovine embryos derived from in vitro maturation and fertilization but followed by in vivo culture up to the blastocyst stage in ligated sheep oviducts. When compared with embryos derived from the SOF culture system and those from superovulated donor cows, it was found that these embryos were similar to the ones derived from complete in vivo origin but still showed few differences with regard to gene expression patterns. However, these embryos were significantly different from those derived from a complete in vitro production process with regard to gene expression. These data correlate with differences found between embryos derived from the temporary culture in sheep oviducts versus those derived from the culture in SOF-BSA or SOF plus serum with regard to cell numbers and allocation of ICM and trophoblastic cell numbers.
Preliminary evidence was also accounted for a possible link between the cellular and molecular markers analyzed in the embryos as potential parameters and an accelerated development as well as an early feature of the Large Offspring Syndrome (LOS) (Lazzari et al., 2002).
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