Preservation of Bull Semen at Sub-Zero Temperatures Part 4

Good results usually can be obtained in freezing bull s.e.m.e.n if care is taken in collecting, diluting and processing the s.e.m.e.n. Occasionally the s.e.m.e.n from certain bulls will not withstand freezing well. The reason for this is not understood at present. However, carefully following the directions and suggestions given below will usually produce satisfactory results with s.e.m.e.n samples that are of good quality at the start.

Experience in the field has shown that fertility results with frozen s.e.m.e.n are usually slightly lower during the first few months than with liquid s.e.m.e.n stored at 5 C. (41 F.). Most units that have worked with frozen s.e.m.e.n over a period of a few months are able to improve and do get fertility results as good as, or better than, obtained in their liquid s.e.m.e.n program.

=Collection of the s.e.m.e.n.= In order to obtain the best possible s.e.m.e.n for freezing, care and cleanliness should be exercised in making the collection. The artificial v.a.g.i.n.a, and the gla.s.sware used should be clean and dry. The underline of the bull should also be clean and dry.

The bull should be restrained near the teaser cow for a minute or two prior to collection in order to excite the flow of secretions prior to e.j.a.c.u.l.a.t.i.o.n. Allowing the bull to mount the teaser once without serving the artificial v.a.g.i.n.a is a good practice to use in properly stimulating the bull before collection of the s.e.m.e.n.

If the bull has not been used for three or four days, the collection of a second e.j.a.c.u.l.a.t.e for freezing may be advisable. The second e.j.a.c.u.l.a.t.e seems to withstand freezing better than the first in many instances. A clean, dry artificial v.a.g.i.n.a should be used for each e.j.a.c.u.l.a.t.e collected. Repeated collections in the same artificial v.a.g.i.n.a may result in contamination of the s.e.m.e.n with bacteria, lubricating jelly and minute particles of dirt. The s.e.m.e.n sample should be protected from contamination and from sudden temperature drops (cold shock).

=Preparation of extender.= A suitable egg yolk-citrate extender for freezing bull s.e.m.e.n can be prepared by the following procedure. One part egg yolk (free of egg white and the membrane surrounding the yolk) is mixed with 4 parts 2.4 to 2.9 percent sodium citrate dihydrate solution.

The citrate is prepared with distilled water and then boiled or autoclaved. The citrate solution should be cooled before it is mixed with the egg yolk. After the egg and citrate are mixed, 1000 units of penicillin and 1000 micrograms of streptomycin are added per milliliter of extender. Sulfanilamide should not be added. This extender can be prepared 12 to 24 hours before use if it is stored at refrigerator temperature. The portion of the extender needed for the original dilution of the s.e.m.e.n should be warmed to room temperature before it is mixed with the s.e.m.e.n.

=Dilution after collection.= As soon as possible after collection, the s.e.m.e.n sample should be diluted with the extender. The extender must be at the same temperature as the s.e.m.e.n (room temperature) when the two are mixed together. At this time the s.e.m.e.n can be partially diluted (1 part s.e.m.e.n to 4 parts of extender) or diluted to a sperm concentration twice the final desired concentration (later in adding the glycerol for freezing, the s.e.m.e.n is diluted further with an equal volume of glycerol containing extender). The diluted s.e.m.e.n is slowly cooled (1-1/2 to 2-1/2 hours) to 5 C. (41 F.). Some units using frozen s.e.m.e.n now allow the s.e.m.e.n to stand at 5 C. for 5 to 6 hours before glycerolization to allow the antibiotics to be more effective against any vibrio fetus organisms that may be present. This step is taken because it has been shown that glycerol inhibits the effectiveness of the antibiotics.[6] After cooling, s.e.m.e.n can be further diluted to twice the desired sperm concentration if that were not done at the start. (Caution: Be sure s.e.m.e.n and diluent are at the same temperature.)

=Adding the glycerol.= The glycerol solution is prepared by adding 14 volumes of glycerol (reagent grade) to 86 volumes of yolk-citrate diluent (same as yolk-citrate used for original dilution). This solution may be added dropwise with constant gentle mixing to the already diluted s.e.m.e.n, or one-third at a time at 10-minute intervals with gentle mixing during each addition. Either method should take about 20 to 30 minutes.

The total volume of glycerol-yolk-citrate solution added should be equal to the volume of the original diluted s.e.m.e.n. In this way a concentration of 7 percent glycerol is obtained in the final mixture that is to be frozen. Care must be taken to keep the temperature at 5 C. (41 F.) during the time the glycerol is being added. (A cold room is best for maintaining a temperature of 5 C., but with care the operation can be carried out at room temperature by using pans of ice water and a refrigerator.)

=Equilibration.= The results presented in this bulletin suggest that little or no time need be allowed after the glycerol is added before freezing. However, results obtained by other workers show improved fertility with at least 12 hours equilibration. Some units getting good fertility results with frozen s.e.m.e.n also are allowing the s.e.m.e.n to stand at 5 C. for 12 to 18 hours before freezing. After the s.e.m.e.n has equilibrated with the glycerol, 1-milliliter portions of the mixture are placed in 1.2- to 2-milliliter vials or ampules which are then sealed.

Ampuling can be done with an automatic syringe or pipette, provided a large gage needle is used. Also, it is important not to force the fluid mixture rapidly through the syringe or the sperm may be injured.

=Freezing.= The vials or ampules of diluted s.e.m.e.n are placed in a bath of isopropyl alcohol which has been cooled to 5 C. (41 F.). This bath can be a wide-mouth thermos bottle or an insulated container of almost any sort with a large opening at the top. The size needed depends on the number of ampules being frozen. Some sort of convenient tray for holding the ampules in an orderly fas.h.i.+on and enabling the samples to be completely submerged is desirable. A few ampules can be kept together easily by placing them in a polyethylene freezer bag that has had many small holes cut in it to let the alcohol of the bath contact the ampules. The ampules must be completely covered by the alcohol to insure uniform cooling.

The alcohol of the bath and the ampules of s.e.m.e.n are cooled by adding chipped or ground dry ice in sufficient amounts to lower the temperature of the bath 2 C. (3.6 F.) per minute from +5 to -20 C. From -20 down to -79 C., the rate of cooling can be doubled (4 C. or 7.2 F.).

Electrical equipment that regulates the cooling rate to the desired temperatures is available commercially, but the cost may be too high for some small operations. The samples should be held at -79 C. (-110 F.) until they are thawed. This can be done by using an alcohol bath and dry ice or by special mechanical refrigerating equipment. At no time prior to thawing should the samples be exposed to warmer temperatures.

=Thawing.= The ampules of frozen s.e.m.e.n can be thawed by removing them from the dry ice storage box and dropping them into a water bath at 5 C. (41 F.). Thawing temperatures up to body temperature, 38 C. (100 F.), can be used but extreme care must then be taken not to pa.s.s the s.e.m.e.n through a cold inseminating tube; for this would subject the sperm to cold shock. The s.e.m.e.n should be used for breeding within a few minutes after thawing.

LITERATURE CITED

[1] DAVENPORT, C. B. Effect of chemical and physical agents upon protoplasm. Macmillan and Co., New York. 1897.

[2] POLGE, C., and PARKES, A. S. Possibilities of long-term storage of spermatozoa at low temperatures. Anim. Breeding Abs.

=20=:1-5. 1952.

[3] EMMENS, C. W., and BLACKSHAW, A. W. The low temperature storage of ram, bull, and rabbit spermatozoa. Austral. Vet. Jour.

=26=:226. 1950.

[4] SMITH, AUDREY W. Effects of low temperatures on living cells and tissues. In biological applications of freezing and drying.

Ed. R. J. C. Harris. Academic Press, Inc., New York, 1954.

[5] EMMENS, C. W., and BLACKSHAW, A. W. Artificial insemination.

Physiol. Rev. =36=:277-306. 1956.

[6] Proceedings of the National a.s.sociation of Artificial Breeders, 1953, 1954, and 1955.

[7] Proceedings of the American Dairy Science a.s.sociation, 1953, 1954, and 1955. Published in the June issue of the Journal of Dairy Science for each year.

[8] BARKER, C. A. V. Low temperature preservation of bovine epididymal spermatozoa. Canad. Jour. Comp. Med. =18=:390-393.

1954.

[9] SAROFF, JACK, and MIXNER, J. P. The relations.h.i.+p of egg yolk and glycerol content of diluters and glycerol equilibration time to survival of bull spermatozoa after low temperature freezing.

Jour. Dairy Sci. =38=:292-297. 1955.

[10] CRAGLE, R G., MYERS, R. M., WAUGH, R. K., HUNTER, J. S., and ANDERSON, R. L. The effects of various levels of sodium citrate, glycerol, and equilibration time on survival of bovine spermatozoa after storage at -79 C. Jour. Dairy Sci.

=38=:508-514. 1955.

[11] BRATTON, R. W., FOOTE, R. H., and CRUTHERS, JOAN C.

Preliminary fertility results with frozen bovine spermatozoa.

Jour. Dairy Sci. =38=:40-46. 1955.

[12] HAFS, H. D., and ELLIOTT, F. I. The effects of methods of adding egg yolk and monosaccharides on the survival of frozen bull spermatozoa. Jour. Dairy Sci. =38=:811-815. 1955.

[13] MILLER, W. J., and VANDEMARK, N. L. The influence of glycerol level, various temperature aspects, and certain other factors on the survival of bull spermatozoa at sub-zero temperatures. Jour. Dairy Sci. =37=:45-51. 1954.