THE AMERICAN SOCIETY OF ANIMAL PRODUCTION
81
STALLION SEMEN STUDIES AT
MICHIGAN STATE COLLEGE
G E O R G E K. D A V I S and C. Lo COLE
Michigan State College
Since April 1939 studies of the chemical and physical properties of
stallion semen have been in progress at Michigan State College. It was
considered probable that, until more knowledge was available concerning
the chemical makeup and behavior of semen, progress towards long
term preservation o f spermatozoa for use in artificial insemination would
be definitely limited. Consequently this project was set up with a view
to ascertaining by chemical treatment and observation, the constituents
of stallion semen, their effect upon the horse spermatozoa, and the value
of diluting and preserving fluids for preservation. As set up at present
the use of semen for artificial insemination is not contemplated except
as an eventual test of preservation methods.
The semen samples
kept for the purpose.
tain irregularities due
were made by means
horse*.
were collected from a two-year,old Belgian stallion
The collections were made twice weekly with certo circumstances of time and place. All collection~
of an artificial vagina (Cambridge) and a dummy
One of the first observations made was the extreme variation in the
quantity of ejaculate and in the number of spermatozoa present in a
single ejaculate. These observations were coupled with observations on
the variability in the amount of the different parts of the ejaculate p a y
ticularly the thick gelatinous fraction.
The variation in the amount of semen collected from the stallion is
shown in Figure 1. Presumably because we were working with a young
animal the amount of ejaculate was quite small at first and gradually
increased until a maximum of 170 grams (approximately six ounces)
was obtained 49 days after collections were first begun. The particular
sample mentioned was obtained following the first failure to obtain a
sample, five days before. The failure was directly attributable to the
fact that until that time the horse had been stabled alone but betvceen
collection periods it became necessary to keep the horse in the regular
horse barn with other horses. Following this period it was necessary to
$ Authorized for publication as Journal Article No. 408 n.s. of the Michigan
interest of Mr. Jack McAllen, who did most of the construction work.
* Credit for the construct/on of the dummy mare is due largely to the help and
Agricultural Experiment Station.
82
THE AMERICAN SOCIETY OF ANIMAL PRODUCTION
have another horse in the vicinity of the
to be made. The presence of a gelding, or
means of teasing as did the presence of
amount of ejaculate ha~e continued to be
be less than 100 to 125 gram in amount.
@rams
dummy when collections were
a pony worked just as well as a
a mare in heat. Variations in
marked but with a tendency to
FI @URE i
QUANTITY OF S E E N
BELGIAN STALLION B1939
200
180
160
o~ 14o
iso
I00
80
o
I
i0
I
20
I
SO
I
40
I
50
I
60
I
70
I
80
I
90
I00
Days from first collection
An observation as to the number of collections which could be made
from this horse should be noted. Twenty-five collections were made between April 14th and August 1st at which time the samples were considered fairly normal in that they were of average amount and were
obtained with little difficulty. Following this period only five samples were
obtained, as of November 15th. Live spermatozoa were present in the
ejaculate but in number of spermatozoa and quantity of ejaculate the
samples were decidedly inferior to those obtained up to August.
THE AMERICAN SOCIETY OF ANIMAL PRODUCTION
83
The variation in numbers of spermatozoa present is shown in Figure 2.
As may be seen the variation was quite marked and apparently not related to the volume of ejaculate. A small volume of ejaculate was no
indication of a small total amount of spermatozoa or vice versa. The
range in count for a single collection was from a few hundred thousand
to a maximum of 47 hundred million.
O0
FIGURE 2
TOTAL NUMBER
SPERMATOZOA
4-5
IN S E M E N
BELGIAN STALLION B1959
0
40
35
0
0
~0
25
0
r.b
20
15
10
5
0!
Av
20
30
40
50
I
80
I
90
Days from first collection
The count was made by means of a bright-line hemocytometer using
regular diluting pipettes. The diluting fluid was a solution of physiological
saline containing 0.1% glacial acetic acid. This solution was usually quite
suitable in that it killed the spermatozoa and prevented clumping.
In making a count of spermatozoa the presence of the gelatinous mate.
rial was a definite interfering factor. With the exception of the very
first counts made when the whole sample was considered as a unit,
counts have been made upon the fractions of the ejaculate. So far
as this one animal is concerned, at least, the gelatinous material has
contained but few spermatozoa (all dead) while fractions collected be-
84
THE AMERICAN SOCIETY OF ANIMAL PRODUCTION
fore and following the ejaculation of the gelatinous material have been
rich in spermatozoa. Walton (1938) has likewise noted the lack of sperm
in this portion of the semen.
Following the appearance of gelatinous material in the semefi of this
stallion which occurred at the fourth collection, attempts were made at
fractionation of the ejaculate at the time of collection. This was accomplished by changing the collection bottles which fitted over the end of a
glass tube fashioned to fit on the artificial vagina by means of a rubber
tube and tapered to enable collection in four ounce bottles.
The success of this method of fractionation was far from uniform
and we are now working on a new type of vagina in which we are including a transparent window in an attempt to obtain a better, more
accurate fractionation.
A feature of stallion semen worth noting is the variation in amount
of gelatinous material which occurs. In our studies variations of from
no gelatinous material to nearly orie-third of the total ejaculate have
been noted. The gelatinous material quickly breaks down upon storage
usually within four or five hours at 15~ and when centrifuged decreases in volume or if centrifuged long enough may be completely disintegrated.
The chemical studies upon the semen have been chiefly concerned with
the effects of temperature upon the spermatozoa and seminal plasma,
with the type of metabolism present in the spermatozoa.
The temperature at which undiluted semen was maintained had a
striking effect upon the hydrogen ion content of the semen beyond that
which would be expected due to the slight shift noted in ordinary buffer
solutions with changes in temperature (Clark, 1928).
In the semen studied, a definite drift in pH with changes in temperature was found*. Furthermore the results indicated that this drift was
not entirely reversible. Once the p H of a sample had shifted to the
alkaline because of lowered temperature it was not possible to cause the
pH to shift back to the original pH by raising the temperature again.
In table 1 is listed an average of the pH shift with temperature change
along with maximum and minimum values for the 25 semen samples
included in these values. In order to demonstrate the shift within given
samples two, B22 and B9, are included to demonstrate individual shift.
Walton, A., 1938. Proc. American Soc. of Animal Prod. 238-241.
Clark, W. M., 1928. The Determination of Hydrogen Ions. Williams and Wilkins,
212.
* All measurements were made with a Beckman Glass Electrode pH Meter.
THE AMERICAN SOCIETY OF ANIMAL PRODUCTION
85
It may be seen that at a temperature of 37.5~ the semen of this stallion
had pH values ranging from a low of 6.9 to a high of 7.7 and at a temperature of 24~ values of from pH 7.3 to pH 8.0. The striking thing
however, has been the shift which occurs in a sample of semen when it
is cooled from body temperature to room temperature and to a lesser
degree to a cold storage temperature of 15~ or lower. Every sample
from this animal has exhibited this shift although not all to the same
extent. The average shift has been of from 0.6 to 1.2 pH units. The
exact influence which such changes have upon the spermatozoa is not
entirely clear although they are known to be quite sensitive to hydrogen
ion change. There are reports in the literature of the fact that stallion
semen exhibits an alkaline shift upon ageing. (Shergin, 1935). Our work
indicates that temperature has a major role in such changes.
TABLE I
VARIATION OF pH OF STALLION SEMEN WITH CHANGE
IN TEMPERATURE
14
Ave. pH Values
Maximum Value
Minimum Value
B 22 Max. Shift
B 9 Min. Shift
8.1
8.4
7.8
8.4
Temp. of Semen ~
20
24
28
7.8
8.2
7.5
8.2
7.6
8.0
7.3
7.4
7.5
7.9
7.2
30
34
37.~
7.4
7.8
7.1
7.3
7.7
6.9
7.1
7.2
7.7
6.9
6.9
7.0
Another feature of stallion semen which has attracted our attention
and of which the alkaline shift is an indication is the apparently different type of metabolism which occurs as compared either with muscle
metabolism or with the metabolism of spermatozoa from other species.
Stallion semen differs from semen of other animals in the low glucose
content and in the low lactic acid content of semen which has aged.
Samples which we have observed have contained very little glucose
ranging, from 4 rag. percent to 18 mg. percent. This small amount of
glucose disappears very rapidly but the content of lactic acid which is
also low is not measurably increased during the period of activity on the
part of the sperm. This points to a different type of metabolism from
that exhibited by muscle and other spermatozoa. A t present we are attempting to work out this system of metabolism in order that we may
have a better basis for obtaining conditions suitable for preservation of
stallion sperm over long periods of time. Preliminary results have indicated that probably the source of energy used by stallion sperm is, indirectly at least, tied up with protein metabolism.
Shergin, N. P., 1935. Proc. Animal Husbandry (U. S. S. R.) No. 12, 100-122.