Fig. 3Percent of motile sperm after freezing and thawing semen in diluents containing various levels of eggyolk and various percentages of sodium citrate(Fig. 3)
In another trial, a similar diluent (1:1 yolk to citrate with 1000 units of penicillin and 5000 units of streptomycin) was prepared and stored in the freezer compartment of a refrigerator at -15° C. Upon thawing, it was whitish in color and more viscous than freshly prepared diluent. Except for the fact that the viscosity seemed to reduce the rate of sperm motility, this frozen diluent stored for 65 days compared favorably with freshly prepared diluent for freezing semen.
Other diluents.Without the protective action of egg yolk or milk, few bull sperm will survive freezing. Several diluents were compared on a limited scale for freezing bull sperm. The results of these trials are compiled inTable 5. In this trial the yolk-citrate extender served best in maintaining sperm motility during freezing. Yolk-phosphate and homogenized whole milk were slightly less protective and yolk-saline seemed to furnish the least protection to sperm during freezing.
A number of investigations in other laboratories have now proven that milk can be used as effectively as the yolk-citrate diluent for freezing bull sperm.[6],[7]
[E]Stored at 5° C. for 7 hours after thawing.
[E]Stored at 5° C. for 7 hours after thawing.
The first trials by the British at freezing bull semen were made with samples containing many millions of sperm cells. In routine artificial breeding, it is common to add extenders to semen so that one milliliter of diluted semen may contain only 10 million living sperm cells. (This number still insures optimal fertility.) Frequently the addition of 100 or more parts of the yolk extender to each part of the original semen sample is possible without reducing the sperm numbers below 10 million per milliliter. No one knew if this process of dilution would affect the resistance of bull sperm to freezing. The effect of various rates of dilution on the freezability of bull sperm was tested with 10 semen samples. The results, presented inTable 6, show that the numbers of sperm between 10 and 90 million per milliliter did not influence the percentage of sperm that survived freezing.
In a later trial it was found that sperm survival was slightly better at lower dilution rates than in the same samples frozen following dilution to 15 million sperm per milliliter. However, field trials with frozen semen carried out by others, using sperm numbers as low as 15 million per milliliter of semen inseminated or even lower, have been highly satisfactory.[11],[12]
During the early studies in the Illinois laboratory, the effects of glycerol level were also tested.[13]These effects are discussed in the section onglycerol additionsbeginning onpage 17.
Effect of further dilution and refreezing after the initial freezing.Under some circumstances it might be advantageous to freeze semenwith a high concentration of sperm cells and then extend it further after thawing. With such a procedure less storage space is needed than when dilution is carried to the maximum before freezing. Two experiments were conducted to test the effects of dilution and storage at 5° C. and dilution and refreezing following an initial freezing of concentrated samples.
(Average of 10 ejaculates)
[F]Mean initial motility of sperm before freezing was 55 percent.
[F]Mean initial motility of sperm before freezing was 55 percent.
Four semen samples were split and extended at rates of 1:1 (semen to extender) and 1:10. These were frozen, then thawed and halved. One half was further extended to a level of 15 million sperm per milliliter; the sperm numbers in the other remained unchanged. Each of these halves was split again, and one portion of each was stored at 5° C. for 3 to 7 hours. The other two portions were refrozen.
[G]Stored at 5° C. for 3 to 7 hours after first thawing.[H]Refrozen following first thawing.
[G]Stored at 5° C. for 3 to 7 hours after first thawing.
[H]Refrozen following first thawing.
[I]The control differed from the 0-glycerol treatment in that no additional citrate or glycerol solution was added.
[I]The control differed from the 0-glycerol treatment in that no additional citrate or glycerol solution was added.
A similar trial was carried out with seven samples; one portion was diluted 1:9; the other was extended at the outset to 15 million sperm per milliliter. Results for both tests are summarized inTable 7.
FromTable 7it can be seen that refreezing following an initial freezing further reduced the number of surviving sperm. The second freezing was more detrimental to the portion of the samples extended to 15 million sperm per milliliter than to the portion that was refrozen at a higher sperm concentration. The percentage of motile sperm remained fairly high in the portions that were diluted to 15 million sperm and stored at 5° C. However, in all cases, survival was best in the samples at the lower dilution levels.
When the British procedure for freezing bull semen was first tried in this country, many of the refinements of the technique still had not been defined. It was known that glycerol worked well in protecting sperm during freezing. The effects of glycerol on sperm at 5° C., the appropriate levels to use in freezing, and the manner of adding it were not well established. Therefore, a number of trials were conducted in an attempt to establish the best procedures.
Effect of glycerol on sperm survival at 5° C.Since early work indicated the need for adding glycerol to diluted semen in order to protect the sperm during freezing, it was considered important to determine the levels of glycerol that sperm would tolerate at 5° C. Ten semen samples were extended 1:9 (semen to diluent) in a 1:1 yolk-citrate diluent (yolk to 2.9 percent sodium citrate dihydrate). Each sample was then split into 6 portions and an equal volume of citrate solution containing glycerol was added slowly to each to bring the glycerol in the final mixture to 0, 5, 10, 20, or 30 percent (by volume). These samples were stored at 5° C. and examined for motile sperm after 1, 3, and 7 days. The effects of glycerol levels on the percentage of sperm surviving and the rate (or speed) of their forward motion (0 = no forward motion; 4 = extremely rapid progressive motility) are presented inTable 8.
The percentage of motile sperm decreased slightly at the higher levels of glycerol. The most noticeable effect of the increase in glycerol level was the reduction in the rate of forward motion of the sperm. At the 30-percent level, the sperm moved slowly and could be seen torotate as they moved forward. Some samples were checked after slowly bringing the diluent up to a level of 40 percent glycerol; the sperm seemed to be immobilized completely in this solution.
Glycerol levels for freezing semen.The British procedure called for the use of 10 percent glycerol in the final mixture of semen and extender prior to freezing. Yet, as shown inTable 6, in our laboratory 5 percent glycerol resulted in the survival of a higher percentage of sperm than did 10 or 15 percent. In order to define more clearly the optimum glycerol level, several ejaculates of semen were subsampled and portions were frozen after the addition of yolk-citrate extender and glycerol in varying quantities. FromTable 9it can be seen that glycerol levels of 6 and 8 percent in the final mixture resulted in maximum sperm survival during freezing. These results were confirmed in tests on the survival of sperm at 5° C. storage for 3 days following freezing and thawing with varying glycerol levels (seeTable 10).
The results shown inTables 9and10were confirmed also in later experiments. Thirty-six samples were subjected to various levels of glycerol and no significant difference in freezability was found between 6 and 8 percent. Based on these findings, a glycerol level of 7 percent was adopted for use in all experiments described in this bulletin, unless otherwise indicated. Results in a number of other laboratories have agreed with our findings regarding the use of approximately 7 percent glycerol with the yolk-citrate diluent.[5],[6],[7],[9],[10]With milk as the extender, 10 to 13 percent glycerol has been preferred by some.[5],[6],[7]
(Average of 13 ejaculates)
(Average of 12 ejaculates)
Rate, temperature, and method of adding glycerol.Closely associated with the question of how much glycerol should be added is that of how the additions should be made. Originally it was believed that the glycerol should be added in stages so that changes would occur gradually. However, there would be a saving in time if the entire amount could be added at once. Also, if the glycerol addition could be made soon after the dilution with egg yolk-citrate extender at room temperature, time would be gained in processing the semen for use. Since agingin vitrois known to reduce the fertilizing ability of sperm, every effort should be made to keep the processing time at a minimum. The results of an experiment involving these items, along with that of how much time should be allowed after the additions before freezing (equilibration time), are presented inTable 11. One can see that sperm survived freezing better when the diluted semen was cooled to 4.5° C. before the glycerol was added. The survival at 10° and 15.5° C. wasreduced with each rise in temperature. Thus, it appears that cooling to refrigerator temperature (4-5° C.) before adding the glycerol should be a part of the routine procedure.
A comparison of the results from adding the glycerol in 5, 3, and 1 equal portions is given also inTable 11. Little difference in survival during freezing was noted between the three rates of addition. Using 3 equal additions resulted in slightly better results, but the advantage was not statistically significant. While little difference was evident from adding the glycerol in 3 portions as compared to 1, many still use 3 additions in the hope of obtaining a slightly better sperm survival. In fact, some have gone to a procedure of adding the glycerol dropwise with constant gentle agitation. This method has not been tested in this laboratory.
Allowing sperm to equilibrate with the glycerol.Allowing sperm to stand in the presence of glycerol is considered by some to be necessary in order that the glycerol penetrate the sperm heads before freezing. From the first successful attempts at freezing bull sperm came the practice of allowing 12 to 20 hours for this process of equilibration. A long equilibration time results in aging the sperm. Data from a number of sources indicate that a drop of approximately 5 percent in fertility in the field occurs with each 24 hours of aging in the test tube. Thus it would seem desirable to reduce the equilibration time to a minimum commensurate with good freezability in order to reduce the effects of aging (at 5° C.). Results of attempting to reduce equilibration time are given inTable 11. At 4.5° C., little variation in motility following freezing and thawing was found after equilibration times of 2, 6, and 18 hours. At the higher temperatures of 10° and 15.5° C., the shortest equilibration time—2 hours—was slightly more detrimental with the differences significant at the 5-percent level at 15.5° C. For all temperatures combined, 6 hours was significantly better than 2 or 18 hours.
Sugar additions and equilibration time.Early in their experiences in freezing semen, the Australian workers found a short equilibration time—30 minutes—to be satisfactory if sugars were added to the diluent.[5]This protective action of sugars during the equilibration period was confirmed in our investigations. The results of one phase of this study are shown inTable 12. From these data it can be seen that the presence of glucose or rhamnose at a level of 1.25 percent improved sperm survival during the period of equilibration. In another trial these sugars and two others, arabinose and xylose, were tested for their protective action in freezing semen. The percentages of surviving sperm remaining after the various steps in the freezing procedure with and without the presence of these sugars are shown inTable 13.
[J]Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent.
[J]Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent.
Three of the sugars—glucose, arabinose, and rhamnose—protected the sperm during equilibration and freezing. Xylose was less effective, but its addition resulted in slightly better sperm survival than glycerol alone. It was found also that the methylene-blue reduction time (metabolic test for semen quality) was faster in samples to which the sugars had been added—after glycerolization, after equilibration, and after freezing the samples. This is confirming evidence for the presence of more living and actively metabolizing sperm in the portions to which sugars had been added.
(Average of 10 ejaculates)
[K]Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent.
[K]Glycerol level in the final frozen mixture was 7 percent. Sugars were added to a level of 1.25 percent.
Substitutes for glycerol.Since glycerol was so effective in protecting sperm during freezing, many have assumed that related compounds might be even better. Several compounds, some related to glycerol and some not, have been tried as substitutes for glycerol in the freezing procedure. They include ethylene glycol, propylene glycol, trimethylene glycol, mannitol, sorbitol, dextrans, and seminal-plasma proteins. None of these materials has been as effective as glycerol in protecting sperm during freezing. In fact, several of the materials proved to be injurious to sperm prior to attempts to freeze the samples. While the work in our laboratory with these substances as glycerol substitutes was by no means finally conclusive, because of the many possible interactions of experimental conditions, sufficient data were gathered to lead us to abandon further study until greater promise of success might be evident.
Effect of freezing rate on sperm survival.Reports by one group of British workers in early trials on freezing bull semen indicated that the rate of cooling in freezing should not exceed 2° C. per minute between +5° and -15° C., although below -15° C. the rate could be faster. Another group expressed the view that semen could be plunged into dry ice at -79° C. after it had been cooled to -15° C. To clarify this part of the freezing procedure, 11 samples of semen were subdivided and portions of each were frozen at rates of 0.25°, 0.5°, 1.0°, 2.0°, and 4.0° C. drop per minute between +5° and -20° C. and then twice these rates between -20° and -79° C. Vials of each ejaculate at +5° C. were also plunged directly into an alcohol bath at -79° C. The samples which were cooled at the rates of 0.25°, 0.5°, 1.0°, 2.0°, and 4.0° C. per minute had the following percentages of motile sperm after thawing: 30, 40, 46, 44, and 44. A mean of 32 percent of the sperm in the samples that were plunged directly into an alcohol bath at -79° C. were motile after thawing. There were no statistically significant differences among the samples frozen at 1.0°, 2.0° or 4.0° C. per minute. All of the others had significantly lower survival rates. Thus, it is obvious that too slow a cooling rate and plunging the samples directly into a -79° C. bath from a temperature of +5° C. cause greater harm to the sperm than cooling at a rate between 1.0° and 4.0° C. per minute.
Some investigators have suggested that rapid cooling below -20° C. is not detrimental to frozen semen. This idea was tested in conjunction with other experiments. Twenty-five samples cooled slowly (2°C. per minute to -28° C., then 4° C. per minute to -79° C.) showed 62 percent sperm survival compared with only 45 percent when cooled rapidly below -28° C. (2° C. per minute to -28° C. then plunged into bath at -79° C.). Thus, rapid cooling was detrimental even after the critical temperature range of +5° C. to -20° C. had been passed.
Fig. 4Cooling rates of diluted semen samples in plastic vials and in glassampules(Fig. 4)
Rate of cooling in plastic and in glass.Plastic vials do not conduct the cold as rapidly as glass ampules do. The temperature in both glass and plastic containers tends to lag behind the change in the bath in which they are immersed as is shown inFigure 4.
Temperatures in the immersion bath were recorded in a 2-milliliter glass ampule containing 1 milliliter diluted semen and in an 8-milliliter plastic vial containing 2.5 milliliters of diluted semen. A second plastic vial and glass ampule filled to capacity with diluted semen showed a cooling rate almost identical to that shown inFigure 4. It was obvious from the comparison that samples in the plastic vials cooled slower than those in glass and that the volume of semen (at least the small volumes used) in the vials had little effect on the rate of cooling. In another experiment, it was shown that the volume of diluted semen in the ampule to be frozen (0.2, 1.0 or 5.0 ml.) had little or no effect on the survival of the sperm.
In freezing and storing bull sperm, an alcohol bath containing dry ice at a temperature of -79° C. has been used as a cooling agent. In many areas, the availability of dry ice is limited and the cost is rather high. Mechanical means are available for obtaining temperatures as low as, or lower than, -79° C. but for the most part they are expensive. If warmer temperatures were suitable for storing frozen semen, the ordinary deep-freeze, which operates at -15° to -25° C., might be used.
Storage at temperatures from -23° to -79° C.In testing the effects of storage temperatures on the survival of frozen bull sperm (in a diluent containing 7 percent glycerol), 9 ejaculates were frozen and kept at -23°, -37°, -51°, -65°, and -79° C. The desired temperatures were maintained by dropping pieces of dry ice into ethyl alcohol baths as needed. Samples were thawed after 1 hour, 1 day, 3 days, and 5 days. After 1 hour, the samples maintained at the various temperatures exhibited approximately equal motility (Fig. 5).
Fig 5Effect of freezing and storing bull sperm at various temperatures on the sperm motility at thawing(average of 9 ejaculates)(Fig. 5)
At the end of 1 day, samples stored at -79° C. exhibited approximately the same motility as did similar samples stored for 1 hour. The samples stored at -65° C. had declined slightly in motility and those maintained at -51° C. had only one-third the motility which they had displayed at 1 hour. The samples at -23° and -37° C. exhibited practically no motility after 1 day in storage. After 5 days, only 3 of the 8 ejaculates stored at -51° C. showed motility upon thawing. Apparently detrimental changes take place more rapidly when the samples are stored at temperatures warmer than -65° C. The nature of these changes has not been determined. Reports from other laboratories indicate that storage temperatures much lower than -79° C. are just as satisfactory as -79° C.
No tests of the effects of storage at -79° C. for periods longer than 51 days have been conducted in this laboratory. Portions of 12 ejaculates were frozen and stored at -79° C. for various periods. One portion of each of these was examined on the second, ninth, 16th and 51st day of storage. The percent of motile sperm and rate of motility at each of these examinations were as follows:
The average prefreezing motility percentage for the above samples was 58, with an average rate of motility of 2.9. It is apparent from these results that the loss in motility was greatest due to the initial freezing, and after that the drop was most pronounced during the first 16 days of storage.
The British and the Australians have both reported the successful maintenance of fertility in frozen semen stored at -79° C. for over two years.[5]
Use of higher glycerol levels and a -20° C. storage temperature.In 1953, a report from Arkansas suggested that warmer storage temperatures could be used if a high percentage of glycerol were included in the freezing mixture.[7]To test the effectiveness of various glycerol levels on protecting sperm stored at deep-freeze temperatures, glycerol levels of 3.5, 5.5, 7.5, and 9.5 percent were used with portions of 4 semen samples. Survival in the portions frozen and stored at -20° C. was poor compared with the portions reduced and held at -79° C. In a second experiment, 4 samples were subdivided and frozen with a final concentration of 7, 11, 15, and 19 percent glycerol in the semen-diluent mixture. In this trial, poor results were obtained at -20° C.except that glycerol at a level of 19 percent protected the sperm more effectively than at lower levels. Maximal survival at -79° C. was obtained at the 7-percent glycerol level. A final trial was run, using glycerol levels of 7, 11, 15, 19, 23, 27, and 31 percent. The percentages of motile sperm present after storage at -79° C. and -20° C. are shown inTable 14.
(Average of 8 ejaculates)
While survival was fair over a short period of time with 19 percent glycerol at -20° C., deterioration was rapid during storage. After 18 hours of storage, the samples at -20° C. (19 percent glycerol) contained only one half as many motile sperm as were still present in the samples at -79° C. (7 percent glycerol). After 42 hours of storage, the best samples at -20° C. contained only one-third the number of motile sperm still present in the samples stored at -79° C. These trials leave little doubt that under the present system of freezing and storing, storage at ordinary deep-freeze temperatures is far inferior to storage at dry-ice temperatures.
The importance of carefully controlled cooling and storage has been emphasized in the foregoing sections. The need for controlling thawing rates and the temperature of thawing was not clearly defined in the early work on freezing bull semen. The British used a thawing temperature of 40° C., which was satisfactory. If there is a need to hold the semen for a time after thawing, then a lower thawing temperature might be more desirable so that cooling again will not be necessary.
Comparison of thawing temperatures of 5° C. and 38° C.The effects of thawing at temperatures of 38° (body temperature) and 5° C. (refrigerator temperature) were investigated. The first trial involved thawing as rapidly as possible by dropping glass ampules of frozen semen into water baths at the two temperatures. The frozen semen samples contained glycerol levels of 4, 6, 8, and 10 percent. The mean percentages of motile sperm found after thawing thirteen diluted semen samples treated in this manner are shown inFigure 6.