Part 5

============================================================ Sperm motility (percent) ------------------------------------- Stage when observed Glycerol Glycerol Glycerol only and glucose and rhamnose ------------------------------------------------------------ Fresh diluted semen 56 56 56 After glycerolization 54 54 54 After equilibration 2 hours 51 53 53 6 hours 48 52 53 12 hours 46 50 51 18 hours 40 46 46 ------------------------------------------------------------

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.

Table 13.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm Motility During the Freezing Procedures

(Average of 10 ejaculates)

====================================================================== 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 -----------------------------------------------------------------------

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.

=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. 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.