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

FILM FREEZING OF SEMEN®

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

E. F. Graham
University of Minnesota, St. Paul, Minn.

Temperature profile differences exist at various sites within an ampule of semen while
it is being frozen (1). A similar variation occurs while the frozen ampule is thawed. In
other words the position of the thermocouple within the ampule will have an influence in the
cooling and thawing profile recorded.

Cryo-research on freezing and thawing rates for maximum survival will become more mean-
ingful if the entire semen mass was exposed to one cooling or warming influence. Graham has
reported on a technique to evaluate semen freezability by freezing semen in glass slides (2).

To be able to expose a given volume of semen to a specific cooling rate the semen should
have the maximum surface area. To be able to control and monitor this freezing and thawing
rate the technique should be a motionless one. The film freezing described here was developed
as a research tool for semen freezing and thawing studies. It fulfilled within very narrow
limits conditions of uniformity in cooling and thawing of the entire semen mass. The tech-
nique also allowed semen to be frozen in dialysing bags stretched flat if one wished to dia-
lyze glycerol after a freeze-thaw operation.

MATERIALS AND METHODS

Bull semen was extended in Minnesota GO (3) and Raffinose-yolk-glycerol (4) according to
directions and frozen four hours after addition of the glycerol portion of the extender. Se-
men was introduced into plastic bags and a thermocouple was inserted into it and the bag was
sealed.

FILM FREEZE METHOD

Plastic bags Il inches by 2 inches were used as caotainers. The semen to be frozen was
taken in amounts of 10 cc per bag. All the air bubbles were excluded and the bags were heat
sealed. The bags were then sandwiched between | mm thick aluminum metal strips of same size
as the bags and held together by strong paper clips. The sandwich was lowered into the va-
pour phase of an M.V.E. 9000 freezer unit and frozen. The frozen sandwiches were stored in
vapour phase of large storage containers. It was pssible to store the frozen semen in plas-
tic bags alone if care was exercised in handling. |t was possible to change the cooling ve-
locity to which the semen wws subjected by using thicker metal sheets or by experimenting
with metal strips of different heat transfer characteristics.

Glass sheets were used for sandwiching the plastic bag only to visually observe the
freezing pattern. Semen can be seen to go into. the opaque solid state almost simultaneously
in the entire plane of the semen. Special low temperature glass is best, otherwise it cracks
at liquid N temperatures.

RESULTS AND DISCUSSION

Typical freezing and thawing profiles occurring at the middle and outside of the semen
mass, when it is frozen in an ampule, is shown in Figure |. It also shows a freezing and
thawing profile occurring during a film freeze and thaw operation. One can observe the lag
at the thawing and freezing phase in the ampule profiles and only a slight pause in the film.
There were obvious differences in freezing and thawing rates between the core and the shell
of semen mass frozen in the ampule. Results comparing a regular freezing and a film freezing
are shown in table I. There was a significant improvement in the freezability of semen at
the freezing and thawing rate shown in Figure | with the film freezing technique. The semen
was also available after thawing for further studies in practical amounts. The technique is

* Scientific Journal Series 6584

1141

21