Vie CONG. INTERN. REPROD. ANIM. INSEM. ARTIF, PARIS, 1968, VOL. |

FREEZING AND FERTILITY STUDIES WITH STALLION SEMEN

A.H.J. Rajamannan
Eastern A.l. Cooperative, Inc., Ithaca, N.Y.

R. Zemjamis and J. Ellery
University of Minnesota, St. Paul, Minn.

Freeze preservation techniques of stallion semen have not kept pace with that of bull
semen even though very encouraging reports have appeared on methods of preservation and fer-
tility testing (I, 2, 3, 4, 5, 6, 7). Glycerol has been known to lower fertility in other
species (8, 9). Compared to bull semen, semen from stallion does not live long in"storage at
+5% ¢ (10). Seminal plasma has been reported to have an adverse effect a the length of
liquid storage of stallion semen (10). Seasonal effects on stallion semen preservation have
also been experienced (3).

The object of this study was to develop techniques that would allow stallion semen to be
frozen with minimum of glycerol and minimum of in-vitro handling time and glycerol equilibra=-
tion time. The effects of removing seminal plasma were studied on the freezability of stal-
lion semen. Semen was collected from stallions around the year to study the effect of season
on the freezability and storage of the semen.

MATERIALS AND METHODS

Six stallions were collected using the Missouri model A.V. once every two weeks or often-
er throughout the year. The semen was used for the freezability, storage, checmical and fer-
tility studies.

Four extenders were used in the experiment and they were made up as follows:

A. 85 cc of Glucose 5.6 gram % solution

15 cc of skim milk (fresh or reconstituted)
B. 85 cc of Lactose 12 gram % solution

16 cc of skim milk (fresh or reconstituted)
C. 95 cc of Lactose 12 gram % solution

5 cc of fresh egg yolk *
D. 80 cc of Glucose 5.6 gram % solution

20 cc of fresh egg yolk *

* Centrifuge the extender at 5000 R.P.M. for 5 minutes in a clinical

centrifuge and use only the supernatant.

The osmotic pressure of the extender was eventually adjusted to 330 milliosmoles with
concentrated glucose or Lactose solutions. (In the preliminary experiments the 0.P. was
varied from 290 - 350 milliosmoles.)

The semen was filtered immediately after collection through many layers of cheese cloth.

Aliquots of semen was taken for pH, osmotic pressure measurements and chemical analysis.
Three different processing times of 15 minutes, |4 hour and 2} hour from collection to freez-
ing were utilized. Extender D was used with the |5 minute processing and A, B and C with |4
and 2; hour proc:esing.

Two methods, one a centrifugation method and other a dilution method were developed dur-
ing this study.

CENTRIFUGATION METHOD

When the semen was of low concentration (less than 200 million sperm cells per c.c.)
20% of the semen's volume of glicose 5.6% solution at 35°C was added to the semen and mixed.

The semen was centrifuged in a clinical centrifuge at 2500 R.P.M. for 3 minutes. The
seminal plasma was poured off and the packed sperm cells were resuspended in the freezing
extender.

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