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

A NEW MEDIA AND TECHNIQUE FOR FREEZING TURKEY SEMEN

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

Turkey semen permits very little time for in-vitro manipulation, before it becomes infer-
tile (1, 2). Egg yolk incorporated extenders have been shown to be detrimental to fertility
(3). Glycerol has also been reported as harmful to fertility (4, 5). Dialysis was proposed
as a means of removing the glycerol (6). Since the time of in-vitro handling in itself has a
deleterious effect on fertility this study was an attempt to freeze turkey semen soon after
collection for recovery of usable motile cells. Since soy protein afforded good freeze pro-
tection in the freeze preservation of Bovine semen in our laboratory it was decided to substi-
tute egg yolk and egg white with the sodium salt of soy protein (Promine D)*. Zero percent
glycerol and two percent glycerol concentrations were used to study the freezability. Dialy-
sis was chosen as the means of removal of glycerol prior to insemination whenever semen was
frozen with glycerol.

MATERIALS AND METHODS

Freezing extender was prepared by dissolving four grams of sodium proteinate obtained
from soy bean in 2.9 gram percent solution of Sodium Citrate dihydrate. The soy protein did
not go completely into solution. The insoluble material was filtered using coarse grade fil-
ter paper. Antibiotics were added at 500 |.U. penicillin per cc and 500 ug of streptomycin
per cc of extender. The extender was used with and without glycerol. The extender was main-
tained at 15° ¢ during extension with semen.

Since turkey semen coagulates quickly after collection, pooled semen was collected by
massage directly into measured amounts of extender. The tube containing the extender was
constantly shaken after each collection.

The semen was extended to five times its original volume. All semen was frozen in
liquid nitrogen vapour in a portable freezing unit (9). Vapour freezing, spin freezing (7)
and film freezing (8) were selected as the freezing techniques. Film freezing has given
better freeze recovery than conventional methods and was selected specially since semen
could be frozen in stretched dialysing tubes. Thawing and dialysing out of the glycerol was
accomplished in one step. :

FREEZING FRAME

A metal frame was designed as shown in Figure 1. Dialysing tubes could be stretched
flat and wound around it. It was rigid and allowed the film of semen to be frozen from both
sides. It was convenient for handling during freezing, storage, thawing and dialysing.

Where glycerol was used the initial glycerol concentration of the extender was 2.5% making
the final glycerol content 2% in the diluted semen.

Soon after dilution the extended semen was ampuled and the ampules left in liquid nitro-
gen vapour of a field semen freezing unit (9). The entire operation from the first dilution
to freezing took 5, 10, and 15 minutes. For film freezing the semen was introduced into a
prepared dialysing tube. The end of the tube was tied, and the tube was stretched flat and
wound around the frame. The entire frame was lowered into the liquid nitrogen vapour. The
operation was conducted within 5, 10, and |5 minutes. Semen filled in ampules were also
spin frozen (8) to evalnate the effect of spin freezing on turkey semen at 5, 10, and 15 min-
utes after extension. Semen frozen in ampules were thawed in water at 40° C and only those
frozen without glycerol in ampules were used for insemination. Only 0.1 cc of semen was
used per insemination. Semen frozen in dialysing tubes were thawed by immersing the entire

* Central Soya, 1825 N. Laramie Ave., Chicago, I11inois 60639, U.S.A.

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