The in vitro culture of various cell types is an important scientific tool and is becoming increasingly acceptable as a viable alternative to animal experiments. Fetal calf serum (FCS) is a supplement used in many cell culture media, and provides cells with growth factors and cytokines necessary for successful culture. In view of the animal welfare issues surrounding the production of FCS, an alternative agent allowing the replacement or reduction in the use of FCS is desirable. A yolk extract factor (EYF-X) obtained from chicken eggs is described, which facilitates the in vitro culture of a variety of cell types. When the extract was added to a culture medium used for in vitro fertilisation, the number of successful fertilisations was significantly increased. In a further in vitro model (permanent neuronal cell line N2A), the yolk extract significantly stimulated cell proliferation as well as the growth of cell processes. A set of specific antibodies against different parts of the prepro-cholecystokinin reacted with the extract. The intensity of the reaction depends on the age of the egg (time after the laying date). Analysis by gel chromatography recorded a main protein fraction with an apparent molecular mass of 20-30kDa. This fraction was labelled by Western blot with an antibody with specificity against CCK-octapeptide. These findings suggest that the yolk factor may be a CCK/gastrin-like molecule. Since CCK/gastrin-like molecules have also been detected in the spermatozoa of mammals, the influence on in vitro fertilisation could be explained by the yolk factor replacing the endogenous CCK/gastrin-like molecule destroyed in sperm freezing. The results of this study suggest that it might be possible to replace FCS with EYF-X. The application of the yolk factor to a broad spectrum of cell types remains to be investigated.

Animal Use Alternatives↗

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Human haemoclassification by use of specific yolk antibodies obtained after immunisation of chickens against human blood group antigens.

Polyclonal antibodies, widely used in research and diagnostics, are conventionally isolated from the blood of immunised mammals, especially rabbits. The fact that antibodies can also be detected in the yolk of eggs laid by immunised hens, led to the development of yolk antibody technology (IgY-technology) as an alternative method that is less stressful to animals. This technology has become a worthwhile alternative to the blood-dependent techniques. Furthermore, because of the phylogenetic distance between birds and mammals, avian antigens have a very specific immune response to highly conserved antigens of mammals, such as human erythrocyte antigens. To evaluate the humoral immune response of hens immunised with human red erythrocyte antigens, 22 White Leghorn hens were kept in cages and immunised with total red blood cells or stroma of the human rhesus positive (Rh+) system (D antigen) by weekly intramuscular and intravenous injections, without the use of an adjuvant. The haemagglutination assay was used to evaluate the dynamics of the production of IgY antibodies against human erythrocyte antigens, and single radial immunodiffusion was used to evaluate the amount of total IgY in de-lipidated supernatants from egg yolk. The highest titres were observed four weeks after the first immunisation, and these remained stable for up to seven weeks for the intravenous route. Positive reactivity against human erythrocyte antigens A, B and O was demonstrated in de-lipidated supernatants from the egg yolks of immunised hens. The strongest reaction was observed against blood group O Rh+ (O+).

ABO Blood-Group System↗