9. THE EFFECT OF AGE OF THE EGG ON 

 EMBRYONIC DEVELOPMENT 



PURPOSE: To determine the effect on early development of the ageing of the amphibian 

 egg prior to insemination. 



MATERIALS: 



Biological : Ovulating females of Rana pipiens, sexually mature males. 



Technical : Controlled temperatures at 10°C. and 20°C. 



METHOD: 



Precautions : The ovulating females must be kept moist and at the constant temper- 

 atures designated. 



Control : Eggs fertilized within 24 hours of the time the first ones reach the uteri 

 may be considered as the control eggs. 



Procedure : 



1. Inject two freshly captured, sexually mature females of Rana pipiens each 

 with the pituitary glands from 5 or 6 adult female frogs (or from twice as 

 many male frogs). Place one at laboratory temperatures, the other at 10°C. 



2. Beginning 24 hours after the pituitary injection, test each female by gentle 

 stripping to determine when the first eggs reach the uteri. (Expect the frog 

 at the laboratory temperature to have uterine eggs within 24-48 hours, the 

 other within 4 to 5 days. ) 



3. As soon as eggs appear in the uteri and are easily expressed from the cloaca, 

 consider this the initial time in the ageing process of the eggs. Fertilize 

 200-300 eggs from each female immediately. These will be considered the 

 control eggs. Determine the percentage cleavage and separate the dividing 

 eggs into appropriate receptacles with adequate surface and 2 cc. of fluid 

 per egg. Keep them at laboratory temperatures, not higher than 23°C. 



4. At daily intervals with the 20°C. female, and every second day with the 10°C. 

 female, strip and fertilize about 100 eggs from each frog into fresh standard 

 sperm suspension. Determine the number of eggs fertilized, segregate, and 

 care for these eggs as under "3" on preceding page. (It can be expected that 

 the uterine eggs at 20 C. may be fertilizable for about 5 to 7 days, while 

 those uterine eggs at 10°C. may be fertilized for 10 to 14 days. 



OBSERVATIONS AND DATA: 



There are three sets of data which are to be collected from these observations: 



1. The percentage cleavage at the different ages and temperatures. 



2. The percentage of cleaving eggs which gastrulate, neurulate, and hatch. 



3. The variety of abnormalities that are produced, correlated with the age of the 

 egg- 



- 125 - 



