123 



FERTILIZATION OF THE FROG'S EGG 



time/concentration - CLEAVAGE i 



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

 hlunt 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 manner 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 he re- 

 peated from 0.1 Stender to 0.01 N Stender, and so on. This entire preparatory procedure 

 should he completed within 10 minuteR of the time when the sperm suspension is ready. 

 The timing of the sperm suspensions should hegin with the time of dilution from "N", and 

 should not he more than 10 minutes after testes maceration. 



Again, this set of observations need not he carried heyond the li-cell stage, or ahout 

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

 that they he 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/'*-9 - 50^ would mean that 98 eggs were inseminated, of which '+9 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 suspension. 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 hQ hours later. 



Covered finger bowls should be placed at each of the following temperatures: h°C, 

 (refrigerator); IQOC. ; 20OC.; and 50°C. Four temperatures covering a range of 26OC. 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 

 accurately. 



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

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

 four temperatures.' 



At the time Intervals of 50 and 60 minutes, 6, 2U, 1*8, and 72 hours, remove exactly 

 5 cc. 6f 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 con- 



