Assessment of development and reproductive toxicology (DART): an in vitro approach

Abstract

Reproductive toxicity can be studied by a holistic approach on experimental animals in extensive and expensive trials. However, using an integrated in vitro approach, the reproductive cycle can be splitinto its main biological components integrating results and providing comprehensive information on the potential effects of chemicals on gametes and embryonic development, decreasing the number ofanimal studies and providing a more detailed toxicological profile. This work aims to introduce the setting up of an in vitro platform for DART testing, applying a battery of assays based on standardizedreproductive biotechnologies. To evaluate the toxicity on male gametes, bovine spermatozoa were exposed to increasing concentrations of the test substance, studying viability with vital stains alongwith motility and velocity parameters assessed by computer-assisted sperm analysis (CASA). The bovine oocyte in vitro (bIVM) was applied to assess the effect of substances during oocyte maturation.Cumulus-oocyte complexes (COCs) obtained from ovaries of slaughter cows were selected and incubated in IVM medium with increasing compound concentrations. After 22 h, the viability andnuclear maturation were evaluated. The obtaining results allowed determining the concentration that reduced the IVM of oocytes by 50% compared to the untreated control. In vitro fertilization of bovineoocyte (bFIV) and early embryonic development were used as a model to evaluate the toxic effect of the fertilization process. Bovine gametes were co-incubated with increasing concentrations of thetest substance. After 18 h, pronucleus formation was registered to determine the concentration that reduced the bFIV by 50% compared to the untreated control. This initial battery of in vitro modelsis part of developing a biotechnological platform for DART testing, promoting the technological cooperation between academia and industry partners within a 3Rs approach.