Part 4

A marked increase in the time required for the sperm to reduce methylene blue occurred when the glycerol was added (Table 17). This increase was greatest in the portions with glycerol alone and with glycerol and glucose. The time increase was less pronounced in the presence of the three pentose sugars used. Following equilibration, the samples regained the ability to reduce methylene blue at a rate only slightly slower than when they were fresh. Freezing and storage of semen resulted in slower reduction of the methylene blue than was shown after equilibration with glycerol. Since freezing usually kills some of the sperm, a slowing of the reduction time after freezing would be expected.

Table 17.--Effect of Freezing Procedures on the Methylene-Blue Reduction Time of Bull Semen With and Without the Addition of Sugars[M]

(Average of 10 ejaculates)

====================================================================== Methylene-blue reduction time (minutes) ------------------------------------------------ Glycerol Glycerol Glycerol Glycerol Glycerol only and and and and glucose arabinose xylose rhamnose ---------------------------------------------------------------------- Fresh semen 5.2 5.2 5.2 5.2 5.2 After glycerolization 26.4 25.2 17.3 14.3 19.4 After 18 hours equilibration 7.4 6.5 6.4 5.3 6.2 Thawed immediately after freezing 11.5 10.5 9.4 9.0 9.4 Thawed 48 hours after freezing 14.3 10.2 11.3 10.1 9.5 ----------------------------------------------------------------------

[M] Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent.

PRACTICAL FREEZING PROCEDURE

Good results usually can be obtained in freezing bull semen if care is taken in collecting, diluting and processing the semen. Occasionally the semen 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 semen samples that are of good quality at the start.

Experience in the field has shown that fertility results with frozen semen are usually slightly lower during the first few months than with liquid semen stored at 5 deg. C. (41 deg. F.). Most units that have worked with frozen semen 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 semen program.

=Collection of the semen.= In order to obtain the best possible semen for freezing, care and cleanliness should be exercised in making the collection. The artificial vagina, and the glassware 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 ejaculation. Allowing the bull to mount the teaser once without serving the artificial vagina is a good practice to use in properly stimulating the bull before collection of the semen.

If the bull has not been used for three or four days, the collection of a second ejaculate for freezing may be advisable. The second ejaculate seems to withstand freezing better than the first in many instances. A clean, dry artificial vagina should be used for each ejaculate collected. Repeated collections in the same artificial vagina may result in contamination of the semen with bacteria, lubricating jelly and minute particles of dirt. The semen 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 semen 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 semen should be warmed to room temperature before it is mixed with the semen.

=Dilution after collection.= As soon as possible after collection, the semen sample should be diluted with the extender. The extender must be at the same temperature as the semen (room temperature) when the two are mixed together. At this time the semen can be partially diluted (1 part semen to 4 parts of extender) or diluted to a sperm concentration twice the final desired concentration (later in adding the glycerol for freezing, the semen is diluted further with an equal volume of glycerol containing extender). The diluted semen is slowly cooled (1-1/2 to 2-1/2 hours) to 5 deg. C. (41 deg. F.). Some units using frozen semen now allow the semen to stand at 5 deg. 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, semen can be further diluted to twice the desired sperm concentration if that were not done at the start. (Caution: Be sure semen 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 semen, 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 semen. 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 deg. C. (41 deg. F.) during the time the glycerol is being added. (A cold room is best for maintaining a temperature of 5 deg. 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 semen also are allowing the semen to stand at 5 deg. C. for 12 to 18 hours before freezing. After the semen 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 semen are placed in a bath of isopropyl alcohol which has been cooled to 5 deg. C. (41 deg. 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 fashion 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 semen are cooled by adding chipped or ground dry ice in sufficient amounts to lower the temperature of the bath 2 deg. C. (3.6 deg. F.) per minute from +5 deg. to -20 deg. C. From -20 deg. down to -79 deg. C., the rate of cooling can be doubled (4 deg. C. or 7.2 deg. 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 deg. C. (-110 deg. 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 semen can be thawed by removing them from the dry ice storage box and dropping them into a water bath at 5 deg. C. (41 deg. F.). Thawing temperatures up to body temperature, 38 deg. C. (100 deg. F.), can be used but extreme care must then be taken not to pass the semen through a cold inseminating tube; for this would subject the sperm to cold shock. The semen 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 Association of Artificial Breeders, 1953, 1954, and 1955.

[7] Proceedings of the American Dairy Science Association, 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 relationship 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 deg. 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.

TEMPERATURE CONVERSIONS

deg.C. deg.F.

+38 +100 +35 +95 +30 +86 +25 +77 +20 +68 +15 +59 +10 +50 +5 +41 0 +32 -5 +23 -10 +14 -15 +5 -18 0 -20 -4 -25 -13 -30 -22 -35 -31 -40 -40 -45 -49 -50 -58 -55 -67 -60 -76 -65 -85 -70 -94 -75 -103 -79 -110