8. Experiments on Early development - Non-Operative 

 7. FERTILIZATION OF THE FROG'S EGG 



PURPOSE: To determine the ideal conditions of concentration, time, and temperature 

 for maximum fertilization of normal eggs of the frog, Rana pipiens. 



MATERIALS: (This experiment can be done best with groups of 4 students) 

 Biological: Mature male frogs 



Ovulating female frogs 



Technical : Finger bowls, paired Petri dishes 



Controlled temperatures at 4°C. , 10°C. , 20°C. , and 28°C. 



(If these exact temperatures are not available, the range should be 

 covered and the temperatures should be stable. ) 



METHOD: 



Precautions : 



1. All glassware must be biologically clean. 



2. Glassware and solutions must be brought to the various temperatures prior 

 to the introduction of spermatozoa, unless otherwise directed. 



3. Separate pipettes (medicine droppers) must be used for the transfer of sperm 

 suspensions from each container. They should be biologically clean and 

 marked for identification. Mark the pipettes at the 1 cc. level. 



4. Eggs from the ovulating female should be tested against normal sperni sus- 

 pensions to determine whether they are fertilizable. 



Control : The control consists of "4" above, and those eggs which are exposed to the 

 "normal" concentration. This is considered as one (1) pair of adult testes per 

 10 cc. of Spring Water. 



Procedure : Three distinct sets of observations are to be made, and the data is to be 

 plotted on graphs. 



A. DILUTION AND FERTILIZATION 



In this first experiment, the temperature shall be that of the laboratory and eggs are to be 

 stripped into the sperm suspensions within 30 minutes of the maceration of the testes. 

 The only variable will therefore be the dilution, or the concentration of the spermatozoa. 



1. Prepare 10 Petri dishes, (5 covers and 5 bases). Label the pairs N, 0. I N. , 0. 01 N. , 

 0. 001 N. , and 0. 0001 N. 



2. Quickly remove and cut into small pieces (with fine scissors) two (2) pairs of testes 

 from adult frogs. These should be further mashed with a flattened end of a glass rod, 

 all in 20 cc. of Spring Water. Note the time of testes excision. 



3. If possible centrifuge by hand the sperm suspension to throw down the larger pieces of 

 testes, or allow them to settle in a tapered test tube. See that the total volume is 20 

 cc. Decant off the homogeneous suspension (do not remove any pieces of tissue) into 

 the two Petri dishes marked "N", 10 cc. in each. 



4. With a clean and dry pipette (medicine dropper), previously marked at the 1 cc. level, 

 remove 1 cc. of the sperm suspension from the bottom Petri dish marked "N" and 

 mix thoroughly with 9 cc. of Spring Water in bottom Petri dish marked 0. IN. In a 

 similar manner transfer 1 cc. of "N" from the top Petri dish to the top Petri dish 

 marked 0. 1 N, adding 9 cc. of Spring Water. In a similar inanner transfer 1 cc. of 

 each thoroughly mixed sperm suspension to 9 cc. of Spring Water in the next dish, 



