2*82 Studies in the Physioloffj/ of Fertilization 



A capital letter (e.g. ^) is used to denote the eggs of a certain 

 female, and a small letter (e.g. 6) the sperm of a certain male. An 

 experiment to test the effect of solution X on the fertilizing power of 

 sperm b would be carried out as follow.s. The object is to inseminate 

 approximately equal quantities of A eggs with (1) a plain dilute 

 suspension of b sperm, and (2) a suspension of b sperm of identical 

 concentration as suspension (1), but to which solution X has been 

 added. To obtain (1) and (2), one sperm-suspension is made up and 

 diluted to the required amount by the further addition of sea-water. 

 Equal quantities are pipetted into two dishes. To the first dish is 

 added a certain quantity (n cc.) of sea- water; to the second an equal 

 quantity (n cc.) of solution X. These two suspensions (1) and (2) are 

 then added to the two lots of eggs and the effect of solution X is 

 judged by the relative numbers of eggs fertilized in the two dishes. 



It is obvious that the whole validity of this method depends on the 

 degi-ee of exactness with which all other conditions are kept identical 

 for the two fertilizations, while the one fiictor alone — here the presence 

 or absence of solution X in the sperm-suspension — is altered. Of especial 

 importance is the identical concentration of the two (or more) sperm- 

 suspensions, and the complete mixing of the eggs and spermatozoa in 

 making the different fertilizations, so that all eggs have an equal chance 

 of being fertilized. The precautions taken to ensure this exactnessi 

 that is to say, the method of making up the sperm-suspensions, of sub- 

 dividing and treating the eggs and sperm, the way in which insemination 

 is effected, together with the method of counting the proportions of 

 fertilized and unfertilized eggs, are given in full in the Part on the 

 " Conditions of self-fertilization in Ciona." 



The results of each series of experiments are tabulated, the numbers 

 in the Tables being the percentages of eggs fertilized, unless otherwise 

 stated. 



Eggs from a single individual and sperm from another single 

 individual were used in each experiment. It goes without saying that 

 the presence of genital products of any other individual was always 

 rigidly excluded. All glassware and instruments were sterilized with 

 hot water before each operation, and the hands were dipped into hot 

 water. All sea-water employed was taken from the circulation and 

 passed through a Berkefeld filter. The method employed in removing 

 the eggs and sperm from Ciona intestinalis has already been described 

 in the part (Part I) on self-fertilization in this form. Similar methods 

 were used in taking the genital products from the other Ascidians used 



