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

OBSERVATIONS ON FREEZING FOWL SPERMATOZOA 1IN LIQUID NITROGEN
P. E. LAKE

A.R.C. Poultry Research Centre, King's Buildings, West Mains Road
Edinburgh 9 Scotland.

Fowl spermatozoa are maintained in the oviduct for about 10 days after
a single insemination (A.I.) with fresh semen, assuming that optimal
conditions exist in the oviduct and that the correct number of good
quality spermatozoa are inseminated. Eggs are fertilized during
this period and it is the minimal requirement if A.I. is to be used
successfully for breeding fowls. Tt has not yet been possible to
produce this pattern of fertility in hens using frozen and thawed
fowl semen. In recent years work to freeze fowl and turkey semen has
resumed and some fertile eggs have been obtained after storage at
ultra-low temperatures for many days (1, 2. 3).

Work has begun in our laboratory to study the problems associated
with storing fowl spermatozoa in liquid nitrogen (-196 C) and so far
they have been stored for 7 days with good revival (68% of the sperma-
tozoa revived in good condition judged by observations on swirling
motility, and morphology in nigrosine-eosin smears) using the
fo%lowing teachnique: Semen was collected at room temperature (15 to
20°C) from males tgat had ejacu%ated on the previous day. The semen
was cooled (2 to 3 C/min) to +5 C and O.15ml aliqugts were pipetted
into test tubes (8mm int. diam.) in a cold room (5 C). 0.45ml cold
(5°C) diluent, containing 11% glycerol,was added to semen with gentle,
rapid mixing. Equilibration for 15 min was allowed and then it was
transferred to ampoules (1ml size)and sealed. The ampoules were cookd
from +5°C to -22°C at the rate of 10C/hin, then plunged into liquid
nitrogen. Thawing was_done in a water-bath at 3 C and samples were
kept for 6h at 3 to 5 C for periodical observations on motility and
morphology.

In experiments, which culminated in the adoption of the above-
ment ioned technique for studies on deep-freezing fowl semen, the
following results were obtained: 1. Glycerol was a better protective
agent than digol, 2-1-4-butane triol,diglycerol,polyethylene glycol
200,erythritol,inositol,sucrose,or dimethyl-sulphoxide; 2.The addition
of egg yolk,milk,bovine plasma albumin,egg albumin,ascorbic acid or
glutathione to our diluents (Table 1) have not improved the survival
of' spermatozoa; 3. 8% glycerol satisfactorily protected spermatozoa
against damage during freezing under our conditions; 4.0Ordinary Iml-
cylindrical ampoules,with the 0.6ml diluted semen, were as suitable
as spherical ampoules (0.6ml capacity); 5.Long equilibration periods
before freezing were unsuitable in our diluents; 6.A high K*content in
the diluents appeared to confer some henefit over a solution of low K*
content; 7.Slow freezing and slow thawing. as outlined above,has so
far produced the best revival of sgermatozo under our conditions.
Plunging directly from +3°C to -79° or -196 C was harmful. Thawing
at 20 to 37°C was harmful, especially if held at these temperatures
for more than 15 min.

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