Part 3

====================================================================== Sperm motility (percent) ------------------------------------------------- Stage of Glycerol Glycerol Glycerol Glycerol Glycerol observation only and and and and glucose arabinose xylose rhamnose ---------------------------------------------------------------------- Fresh diluted semen 63 63 63 63 63 After glycerolization 54 55 54 57 60 After 18 hours equilibration 39 43 44 39 46 After freezing to -79 deg. C. and immediate thawing 28 34 34 29 24 After 4 days at -79 deg. C. 23 26 26 25 27 -----------------------------------------------------------------------

[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.

FREEZING RATE

=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 deg. C. per minute between +5 deg. and -15 deg. C., although below -15 deg. C. the rate could be faster. Another group expressed the view that semen could be plunged into dry ice at -79 deg. C. after it had been cooled to -15 deg. 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 deg., 0.5 deg., 1.0 deg., 2.0 deg., and 4.0 deg. C. drop per minute between +5 deg. and -20 deg. C. and then twice these rates between -20 deg. and -79 deg. C. Vials of each ejaculate at +5 deg. C. were also plunged directly into an alcohol bath at -79 deg. C. The samples which were cooled at the rates of 0.25 deg., 0.5 deg., 1.0 deg., 2.0 deg., and 4.0 deg. 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 deg. C. were motile after thawing. There were no statistically significant differences among the samples frozen at 1.0 deg., 2.0 deg. or 4.0 deg. 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 deg. C. bath from a temperature of +5 deg. C. cause greater harm to the sperm than cooling at a rate between 1.0 deg. and 4.0 deg. C. per minute.

Some investigators have suggested that rapid cooling below -20 deg. C. is not detrimental to frozen semen. This idea was tested in conjunction with other experiments. Twenty-five samples cooled slowly (2 deg. C. per minute to -28 deg. C., then 4 deg. C. per minute to -79 deg. C.) showed 62 percent sperm survival compared with only 45 percent when cooled rapidly below -28 deg. C. (2 deg. C. per minute to -28 deg. C. then plunged into bath at -79 deg. C.). Thus, rapid cooling was detrimental even after the critical temperature range of +5 deg. C. to -20 deg. C. had been passed.

=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 in Figure 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 in Figure 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.

STORAGE TEMPERATURE

In freezing and storing bull sperm, an alcohol bath containing dry ice at a temperature of -79 deg. 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 deg. 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 deg. to -25 deg. C., might be used.

=Storage at temperatures from -23 deg. to -79 deg. 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 deg., -37 deg., -51 deg., -65 deg., and -79 deg. 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).

At the end of 1 day, samples stored at -79 deg. C. exhibited approximately the same motility as did similar samples stored for 1 hour. The samples stored at -65 deg. C. had declined slightly in motility and those maintained at -51 deg. C. had only one-third the motility which they had displayed at 1 hour. The samples at -23 deg. and -37 deg. C. exhibited practically no motility after 1 day in storage. After 5 days, only 3 of the 8 ejaculates stored at -51 deg. C. showed motility upon thawing. Apparently detrimental changes take place more rapidly when the samples are stored at temperatures warmer than -65 deg. C. The nature of these changes has not been determined. Reports from other laboratories indicate that storage temperatures much lower than -79 deg. C. are just as satisfactory as -79 deg. C.

No tests of the effects of storage at -79 deg. C. for periods longer than 51 days have been conducted in this laboratory. Portions of 12 ejaculates were frozen and stored at -79 deg. 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:

Day 2 9 16 51 Percent of motile sperm 49 46 40 38 Rate of motility 2.5 2.3 2.2 2.2

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 deg. C. for over two years.[5]

=Use of higher glycerol levels and a -20 deg. 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 deg. C. was poor compared with the portions reduced and held at -79 deg. 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 deg. C. except that glycerol at a level of 19 percent protected the sperm more effectively than at lower levels. Maximal survival at -79 deg. 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 deg. C. and -20 deg. C. are shown in Table 14.

Table 14.--Effect of Glycerol Level and Storage Temperature on Freezability of Semen

(Average of 8 ejaculates)

================================================================ Storage Glycerol Sperm motility after storage (percent) temperature level -------------------------------------- ( deg.C.) (percent) 18 hours 42 hours ---------------------------------------------------------------- -79 7 61 61 -20 7 2 1 11 3 1 15 14 10 19 30 22 23 29 19 27 25 18 31 21 12 ----------------------------------------------------------------

While survival was fair over a short period of time with 19 percent glycerol at -20 deg. C., deterioration was rapid during storage. After 18 hours of storage, the samples at -20 deg. C. (19 percent glycerol) contained only one half as many motile sperm as were still present in the samples at -79 deg. C. (7 percent glycerol). After 42 hours of storage, the best samples at -20 deg. C. contained only one-third the number of motile sperm still present in the samples stored at -79 deg. 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.

THAWING

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 deg. 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 deg. C. and 38 deg. C.= The effects of thawing at temperatures of 38 deg. (body temperature) and 5 deg. 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 in Figure 6.

The 5 deg. C. thawing temperature resulted in a higher percentage of sperm survival at all the glycerol levels than 38 deg. C., with the difference in favor of 5 deg. 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 deg. C. brought about harmful metabolic activity. The difference in survival of sperm in semen thawed at 5 deg. C. and at 38 deg. C. continued during storage at 5 deg. C. (Table 15). It was also evident that the interaction between glycerol level and thawing temperature continued during storage (Fig. 7).

Table 15.--Effect of Glycerol Level, Thawing Temperature, and Storage at 5 deg. C. After Thawing on Sperm Motility (Average of 13 ejaculates)

===================================================================== Thawing Glycerol Sperm motility (percent) temperature level ------------------------------------------- ( deg. C.) (percent) Post- After storage at 5 deg. C. thawing ---------------------- Average 1 day 3 days --------------------------------------------------------------------- 38 4 28.5 17.3 5.1 17.0 6 31.5 22.4 9.2 21.1 8 33.1 15.0 4.6 17.6 10 19.5 3.6 0.8 8.0 Average 28.2 14.6 4.9 12.2

5 4 29.2 21.7 19.8 23.9 6 37.7 33.8 23.5 31.7 8 41.5 33.1 17.3 30.6 10 33.1 18.5 6.0 19.2 Average 35.4 26.8 16.6 20.6 ---------------------------------------------------------------------

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. Since then, 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 deg. or 38 deg. C. appears to be suitable. However, there is less danger of cold shock due to recooling if 5 deg. 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 deg. C. and placed in water at 5 deg. 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 deg. C. are shown in Figure 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.

EFFECTS OF FREEZING PROCEDURES ON METABOLIC ACTIVITY OF BULL SPERM

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 deg. C. in a Warburg apparatus.

=Effect of glycerol additions on oxygen uptake of diluted semen at 37 deg. 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 10^{8} sperm per hour (ZO_{2}).

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.

With each increase in glycerol level, motility was reduced during the incubation period. This is shown in Figure 10 along 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 deg. 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 in Figure 11.

Oxygen consumption was increased by the presence of added catalase at all glycerol levels and in the control. Sperm survival during the 3-hour period at 37 deg. 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.

Table 16.--Effect of Freezing Procedures on Oxygen Utilization of Bull Sperm in Yolk-Citrate Extender

(Average of 5 ejaculates)

===================================================================== Microliters of oxygen Semen sample tested utilized per 10^{8} sperm -------------------------- First hour Second hour --------------------------------------------------------------------- Fresh diluted semen 10.3 8.1 Fresh diluted semen glycerol tipped in at end of first hour 9.7[L] 12.9[L] Aged 20 to 24 hours at 5 deg. C. 11.2 8.3 Aged 20 to 24 hours at 5 deg. C. glycerol tipped in at end of first hour 11.8[L] 12.9[L] After 20 hours equilibration with glycerol 11.7[L] 7.8[L] After freezing and thawing 9.7 6.3 --------------------------------------------------------------------

[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 deg. 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 the sperm. 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 x 10^{6} cells 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.