900 
Journal of Agricultural Research 
Vol. XXX, No. 10 
is not known. Often there were 
transparent gelatinous masses as much 
as 2 or 3 mm. in diameter. No 
attempt was made at a chemical 
analysis of the liquid or to determine 
its physical properties except such as 
were visible. The liquid was exam¬ 
ined microscopically as soon as recov¬ 
ered. If no active spermatozoa were 
found, the liquid was discarded, but if 
there was a fair number of active 
spermatozoa, the liquid was used. 
Sometimes the liquid was centri¬ 
fuged untreated, but usually in order 
to get a convenient volume it was 
diluted to some degree with Ringer’s 
solution. The only noticeable effect 
of dilution was that it resulted in a 
more distinct separation of the sper¬ 
matozoa and cells from the liquid upon 
centrifuging and fewer of them were 
found in the inside of the centrifuge 
tube. Presumably, therefore, it de¬ 
creased the specific gravity or the 
viscosity of the fluid. 
The centrifuging was done in the 
earlier experiments with a Bausch & 
Lomb hand centrifuge with which it 
was possible to govern the centrifugal 
force by regulating the speed at which 
the operator turned the handle. The 
speed ordinarily used was about 1,600 
r. p. m. The later experiments were 
carried out with a Bausch & Lomb 
electric centrifuge which had a lowest 
speed of about 1,600 r. p. m. and a 
second speed of about 2,000 r. p. m. 
The liquid was placed in the centrifuge 
in glass tubes 12.5 cm. long with an 
inside diameter of about 6.5 mm., and 
therefore a capacity of about 4.15 c. c. 
They were so arranged that when re¬ 
volving the extreme bottoms of the 
tubes were about 30 cm. apart and the 
tops were about 5 cm. apart. There¬ 
fore, the amount of centrifugal force 
applied varied in the different regions 
of the tube. The centrifuge tube con¬ 
taining the liquid was kept in a water 
bath at a temperature of 35° to 37° C. 
until it was placed in the centrifuge 
which was at room temperature. This 
naturally varied somewhat at different 
seasons. After the centrifuging, the 
tubes were again placed in the warm- 
water bath before injection. The 
length of time during which the tubes 
were centrifuged varied from 2 to 10 
minutes. When prolonged much be¬ 
yond 10 minutes, the spermatozoa were 
so nearly all thrown to the bottom of 
the tube that it seemed useless to try 
insemination with the liquid from the 
top. 
INSEMINATION 
The liquid was injected into the 
uterus of the females with another 
catheter which was so long that nearly 
all the liquid must have been deposited 
in the upper end of the uterus or in 
one of the horns. Some females were 
inseminated when not in heat and 
others showed unmistakable signs of 
heat but had not actually copulated 
with the test males. This was done 
to test whether copulation is necessary 
to ovulation in the rabbit as main¬ 
tained by Marshall {29). Some were 
inseminated with the untreated liquid 
just as it was recovered. Others were 
inseminated with liquid which, aside 
from dilution, had received no treat¬ 
ment except that it had been allowed 
to remain in test tubes at low tempera¬ 
ture for periods of from 1 to 48 hours. 
However, the majority were insemi¬ 
nated with centrifuged material and 
these were grouped in two classes ac¬ 
cording to the part of the centrifuge 
tube from which the material was 
taken. The top one-third of the 
liquid in the tube, that which had 
been on the inside while the centrifuge 
was running, was carefully drawn off 
with j.he catheter, and females in¬ 
seminated with this portion were re¬ 
corded as “inside insemination.” The 
middle one-third of the fluid was dis¬ 
carded entirely except that occasionally 
microscopic slides were made from it. 
The bottom or outer one-third, which 
also contained the sediment, was 
stirred up and injected into a single 
female. Such inseminations were re¬ 
corded as “outside inseminations.” 
ISOLATION OF FEMALES 
Females used for breeding were kept 
in individual hutches from which they 
were removed only for testing whether 
they were in heat. All females when 
bred in any manner were left in their 
hutches for 10 davs before being again 
tested. 
CHECKING THE SEX OF THE OFFSPRING 
The sex of the living rabbit can not 
often be identified before it is 3 weeks 
old and sometimes not until it is 8 
weeks old. To make possible the pro¬ 
duction of a larger number of offspring 
from a given number of females, the 
young were often killed immediately 
after birth and the mother bred again, 
as that is one of the surest times to 
