VI® CONG. INTERN. REPROD. ANIM. INSEM. ARTIF., PARIS, 1968, VOL. Il

EFFECT OF SUDDEN COOLING ON THE PROPORTION OF
EOSINOPHILIC BULL, RAM AND RABBIT EJACULATED SPERMATOZOA
IN DILUTED AND UNDILUTED SEMEN

H. M. DOTT

A.R.C. Unit of Reproductive Physiology and Biochemistry,
University of Cambridge, England.

The effect of the sudden exposure of semen to temperatures
between 0°C and 30°C on the proportion of eosinophilic spermatozoa
has been used as a measure of semen quality (1), and also to compare
the susceptibility of epididymal and ejaculated spermatozoa to cold
shock (2, 3). The experiments described below were dome to discover
whether the increase in the proportion of eosinophilic spermatozoa
on exposure to cold shock is entirely due to cold shock or if other
factors are involved as well.

MATERIALS AND METHODS. Semen was collected in an artificial vagina
from bulls, rams and rabbits, subjected to some form of treatment
and stained for five minutes in a nigrosin-eosin mixture (4).

For bull and ram semen the treatments consisted of exposure to
0°c, 7.5°C, 10°C, 20°C or 30° without dilution for 5, 10 or 15
minutes, or after dilution for 10 or 15 minutes. The stain was at
the same temperature. Samples were also stained at 30°C but they
were only held at the treatment temperature for 5 or 10 minutes. To
ensure rapid cooling 5 drops of semen were dropped into precooled
glass tubes or 1 drop of semen into 4 drops of precooled diluent
(0.154M NaCl buffered to pH 7.4 with 0.1M phosphate buffer).

The slides were coded before the experiment and the code was not
referred to until all the slides had been counted. A spermatozoon
was counted as eosinophilic if there was eosin in any part of the
head. The total number of spermatozoa in the first microscope field
(selected at random) was recorded on a hand tally counter and the
number of eosinophilic spermatozoa was written down, the slide was
moved to the adjacent field and the process repeated until 100 sperm—
atozoa had been counted on each slide. The number of eosinophilic

(1) M. W. H, BISHOP, R. C. CAMPBELL, J. L. HANCOCK & A. WALTON, 1954.
J. Agric. Sci., 44: 227,

(2) I. G. WHITE & R. G. WALES, 1960. Internat. J. Fertil., 5: 195.

(3) J. P. BENNETT & H. M. DOTT, 1966. J. Reprod. Fert., 12: 327.

(4) J. L. HANCOCK, 1951. Nature, 167: 323. S

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