The Effects Of Different Concentrations Of Possible Teratogens On Amphibian Embryos

The effects of different concentrations of possible teratogens on the embryonic development, larval growth and survival competence were studied in amphibian embryos. Embryos were harvested then separated into 4 groups with two treatments. Half the embryos were de-jellied and the other half were not to see in what conditions could the embryos survive with unknown treatments being used. Later the treatments were known after the results and the data graphed (averages being taken from the class as a whole). The treatments were (A) 25 uM Ibuprofen in 0.1% ethanol, (B) 1X Holtfreter’s, (C) 250 uM Ibuprofen and (D) 0.1% ethanol with treatment A having a higher survival rate with un-jellied embryos and treatment B being the control thus yielding the normal developmental stages. 1. Introduction Ibuprofen, the most common oral NSAID consumed by people, is discouraged from being digested during the second and/or trimester of a woman’s pregnancy. Ibuprofen is a non-selective cyclooxygenase inhibitor of prostaglandin synthesis, often being the concern for premature close of fetal ducts and delayed labor with experimental studies showing that the drug causes IUGR (intrauterine growth retardation), ventricular septal defects (VSDs) and embryonic malformations, also known as a teratogen. Studies on the effects that NSAIDs have on the human embryo were previously done on model organisms such as rats or mice and were indicated to be developmental toxicants, however the area research on the effects of teratogen on amphibian embryos have yet to plunge into deep exploration.

Axolotl embryos are efficient due to how they develop in liquid, this allows for easy control and manipulation with the teratology factors. The embryos also have easily distinguishable stages and develop externally as well as relatively quickly in comparison to a mouse or human embryo. The objective of this investigation was to determine if Axolotl embryos could function as a predictive test for amphibian teratogenicity.

Axolotls embryos

34 Axolotl embryos were obtained from Dr. Rogers, who acquired them after fertilization 24 to 48 hours in advance from domesticated Axolotls found at the aquarium at California State University, Northridge. The embryos were kept at a temperature of 12°C (embryo stages: 9-13, 20, 24 and 27) before being acclimated to room temperature during BIOL 441 laboratory prior to the experiment.

A solution was made to keep the embryos submerged in mild salty water. This solution, Holtfreter’s solution, was made via 2.5 mL of 400% Holtfreter’s solution diluted with 47.5 mL ddH2O. 16 of the embryos had the jelly coats removed manually under a microscope prior to neurula stage with the use of a tool kit that contained two silver forceps (this was provided in advance). After de-jelly, the embryos were separated into 8ths (with the exception of two dishes) then placed into four 1% agarose jell petri dishes that contained the Holtfreters solution. Treatments were then selected blindly based on letters (A, B, C and D). For the following experiment treatments A and D were selected without knowledge of what letter contained a control or experimental factor.

Treatments

A and D Two petri dishes, one containing the embryos with jelly and one containing the embryos without, were treated with A and labeled. After treatment the embryos were allowed to grow at various temperatures (12°C-23°C) for 2 days in Chaparral Hall room 5435 (Dr. Rogers laboratory) on the California State University, Northridge campus.

Two petri dishes, one containing the embryos with jelly and one containing the embryos without, were treated with D. Afterwards the dishes containing treatment D were labeled and placed into incubation with treatment A at the same varying temperatures for 2 days.2.3 Embryo cleaning Observations were made on the embryogenesis the first day of incubation for 15 minutes during the late afternoon (4:45pm) inside room 5435 in Chaparral Hall at California State University, Northridge. Dead embryos were discarded immediately after recognition. Morphological deformities and/or noticeable phenotypic treats were noted and carefully observed. The images were captured using a basic camera. A total of 2 embryos died, one from treatment A no jelly and another from treatment D no jelly. All four dishes did not have noticeable phenotype.

In order to access any abnormalities in the development involving change due to drug exposure the embryos were taken from their incubation on the second day and placed under a microscope to be analyzed and staged for the final time. Afterwards the data was recorded from every group.3. Results The jelly coated embryos that were exposed to treatment A, which was noted to be the 25uM Ibuprofen in 0.1% ethanol underwent normal development. Treatment A had a 95% survival rate for the class average but a 100% survival rate for the group’s average. The embryos with treatment A that did not contain jelly coats had a class average of 79% but a group average of 67%. Treatment D was revealed to be the 0.1% ethanol yielding a 91.6% success rate with jelly coats and 61.1% success rate without jelly coats on a class average. For the group treatment D yielded 87.5% success with jelly coats and 77.78% success without jelly coats. For treatment D in comparison the A, the embryos were smaller in size but had advanced stages whereas treatment A was normal and large but still lingered around stages 12-21.