STUDIES OF FERTILIZATION 537 



numbers across the clear margin to the ring and are speedily 

 paralyzed by the diffusing CO2; this process continues very rapidly 

 and as a consequence the central aggregation expands, and may in 

 time absorb all the spermatozoa of the suspension. 



The same j:)henomenon in less pronounced form is exhibited by 

 the reaction to a 1/10 dilution of the saturated CO2 sea-water. 

 Here we may give a definite experiment with measurements of 

 growth of the aggregation: July 1, 1912. Into a fresh sperm sus- 

 pension beneath a raised cover a drop each, (a) of 1 per cent, and 

 (b) of 10 per cent CO2 sea-water was injected some distance apart. 

 Drop (a) measured 5 mm. in diameter and drop (b) 3 mm. immedi- 

 ately after injection at 2.28 p.m. The aggregations caused by 

 these drops measured at 2.32 : (a) 3 mm. (b) 5 mm. That is to 

 say, the aggregations formed inside the drop in case of the weaker 

 solution, and outside in the case of the stronger. At 2.35 (a) 

 still measured 3 mm. and (b) now 6 mm. In a repetition of this 

 experiment drops (a) and (b) each measured 3 mm. at 2.37. The 

 aggregations caused by them measured at 2.40, (a) 2 mm., (b) 5 

 mm.; at 2.47, (a) 2 mm., (b) 10 mm. 



It is clear from these observations that the spermatozoa are 

 positively chemotactic in a CO2 gradient where the tension is 

 above a certain point, and that the aggregation caused by the 

 more concentrated drop grows because the diffusion of CO2 from 

 the center forms a widening ring of the necessary concentration. 

 To furnish a gradient the concentration must exceed the CO 2 

 tension in the general suspension, which is a function of the age 

 of the sperm suspension and the activity of the spermatozoa in it, 

 and on the other hand a limit is set to the differential which fur- 

 nishes the reaction by the fact that a concentration of 'about 

 1 per cent of the saturated CO 2 sea-water paralyzes the spermato- 

 zoa. The gradient that furnishes the reaction must, therefore, 

 operate within very narrow limits. 



Greater dilutions of the CO2 sea-water than 1/100 will act 

 positively in the case of fresh sperm suspensions. The ring forms 

 within the margin of the drop in the case of 1/200 dilution, but 

 it remains narrow, and tends to break into bead-like aggregations, 

 proving that the spermatozoa by their own activity have produced 



