21 6 NEIL S. DUNGAY. 



was made to keep from crowding the developing eggs since this 

 seems to be a source of abnormality. Any error which might 

 creep in here was checked by the controls since they always 

 contained at least as many eggs as were kept in the experimental 

 cultures. 



E. Controls. 



Along with each experiment two or more control cultures 

 were kept. One control consisted of a dish of sea water contain- 

 ing a part of the eggs which were not inseminated. The other 

 contained about the same number of eggs as was kept in each of 

 the experimental dishes, inseminated with injured sperm; these 

 were as closely followed during the period of observation as were 

 the experimental cultures. In all experiments in which the sperm 

 cells were subjected to the action of reagents it is impossible to 

 avoid carrying over a trace of the reagent used, to the dishes of 

 eggs. Control experiments were made in such cases by adding 

 to the eggs at the time of insemination, as much of the reagent 

 as was introduced into the experimental cultures. In nearly 

 all cases the eggs and sperm used in the controls came from the 

 same animals which furnished the materials for the experiments. 



F. Treatment of Sperm Cells. 



Attempts to injure the sperm cells as much as possible without 

 preventing their attachment to the eggs proved unsuccessful in 

 many cases. Either the sperm were killed or they were not 

 sufficiently affected to give striking results. With improvement 

 of the technique consistent results were secured, although these 

 results indicated a great variation in the intensity of the injuries 

 to the sperm cells, thus rendering it impossible to repeat an ex- 

 periment with a complete duplication of results. 



The sperm cells were injured in various manners. Inasmuch 

 as some methods gave promise of easier and more striking 

 results or seemed less open to criticism, but few experiments 

 were made along other lines in order to give more time to follow 

 out the more desirable methods. Injury was attempted by 

 soaking the sperm cells in distilled water or in diluted sea water ; 

 by treating them with weak alcohol; by exposing them to the 

 action of dilute acids or alkalis; by freezing; by heating; and 



