10-fold before artificial insemination. The preparation of
deep-frozen semen followed the method of Jones and Martin (4).
Ewes were inseminated once with 100 million spermatozoa and
the spermatozoa in these samples were centrifuged before
insemination to concentrate them to the sperm count of the
undiluted ejaculate (3).

RESULTS. Ribose, xylose, arabinose, glucose, mannose, fructose,
rhamnose, galactose, maltose, lactose, sucrose and raffinose
were tested and had a range of value as components of diluents
and their ranking altered according to the temperature of
storage. Table 1 is a summary of the response to four of the
sugars showing that glucose was one of the best for incubation
at 37 C and for deep-freezing but was the poorest for storage

at 5°C. Arabinose was the least satisfactory for the deep-
freezing but was best for the chilled storage of ram spermatozoda.

The survival of spermatozoa after deep-freezing in
diluents containing increasing amounts of sugar is shown in
Table 2. The proportion of spermatozoa motile after freezing
was not altered by raising the sugar concentration to 247 mM
except where lactose was used.

Treatments to detoxify milk before use as a diluent at
37° and 5°C have been investigated and, where heating
alone was used, 85 C for 10 min. was superior to the other
temperatures and times tested. The addition of 1 mg/ml
cystelne was as satisfactory but a combination of heating at
85°C for 3 min. and addition of 1 mg/ml glutathione or the
addition of 0.25 mg/ml mercaptoethanol gave better preparations
of m11k judging by the survival of ram spermatozoa after 6 hr.
at 37°C or 6 days at 5 . However, all of these preparations of
milk were equal in value as diluents for deep-freezing.

The lyophilised preparation of the non-dialysable fraction
of milk has been shown to increase significantly the survival of
chilled or frozen spermatozoa in synthetic diluents. However
solutions of these preparations equal to that in milk (3 to 3.5%)
were frequently difficult to prepare as some denaturation
occurred during lyophilisation. This preparative step was
avoided by dialysing milk against synthetic diluent until the
contents of the dialysis sac were milk protein at its original
concentration but in the synthetic diluent. Some of these
preparations, particularly those based on glucose, were found
to be equal to skim-milk as diluents for deep-freezing ram
spermatozoa.

(4) {8 213R.C. and Martin, I.C.A. 1965. J. Reprod. Fertil.

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