FERTILIZATION OF THE FROG'S EGG 119 



Remove the testes from 7 adult male frogs, cut into very small pieces and mash with 

 blunt end of glass rod. When thoroughly macerated, dilute to 70 cc. with Spring Water. 

 Decant off the homogeneous suspension from the remaining small pieces of testes, and 

 place 10 cc. of this suspension in each Stender marked "N". In a nnajiner similar to that 

 of the preceding section, remove 1 cc. of the suspension from each of these "N" Stenders 

 and place it in the adjacent 0. 1 N Stender, and add 9 cc. of Spring Water. This will be 

 repeated from 0. 1 Stender to 0. 01 N Stender, and so on. This entire preparatory pro- 

 cedure should be completed within 10 minutes of the time when the sperm suspension is 

 ready. The timing of the sperm suspensions should begin with the time of dilution from 

 "N", and should not be more than 10 minutes after testes maceration. 



Again, this set of observations need not be carried beyond the 4-cell stage, or about 4 

 hours after the eggs are inseminated. It is important that the Stenders be covered, and 

 that they be kept at the same temperature throughout the experiment. 



In recording the data, it is suggested that the total number of eggs stripped, the number 

 of eggs cleaving, and the percentage cleavage all be indicated. For example, 98/49 - 50% 

 would mean that 98 eggs were inseminated, of which 49 cleaved representing 50% of the 

 total. It must be remembered that #2 Stenders are not the ideal receptacle for artificial 

 insemination and such eggs as are stripped must be adequately exposed to the sperm sus- 

 pension. Further, a single female should be adequate for the entire series, if only about 

 50 eggs are removed into each Stender. If more than a single female is available, it is 

 better to use fresh females for the older sperm suspensions. Remember in all cases to 

 flood the eggs 15 minutes after stripping them into the various sperm suspensions. 



C. TIME-TEMPERATURE RELATIONS 



In this experiment only "N" concentration is used, i. e. , two testes per 10 cc. of Spring 

 Water. The variables are time and temperature. 



Prepare two (2) ovulating females in advance so that the uteri are filled with eggs at the 

 time the sperm suspensions are made up. Prepare two (2) additional ovulating females 

 to be ready 48 hours later. 



Covered finger bowls should be placed at each of the following temperatures: 4°C. (re- 

 frigerator); 10°C. ; 20°C. ; and 30°C. Four temperatures covering a range of 26°C. will 

 be adequate. While it is not essential that the four temperatures be exactly those listed, 

 it is important that the temperatures chosen cover this range and be maintained accu- 

 rately. 



Remove the testes from 12 adult males, and make up a homogeneous sperm suspension 

 in a total of 120 cc. of Spring Water. Place 30 cc. in each of the four finger bowls at 

 the four temperatures. 



At the time intervals of 30 and 60 minutes, 6, 24, 48, and 72 hours, remove exactly 5 cc. 

 of sperm suspension from each of the four temperatures into Petri dishes and strip eggs 

 from ovulating females into each suspension. The control for this experiment consists of 

 a fresh sperm suspension into which eggs from the same female are stripped, simultan- 

 eously with the stripping into the experimental sperm suspensions. 



