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Materials and Methods

Franklin and Marshall College

Following the methods described in Cartwright and Smith (1995), we staged several 27 hour chick embryos according to the system by Hamburger and Hamilton (1951) and prepared them for injection by creating a small hole in the blunt end of the egg. A 250 µl injection of 0% (control), 5%, 10%, or 15% ethanol (in Howard Ringer's solution) was introduced directly into the yolk using a 1 ml syringe. Each concentration of ethanol was injected into a set of ten eggs. Eggs which were not viable or died before collection were discarded and eliminated as data.

The eggs were then incubated at 37ºC for 14 days. At day 14 post-injection, the development of the embryos was terminated and the effects of the ethanol were noted. Eggs were opened carefully and chicks were qualitatively compared to one another. Additionally, quantitative data was gathered. The total body mass, head diameter, and beak length were measured for each set of embryos. Head diameter was measured with calipers which were placed immediately behind the eyes. Calipers were also used to measure beak length from the tip of the beak to the end of the beak.

The data was analyzed using Data Desk software. First, an ANOVA test was performed. A Bonferroni post hoc analysis of the data was carried out. The results of these tests were used to determine whether any significant trends in our data existed.

Temporal -dependence
Swarthmore College

We obtained 24 27-hour eggs and 12 51-hour eggs. One egg from each age was opened and staged according to Hamburger and Hamilton (1951). Of the remaining eggs, each was sterilized on the blunt end with 70% ethanol and a small hole was made using beak forceps. Through the hole, 8 27-hour eggs and 5 51-hour eggs, designated the controls, were injected with 250 µl Howard Ringer's solution directly into the yolk using a 1 mL syringe. The remaining eggs were injected in the same manner with 15% ethanol in Howard Ringer's solution, determined by taking the dose that produced the most significant effects in the dose-dependent experiment. Within our experiment, only a few of the eggs were viable at harvesting and it was hypothesized that the needle was injected directly into the blastodisc, terminating development. Allowing the blastodisc to float to the top and rotating the blunt end of the egg to the side to allow for injection away from the blastodisc and into the yolk may reduce this problem. The holes were then covered with Scotch tape to minimize infection and eggs were incubated at 37ºC.

Forty-eight hours post-injection, one experimental and one control egg from each age group was harvested and treated with Neutral Red vital stain to detect apoptosis. Neutral Red stock solution was diluted 1:100 in Howard Ringer's sollution and embryos were incubated in the dye for 20 minutes at 37ºC, after which they were rinsed in Howard Ringer's solution and photographed using the Olympus DP 12.

At ten days post-injection, the development of the eggs treated at 51-hours was examined by carefully opening the eggs and isolating the embryo from the rest of the egg. Mass, head diameter, and beak length were recorded and the embryos were again photographed. In consistency with the above study, head diameters were measured with calipers placed immediately behind the eyes, and beak lengths were measured from the tip to end of the beak.

Eggs treated at 27-hours were harvested 11 days post-injection to be consistent that all embryos were the same developmental age to control differences in size and mass measurements. Data were collected as above.

©Cebra-Thomas, 2000

Last Modified: 10 May 2004

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