Bovine Gamete and Embryo Research Models: Development of Alternative in vitro Screening Assays for Toxicity Testing

Ellen Jorssen, MSc, PhD

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Since the onset of the industrial revolution, the number of chemical substances has increased tremendously. The knowledge about the possible toxic risks of thousands of these and newly produced chemicals is limited. Therefore, the REACH (Registration, Evaluation and Authorization of Chemicals) legislation, based on the 3R principle (to reduce, to replace and to refine animal testing), was introduced in 2007 by the European Commission. This legislation states that all chemical substances should be tested for their toxicity. Consequently there is an urgent need for alternative in vitro toxicity screening tests to reduce laboratory animal use. The bovine model is increasingly promoted as an excellent animal model for human pre-implantation reproductive research, because reproductive physiology in both species shows considerable similarities and bovine slaughterhouse ovaries are excessively available for research purposes. The latter implicates that no additional animals need to be sacrificed exclusively for toxicity testing and laboratory animal use is reduced.

Within this thesis, the in vitro embryo production (IVP) procedure and the in vitro follicle culture are optimized aiming for the development of a fully defined, routinely applicable and sensitive in vitro embryo and follicle toxicity test. This thesis mainly focusses on the description of the test definition, the within laboratory variances and the predictive capacity of these tests.

A fully individual IVP procedure was developed, allowing individual exposure of embryos under serum-free conditions. Results showed that the bovine embryo showed a reaction in relation to the exposure conditions. In contrast to the in vitro embryo production procedure, pre-antral follicle dynamics have not yet been fully elucidated. A staining method, using Neutral Red, was described to consecutively visualize, quantify and evaluate follicles within ovarian fragments. Additionally, a more defined isolated in vitro follicle culture procedure was optimized and several outcome parameters described.

However both in vitro alternative tests require further optimization and standardization for a final application within toxicity testing, the use of these proposed tests will reduce the number of laboratory animals in the future.