Analysis of tail coiling activity of zebrafish (Danio rerio) embryos allows for the differentiation of neurotoxicants with different modes of action.

In (eco)toxicology, there is a critical need for efficient methods to evaluate the neurotoxic potential of environmental chemicals. Recent studies proposed analysis of early coiling activity in zebrafish embryos as a powerful tool for the identification of neurotoxic compounds. In order to demonstrate that the analysis of early tail movements of zebrafish embryos allows for the discrimination of neurotoxicants acting via different mechanisms, the present study investigated the effects of four different neurotoxicants on the embryogenesis (fish embryo toxicity test) and early tail coiling movements of zebrafish embryos. Cadmium predominantly increased the frequency of tail coiling at the late pharyngula stage. Dichlorvos delayed embryonic development and caused convulsive tail movements resulting in prolonged duration of tail coils. Embryos exposed to teratogenic concentrations of fluoxetine and citalopram displayed absence of spontaneous tail movements at 24 h post-fertilization. In contrast, a non-teratogenic test concentration of citalopram decreased coiling frequency at multiple time points. Results demonstrated that the analysis of tail coiling movements of zebrafish embryos has the potential to discriminate neurotoxic compounds with different primary modes of action. In addition, chemical-induced effects on coiling activity were shown to potentially overlap with effects on embryogenesis. Further studies are needed to clarify the interplay of unspecific developmental toxicity of neurotoxic chemicals and effects resulting from specific neurotoxic mechanisms.

Intraindividual Embryo Morphokinetics Are Not Affected by a Switch of the Ovarian Stimulation Protocol Between GnRH Agonist vs. Antagonist Regimens in Consecutive Cycles.

Background: The impact of controlled ovarian stimulation (COS) during medically assisted reproduction (MAR) on human embryogenesis is still unclear. Therefore, we investigated if early embryonic development is affected by the type of gonadotropin-releasing hormone (GnRH) analog used to prevent a premature LH surge. We compared embryo morphology and morphokinetics between GnRH agonist and antagonist cycles, both involving human chorionic gonadotropin (hCG)-trigger. To reduce possible confounding factors, we used intraindividual comparison of embryo morphokinetics in consecutive treatment cycles of the same patients that underwent a switch in the COS protocol. Methods: This retrospective cohort study analyzed morphokinetics of embryos from patients (n = 49) undergoing a switch in COS protocols between GnRH agonists followed by GnRH antagonists, or vice versa, after culture in a time-lapse incubator (EmbryoScope®, Vitrolife) in our clinic between 06/2011 and 11/2016 (n = 49 GnRH agonist cycles with n = 172 embryos; n = 49 GnRH antagonist cycles with n = 163 embryos). Among time-lapse cycles we included all embryos of the two consecutive cycles before and after a switch in the type of COS in the same patient. In-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) was performed and embryos were imaged up to day 5. Data were analyzed using Mann-Whitney U test or Fisher's exact test. The significance level was set to p = 0.05. Patients with preimplantation genetic screening cycles were excluded. Results: The mean age (years ± standard deviation) of patients at the time of treatment was 35.7 ± 4.3 (GnRH agonist) and 35.8 ± 4.0 (GnRH antagonist) (p = 0.94). There was no statistically significant difference in the number of oocytes collected or the fertilization rate. The numbers of top quality embryos (TQE), good-quality embryos (GQE), or poor-quality embryos (PQE) were also not different in GnRH agonist vs. antagonist cycles. We found no statistically significant difference between the analyzed morphokinetic parameters between the study groups. Conclusions: Our finding supports the flexible use of GnRH analogs to optimize patient treatment for COS without affecting embryo morphokinetics.

Time-course of coiling activity in zebrafish (Danio rerio) embryos exposed to ethanol as an endpoint for developmental neurotoxicity (DNT) - Hidden potential and underestimated challenges.

Detection of developmental neurotoxicity (DNT) has been recognized as a major challenge by regulatory bodies and science. In search of sensitive and specific test methods, spontaneous tail coiling of embryonic zebrafish has been recommended as a promising tool for identification of DNT-inducing chemicals. The present study was designed to develop a protocol for a prolonged test to study neurotoxicity during the entire development of coiling movement in zebrafish embryos. Ambient illumination was found to modulate coiling activity from the very onset of tail movements representing the earliest behavioral response to light possible in zebrafish. In the dark, embryos displayed increased coiling activity in a way known from photokinesis, a stereotypical element of the visual motor response. Elevated coiling activity during dark phases allows for the development of test strategies that integrate later coiling movements under the control of a further developed nervous system. Furthermore, zebrafish embryos were exposed to ethanol, and coiling activity was analyzed according to the new test protocol. Exposure of embryos to non-teratogenic concentrations of ethanol (0.4-1%) resulted in a delay of the onset of coiling activity and heartbeat. Moreover, ethanol produced a dose-dependent increase in coiling frequency at 26 h post-fertilization, indicating the involvement of neurotoxic mechanisms. Analysis of coiling activity during prolonged exposure allowed for (1) attributing effects on coiling activity to different mechanisms and (2) preventing false interpretation of results. Further research is needed to verify the potential of this test protocol to distinguish between different mechanisms of neurotoxicity.