Fig. 6Effect of glycerol percentage and thawing temperature on spermmotility after freezing and thawing(Fig. 6)
The 5° C. thawing temperature resulted in a higher percentage of sperm survival at all the glycerol levels than 38° C., with the difference in favor of 5° C. becoming greater as the glycerol level increased. The reason for the interaction between glycerol level and thawing temperature is not known. It may be that the presence of the higher levels of glycerol at 38° C. brought about harmful metabolic activity. The difference in survival of sperm in semen thawed at 5° C. and at 38° C. continued during storage at 5° C. (Table 15). It was also evident that the interaction between glycerol level and thawing temperature continued during storage (Fig. 7).
Fig. 7Effect of thawing temperature on sperm motility during storage at 5°C. following freezing and thawing(Fig. 7)
(Average of 13 ejaculates)
It is obvious that motility falls off rapidly after the semen is thawed. In a field trial in which the initial intent was to test the effect of glycerol levels on fertility of frozen semen, the semen was thawed in the morning and used during the same day. Survival of the sperm with 4 percent glycerol was so poor that only a few breedings were made with these samples. Even at 7 and 10 percent, the fertility results were much lower than with semen that had not been subjected to freezing. At that time it was felt that thawing the samples and using them throughout the day may have caused the low fertility results. Sincethen, a large-scale experiment by Cornell University investigators, in cooperation with the New York Artificial Breeders’ Cooperative, has shown definitely that thawing should be delayed until a few minutes prior to breeding.[11]If the semen is used immediately, a thawing temperature of either 5° or 38° C. appears to be suitable. However, there is less danger of cold shock due to recooling if 5° C is used.
Thawing rate in plastic and in glass.Glass ampules transmit cold or heat more readily than plastic ones. The temperature rise is rapid in both glass and plastic when samples are taken from the storage box at -79° C. and placed in water at 5° C. However, complete thawing occurs more rapidly in glass than in plastic ampules. The changes in temperature that occurred when glass and plastic ampules were thawed in a water bath at 5° C. are shown inFigure 8. The initial temperature rise for the first minute or two was about the same, then the rate of warming in the plastic slowed and actual melting of the frozen sample occurred a little over a minute later in the plastic than it did in the glass. Both were thawed in less than four minutes.
Fig. 8Warming rates of diluted semen samples in plastic vials and in glassampules(Fig. 8)
By finding how methods of handling affect the sperm cells, one can sometimes improve the procedures to avoid harmful effects. Some attempts have been made in this laboratory to determine the effects of the freezing procedures on the metabolic activity of bull spermatozoa. These investigations have been limited in scope, involving the measurement of oxygen-consumption and estimates of sperm motility during and after incubation at 37° C. in a Warburg apparatus.
Effect of glycerol additions on oxygen uptake of diluted semen at 37° C.The effect of adding glycerol to diluted semen on oxygen consumption of the sperm was tested in a Warburg apparatus, using semen diluted with an extender consisting of one part egg yolk and one part 2.9 percent sodium citrate dihydrate. The yolk-citrate extender was added to the semen at a rate which brought the sperm concentration in 0.5 milliliter to 200 million to 500 million. An exact count was used to calculate the oxygen uptake per 108sperm per hour (ZO2).
Fig. 9Effect of glycerol additions on oxygen consumption of sperm at 37° C.(Fig. 9)
Glycerol in various percentages in 2.9 percent sodium citrate dihydrate solution was placed in the sidearm of the Warburg flasks.The diluted semen was held in the main compartment. After a 60-minute preliminary run, in which the rate of oxygen uptake of the sperm in yolk-citrate diluent was determined, the contents of the sidearm were tipped into the main compartment. The resulting glycerol percentages after mixing the sidearm and main compartment contents were 0, 4, 8, and 12 percent. Ten samples of semen were subsampled and the oxygen uptake of each was determined at all four levels of glycerol.
Oxygen uptake was increasingly stimulated during the first 20-minute interval by each increase in the amount of glycerol added (Fig. 9). After the first 20 minutes, the rate of oxygen utilization decreased at the two higher levels of glycerol but persisted at 4 percent. The rate of oxygen consumption for the first 20-minute period at the 4-percent glycerol level was 130 percent that of the control to which only sodium citrate had been added. At 8 and 12 percent the values for the period were 144 and 192 percent, respectively, of the control rate.
Fig. 10Effect of glycerol and glycerol-plus-catalase additions on sperm motility during incubation at 37° C.(Fig. 10)
With each increase in glycerol level, motility was reduced during the incubation period. This is shown inFigure 10along with the effect on motility of adding catalase, which is discussed in the next section.
Effect of glycerol-plus-catalase on oxygen uptake of diluted semen.Certain bacteria have been shown to break glycerol down, forming hydrogen peroxide as follows:
Glycerol + oxygen → lactic acid + hydrogen peroxide.
Hydrogen peroxide is known to be detrimental to sperm. The addition of glycerol to diluted semen first increased oxygen uptake and then reduced it. Since a reduction in sperm survival followed, some harmful action must have taken place with the addition of glycerol at 37° C. To test whether this action could be due to the release of hydrogen peroxide as occurs in certain bacteria, glycerol with catalase—the enzyme which breaks down hydrogen peroxide—was added to a portion of 8 diluted semen samples and the oxygen uptake was recorded. Comparison of the resulting oxygen uptake with glycerol and with glycerol plus catalase is shown inFigure 11.
Fig. 11Effect of additions of glycerol-plus-catalase on oxygen consumption of sperm at 37° C.(Fig. 11)
Oxygen consumption was increased by the presence of addedcatalase at all glycerol levels and in the control. Sperm survival during the 3-hour period at 37° C. also was improved by the presence of catalase (Fig. 10). However, the general trend in oxygen consumption produced by the addition of glycerol was not changed greatly. The higher levels of glycerol still stimulated oxygen uptake during the first 20-minute period after the additions and then slowed the rate of oxygen utilization. The rate of utilization was generally higher during the test period in the presence of catalase than without added catalase. It appeared that a part of the harmful effect of glycerol might be due to the formation of hydrogen peroxide. Still, the detrimental effects of the higher levels of glycerol were not completely removed.
(Average of 5 ejaculates)
[L]Average of 3 ejaculates.
[L]Average of 3 ejaculates.
Effect of freezing procedures on oxygen utilization by sperm.Limited data have been obtained on the effects of some of the freezing procedures on the oxygen utilization of bull sperm. The results obtained in these experiments confirmed the earlier findings that tipping glycerol directly into the diluted semen at 37° C. caused an increase in oxygen consumption (Table 16). All other steps in the freezing procedure had little effect on oxygen consumption by the sperm. Except where glycerol was added during the determination, the rate of oxygen utilization was lower the second hour than during the first. The oxygen uptake of semen that had been frozen and thawed seemed to drop faster than that of unfrozen samples.
Effect of freezing procedures on methylene-blue reduction time.The methylene-blue reduction test has been used as a means of measuring semen quality and is dependent on the metabolic activity of thesperm. The effects of various freezing procedures on the ability of samples to decolorize methylene blue were determined with 10 semen samples. Sperm numbers were standardized to 300 × 106cells per milliliter and the time required for these cells to reduce a 1:40,000 solution of methylene blue was determined on freshly diluted semen, after the addition of glycerol, after equilibration, and after freezing and thawing. Portions of each diluted sample were tested at these stages of the procedure with glycerol alone added and with glycerol and various sugars added.
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.
(Average of 10 ejaculates)
[M]Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent.
[M]Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent.
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° C. (41° 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 andthen 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 (11⁄2to 21⁄2hours) to 5° C. (41° F.). Some units using frozen semen now allow the semen 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, 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° 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 thatlittle 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° 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° 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 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° 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 semen 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 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.
[1]Davenport, C. B.Effect of chemical and physical agents upon protoplasm. Macmillan and Co., New York. 1897.
[1]Davenport, C. B.Effect of chemical and physical agents upon protoplasm. Macmillan and Co., New York. 1897.
[2]Polge, C., andParkes, A. S.Possibilities of long-term storage of spermatozoa at low temperatures. Anim. Breeding Abs.20:1-5. 1952.
[2]Polge, C., andParkes, A. S.Possibilities of long-term storage of spermatozoa at low temperatures. Anim. Breeding Abs.20:1-5. 1952.
[3]Emmens, C. W., andBlackshaw, A. W.The low temperature storage of ram, bull, and rabbit spermatozoa. Austral. Vet. Jour.26:226. 1950.
[3]Emmens, C. W., andBlackshaw, 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.
[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., andBlackshaw, A. W.Artificial insemination. Physiol. Rev.36:277-306. 1956.
[5]Emmens, C. W., andBlackshaw, A. W.Artificial insemination. Physiol. Rev.36:277-306. 1956.
[6]Proceedings of the National Association of Artificial Breeders, 1953, 1954, and 1955.
[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.
[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.
[8]Barker, C. A. V.Low temperature preservation of bovine epididymal spermatozoa. Canad. Jour. Comp. Med.18:390-393. 1954.
[9]Saroff, Jack, andMixner, 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.
[9]Saroff, Jack, andMixner, 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., andAnderson, 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.
[10]Cragle, R G., Myers, R. M., Waugh, R. K., Hunter, J. S., andAnderson, 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., andCruthers, Joan C.Preliminary fertility results with frozen bovine spermatozoa. Jour. Dairy Sci.38:40-46. 1955.
[11]Bratton, R. W., Foote, R. H., andCruthers, Joan C.Preliminary fertility results with frozen bovine spermatozoa. Jour. Dairy Sci.38:40-46. 1955.
[12]Hafs, H. D., andElliott, 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.
[12]Hafs, H. D., andElliott, 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., andVanDemark, 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.
[13]Miller, W. J., andVanDemark, 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.
Transcriber's Notes:The original text has not been modified, except that some minor typographical errors have been corrected silently.Lettered footnotes (with anchors [A], [B], etc.), explaining the text, have been moved to directly below the paragraph or table they refer to. Numbered footnotes ([1], [2], etc.) refer to references, that are listed towards the end of the text.Where a single footnote is referenced more than once, the backwards link to the anchor has not been implemented.Back totopof document.
Transcriber's Notes:
The original text has not been modified, except that some minor typographical errors have been corrected silently.
Lettered footnotes (with anchors [A], [B], etc.), explaining the text, have been moved to directly below the paragraph or table they refer to. Numbered footnotes ([1], [2], etc.) refer to references, that are listed towards the end of the text.
Where a single footnote is referenced more than once, the backwards link to the anchor has not been implemented.
Back totopof document.