228 GREGOR T. POPA. 



some of the follicles had not reached the proper stage of con- 

 centration of the necessary constituents. However, follicular 

 fluid taken from follicles of ten different sizes (cow and sheep), 

 from the smallest to the largest gave a positive result in all cases. 

 The age of the follicle does not appear to be of importance for 

 the reaction. 



(d) Time of reaction. In a series of ten tests (cow and sheep) 

 observed after ten minutes, six were positive and four were 

 negative. In ten tests observed after twenty minutes, all showed 

 the coagulum. In a third series of ten tests, five of which were 

 observed after one hour, five after 24 hours, all were positive. 

 Thus the minimum average time necessary for the reaction lies 

 between ten and twenty minutes. But the reaction is often 

 observable after one minute. When dry sperm is mixed with 

 follicular fluid on a slide, the reaction appears instantly. A 

 delicate layer of coagulum is formed on the surface of the fluid. 



(e) Variations in the time of addition of one of the components. 

 In these experiments two of the three components were mixed 

 and the third constituent added after a time interval, varying 

 from one to twenty minutes. The following are the possible 

 combinations: 



(1) Follicular fluid mixed with salt solution; sperm added later. 



(2) Sperm mixed with salt solution ; follicular fluid added later. 



(3) Sperm and follicular fluid mixed; salt solution added later. 

 No noticeable difference in the time of appearance of coagulum 



was noted in all three cases counting from the time of addition 

 of the third constituent. In the third case the beneficial effect 

 of salt solution on the reaction was again verified. 



(/) Variations of temperature. 



Dry sperm and follicular fluid were brought to various tempera- 

 ture and mixed (with the addition of salt solution as usual). 



The results are summarized on opposite page. 



These results show that there is in the follicular fluid a thermo- 

 labile substance which is responsible for the reaction. If the 

 follicular fluid is heated to 56 C. the reaction fails. If the 

 sperm is heated to a high temperature, even to 100 C. the 

 reaction is obtained as long as follicular fluid is not heated 

 above 55 C. 



