STUDIES OF FERTILIZATION. 25 



variations in results. But if an adequate series of controls is 

 kept it is possible to check these up. 



The following controls were kept: (i) Some untreated eggs. 

 (2) Eggs left in the butyric acid. (3) Eggs left in the sea-water 

 to which transfer was made from butyric acid (unfertilized). 

 (4) When eggs were shaken for superimposed insemination, 

 some were left unfertilized. (5) In some cases samples from 

 control 3 were fertilized without shaking. With these controls 

 one can follow back the history of any given result and determine 

 the percentage and kind of membranes originally formed, the 

 extent to which they were destroyed by shaking, and the capacity 

 for fertilization without shaking. 



The eggs used for any given experiment were selected usually 

 from several lots representing different females ; those eggs that 

 gave the best reaction to insemination being selected. 



As Loeb and others have noted, the eggs that have received the 

 butyric acid treatment do not segment without subsequent treat- 

 ment, but invariably cytolyze, or die from other causes, sooner 

 or later. This happened invariably in my controls (3) and not 

 a single segmenting egg was recorded from such controls. 



The membranes when first formed are soft, and are easily 

 destroyed by shaking within two minutes (approximately) after 

 transfer to sea-water from the butyric acid; but they soon 

 harden and thereafter it is difficult to destroy them by shaking. 

 Moreover, the membranes do not form instantaneously after 

 transfer to sea-water, but require about the same time (approxi- 

 mately 30 seconds at 15 C.) as for their formation by insemina- 

 tion. The time for effective results from shaking is therefore 

 cut down accordingly. 



The exposure to butyric acid thus acts like insemination only 

 in the sense that it prepares the egg for membrane formation, 

 but the reaction does not begin until the butyric acid is removed ; 

 and, after that, the time required for membrane formation is the 

 same as for membrane formation of control eggs by insemina- 

 tion. So long as the eggs are in butyric acid, therefore, they 

 presumably maintain a f ertilizable condition, and it requires about 

 30 seconds after transfer for this fertilizable condition to become 

 lost bv occurrence of the full membrane reaction. 



