TheEffectofpHLevelsByVaryingtheAcidityoftheExtracellularSolution

Background

Zebrafish, Brachydanio rerio, have a distinct embryonic development. The embryo goes through a process called epiboly. Epiboly, as defined in the sixth edition of Developmental Biology(Gilbert 2000), is the movement of epithelial sheets (usually of the ectoderm cells) that spread as a unit, rather than individually, to enclose the deeper layers of the embryo. Epiboly enables an embryo to protect the inner cells from extracellular variations. According to Professor Richard Fluck, who works with medaka and is a Dr. E. Paul and Frances H. Reiff Professor of Biology at Franklin and Marshall College, it is his experience that medaka embryos are more susceptible to exteriorenvironmental change prior to complete epiboly. Zebrafish are supposed to be even more sensitive to environmental change than medaka.

Objective

The study of acidic pH levels effecting the development of zebrafish. This is being done to determine if acid rain has any major effect on developing embryos. Rainfall (pH 5.6) has a more acidic value than that of normal water (pH 7 or neutral).
The Embryo medium, which is used for developing dechorionated embryos, as well as the other Hank's standard solutions can be seen here. Adding less NaOH buffer so that it will be less than a pH 7.2 level will change the embryo medium. This is supposedly only pertinent if the chorion has been removed. Hopefully the chorion will not hinder pH interactions. The slow addition of NaOH will enable for a fair range of acidic to optimal pH reading levels. By taking pictures of the embryos prior to exposure to various pH solutions and comparing these to pictures of the same embryos further along development it will hopefully show that pH level has en effect on the development of theembryos.

Procedure
1) Zebrafish were marbled the previous night before collection of embryos
-The marbles in the tank bottom promote spawning when the next dawn comes
2) Embryos were selected for two varying cell stages of development
-first set were between the 2-4 cell stage of development
-second set was of embryos between 64-128 cells in development
3) pH levels used were: 7.2, 6.6, 6, 5, and 4.
-pH 7.2 is the control experiment

Note: Each of these pH levels was applied to each of the two cell stages previously mentioned. An image was taken of the initial embryos to compare with the developing embryos in the various pH solutions. Embryos were observed over a 48 hour period.

Results

It was found that the difference in pH levels that were used did not have stop development. The only significant effect was that the lower pH levels caused the zebrafish to develop slower. This was observed over a period of 48 hours.

Discussion

The zebrafish showed no signs of altered development due to the levels of pH used in this experiment. This may have occurred because the embryos were not dechorionated. Perhaps dechorionating them would have produced viable results. As it stands zebrafish are impervious to the pH levels that were used in this experiment. It almost certain that much more acidic pH levels would have drastic effects on the development of zebrafish because the protein layers of the embryos would be disturbed.