Forcipulata 551 



normal upper limit for seawater if seawater of an initial salinity of 27-28 

 parts per thousand is used. 



ARTIFICIAL FERTILIZATION 



Fill two finger bowls with seawater. Rinse the ovaries from a ripe 

 female in one and place them in the second (3 liter capacity) . Some eggs 

 will be lost but the amount of coelomic fluid carried over is greatly 

 reduced. The toxic effect of the body fluids is well known. Let the 

 ovaries stand for about five minutes. This procedure allows a large num- 

 ber of eggs to pass out of them without mechanical pressure. More eggs 

 may easily be gotten out of the gonads by drawing them up into a wide 

 mouthed medicine dropper. Agassiz advocates cutting them into small 

 pieces ; Gemmill speaks of shredding. It is very difficult to remove every 

 piece of follicular debris after cutting or shredding, but greater damage 

 is done by the liberation of substances from the mutilated eggs and 

 ovarian tissues which will retard or cause abnormal development. After 

 the gonads are removed, the eggs settle to the bottom of the dish. Decant 

 about % of the water and refill with fresh, agitating the eggs slowly in 

 order to wash them. Repeat this procedure three times, as quickly as 

 practicable. Meanwhile examine the eggs microscopically at intervals of 

 ten minutes to observe the beginning of maturation. The time required 

 for maturation varies with the stage of ripeness of the female. In May 

 this will sometimes require two hours. Later, after the normal peak of 

 the spawning season (from the first to the second week in July at Milford, 

 Conn.) sperm should be added to the suspension immediately upon the 

 liberation of the eggs, for maturation is then extremely rapid. Agassiz 

 placed pieces of ovaries and testes in the same dish simultaneously. This 

 adds an unnecessary amount of foreign tissue juices to the egg suspen- 

 sion. Gemmill did not fertilize the eggs of Asterias rubens until they 

 had been liberated for two hours, which is often too late. The eggs 

 quickly lose their power of fertilization after the liberation of the second 

 polar body. 



Use only very motile sperm to fertilize the eggs. Examine a portion 

 of the sperm from a gonad under the microscope in a little seawater. The 

 whole microscopic field should shimmer. Wash the examined gonad in a 

 small finger bowl full of water and transfer to another. Press the sperm 

 out by means of a pipette, then remove the gonad. Stir the water until 

 a uniform suspension is obtained. Add 10 cc. of this suspension to the 

 bowl of eggs when the germinal vesicle begins to break down and stir 

 gently to mix the suspensions evenly. 



The fertilized eggs rapidly settle to the bottom of the dish. Decant 

 and add fresh seawater at intervals of % hour for two hours. There 

 should be about seven washings altogether, so that if maturation occurs 



