Preservation of Bull Semen at Sub-Zero Temperatures Part 2

Table 9.--Effect of Glycerol Level on Sperm Motility After Freezing to -79 C. and Thawing

========================================================== Glycerol Number Pre- Post- Survival level of freezing thawing (percent) (percent) samples motility motility (percent) (percent) ---------------------------------------------------------- 2 10 53 2 4 4 19 55 29 53 6 19 55 34 62 8 19 55 35 64 10 19 55 24 44 12 10 53 13 25 ----------------------------------------------------------

Table 10.--Effect of Glycerol Level and Storage at 5 C. After Thawing on Sperm Motility

(Average of 13 e.j.a.c.u.l.a.t.es)

================================================ Sperm motility (percent) Glycerol ---------------------------------- level Post- After storage at 5 C.

(percent) thawing ----------------------- 1 day 3 days ------------------------------------------------ 4 29 22 20 6 38 34 24 8 42 33 17 10 33 18 6 ------------------------------------------------

Table 11.--Effects of Temperature, Rate of Addition of Glycerol, and Equilibration Time on Sperm Motility

(Average of 12 e.j.a.c.u.l.a.t.es)

=================================================================== Temperature Post-thawing motility (percent) during Equilibration ---------------------------------- addition time Glycerol additions of glycerol (hours) ---------------------------------- ( C.) 5 3 1 Average ------------------------------------------------------------------- 4.5 2 48 48 45 47.4 6 49 51 47 48.8 18 46 47 46 46.3 Average 47.8 48.6 46.0 47.5

10.0 2 44 43 45 43.9 6 48 50 46 47.9 18 43 46 42 44.0 Average 45.0 46.5 44.3 45.3

15.5 2 41 38 38 39.1 6 42 45 43 43.6 18 42 43 42 42.5 Average 42.0 41.8 41.4 41.7 -------------------------------------------------------------------

=Rate, temperature, and method of adding glycerol.= Closely a.s.sociated 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 s.e.m.e.n for use. Since aging _in vitro_ is 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 in Table 11. One can see that sperm survived freezing better when the diluted s.e.m.e.n was cooled to 4.5 C.

before the glycerol was added. The survival at 10 and 15.5 C. was reduced 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 in Table 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 in Table 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 s.e.m.e.n, 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 in Table 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 s.e.m.e.n. The percentages of surviving sperm remaining after the various steps in the freezing procedure with and without the presence of these sugars are shown in Table 13.

Table 12.--Effect of Adding Sugars to Yolk-Citrate Diluent on Sperm Motility During Equilibration With Glycerol[J]

============================================================ Sperm motility (percent) ------------------------------------- Stage when observed Glycerol Glycerol Glycerol only and glucose and rhamnose ------------------------------------------------------------ Fresh diluted s.e.m.e.n 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 ------------------------------------------------------------

[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 s.e.m.e.n 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[K]

(Average of 10 e.j.a.c.u.l.a.t.es)

====================================================================== Sperm motility (percent) ------------------------------------------------- Stage of Glycerol Glycerol Glycerol Glycerol Glycerol observation only and and and and glucose arabinose xylose rhamnose ---------------------------------------------------------------------- Fresh diluted s.e.m.e.n 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 C. and immediate thawing 28 34 34 29 24 After 4 days at -79 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.

=Subst.i.tutes for glycerol.= Since glycerol was so effective in protecting sperm during freezing, many have a.s.sumed that related compounds might be even better. Several compounds, some related to glycerol and some not, have been tried as subst.i.tutes 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 subst.i.tutes 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 s.e.m.e.n 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 s.e.m.e.n 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 s.e.m.e.n 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 e.j.a.c.u.l.a.t.e 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 s.e.m.e.n. 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 pa.s.sed.

[Ill.u.s.tration: Cooling rates of diluted s.e.m.e.n samples in plastic vials and in gla.s.s ampules (Fig. 4)]

=Rate of cooling in plastic and in gla.s.s.= Plastic vials do not conduct the cold as rapidly as gla.s.s ampules do. The temperature in both gla.s.s 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 gla.s.s ampule containing 1 milliliter diluted s.e.m.e.n and in an 8-milliliter plastic vial containing 2.5 milliliters of diluted s.e.m.e.n. A second plastic vial and gla.s.s ampule filled to capacity with diluted s.e.m.e.n 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 gla.s.s and that the volume of s.e.m.e.n (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 s.e.m.e.n 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 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 s.e.m.e.n, 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 e.j.a.c.u.l.a.t.es 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).

[Ill.u.s.tration: Effect of freezing and storing bull sperm at various temperatures on the sperm motility at thawing (average of 9 e.j.a.c.u.l.a.t.es) (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 e.j.a.c.u.l.a.t.es 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 e.j.a.c.u.l.a.t.es 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:

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 p.r.o.nounced during the first 16 days of storage.

The British and the Australians have both reported the successful maintenance of fertility in frozen s.e.m.e.n 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 s.e.m.e.n 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 s.e.m.e.n-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 in Table 14.

Table 14.--Effect of Glycerol Level and Storage Temperature on Freezability of s.e.m.e.n

(Average of 8 e.j.a.c.u.l.a.t.es)