74 
BULLETIN OF THE UNITED STATES FISH COMMISSION. 
Tlie results of this experiment are too varying to he of any practical value. It 
seems probable that both spermatozoa and ova remain passive in the salt solution 
and that fertilization takes place only after the addition of fresh water in washing. 
Where a considerable quantity of fresh water was added and the ova mixed well with 
it before it was poured off the percentage of fertilization was high. When the water 
was poured off immediately and without mixing the ova well the fertilization was 
incomplete. Normal salt solution apparently pi*eserves the vitality of the sperma- 
tozoa longer than fresh water. 
Vitality in air . — It was found that milt kept in an open, large-month bottle for 
24 hours fertilized 74 per cent of the eggs it was mixed with. Milt that had been 
so exposed 48 hours did not fertilize any eggs, nor did that kept in a tightly corked 
vial for 24 hours. 
“Watery” milt . — Milt when taken from the fish varies greatly in consistency. 
That from some fishes is very thin and is known as watery milt. Experiment 
proves that it fertilizes eggs as well as any and that no larger quantity is needed. 
Amount of milt required in artificial fertilization . — In taking spawn it was 
the custom at Battle Creek to express the eggs from one female into a pan con- 
taining about a pint of water and add enough milt to make the water distinctly 
whitish. The amount of milt necessary for this varies, depending on the amount 
of abdominal fluid mixed with it, but is never less than 3 or 4 fluid ounces. This 
method gives good results and should be followed when there is an abundance of 
males, which is always true at Battle Creek after the first few days of the season. 
A smaller amount of milt, however, will suffice. Ninety-six per cent of the eggs from 
one female were fertilized by a tablespoonful of milt; 85 per cent were fertilized by 
a teaspoonful; 35 and 57 per cent were fertilized by spawning fishes in the creek and 
letting the milt float over the eggs, which had been caught on a screen. 
Of course, all that is necessary is to bring a very minute quantity of milt in 
contact with each egg. A single drop of milt if thoroughly disseminated through 
the water would be sufficient to fertilize all the eggs from one female. In the experi- 
ments above noted the milt could not have been thoroughly mixed until after it had 
become inactive. It is not advisable to use less than a fifth of an ounce of milt to 
fertilize 1,000 eggs. More water is necessary where a small amount of milt is used 
in order to facilitate thorough mixing. 
EXPERIMENTS WITH OVA. 
How to test fertilization. — The quickest and surest way to determine whether ova 
have been fertilized is to put them into a dilute (5 to 10 per cent) acetic acid. This 
can be made from commercial acetic by adding from two to five parts of water. A 
few minutes after the ova have been placed in the acid the embryos turn white, 
while the yolk remains clear. The embryo can be distinguished in this manner 
within 15 hours after the ovum lias been fertilized. In making this test for the first 
time it is best to make a comparative test with unfertilized ova that have been kept 
in water during the same period. 
Short exposure to water detrimental . — A quantity of eggs were spawned into a 
pan of water and some were removed and spermatized every half minute for several 
minutes, and for various periods up to several hours. The milt was, of course, 
taken fresh each time. 
