Part 2

=Citrate level in the final diluent.= The early work of the British indicated that a final citrate level near 2 percent in the diluent was satisfactory for freezing bull sperm. Later, in a personal communication, Polge of the British group suggested that a citrate level of about 2.35 percent might be best with a final glycerol concentration of 7 percent. Some of the first attempts in this laboratory at establishing the optimum yolk-to-citrate ratios are shown in Fig. 3. In these experiments, the optimum levels of citrate appeared to be lower than anticipated from the British work. Thus a more complex experiment was set up to test a wider range of citrate levels using 16 and 24 percent egg yolk in the final freezing mixture. The average percentages of motile sperm found after freezing 10 semen samples at each of the citrate and yolk levels in this experiment are shown also in Fig. 3. Little difference in freezability was found between citrate percentages of 1.55 and 1.95. When the rate of sperm motility following freezing and thawing was considered along with the percent of motile sperm, a slight advantage was found with 16 percent yolk and a citrate concentration of 1.55 percent.

From the results of these experiments, and from several reports in the literature,[5],[6],[7],[9],[10] it appears that a diluting medium resulting in a final concentration of 16 to 25 percent yolk and 1.55 to 2.2 percent sodium citrate dihydrate is highly satisfactory for freezing.

=Storing and freezing diluent.= In some instances it would be advantageous to have prepared diluent on hand for use at any time. The suitability of stored diluent was tested with a yolk-citrate (equal parts yolk and citrate without antibiotics added) diluent prepared and stored at 5 deg. C. for 0, 2, 5, 7, and 9 days. Seven semen samples were diluted and frozen in these diluents. No difference was noted in the survival of sperm that could be attributed to the age of the diluent.

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 deg. 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 in Table 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]

Table 5.--Comparison of the Freezability of 4 Semen Samples in Different Extenders

====================================================================== Dilution Pre- Post- Motility Extender rate freezing thawing Survival after (semen: motility motility (percent) storage[E] extender) (percent) (percent) (percent) ---------------------------------------------------------------------- Yolk-citrate 1:1 60 49 82 46 1:10 53 45 85 36 Yolk-saline 1:1 57 29 51 28 1:10 60 31 52 24 Yolk-phosphate 1:1 55 33 64 25 1:10 60 43 72 25 Whole milk 1:1 60 40 67 35 1:10 60 35 58 16 ----------------------------------------------------------------------

[E] Stored at 5 deg. C. for 7 hours after thawing.

DILUTION RATES

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 in Table 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 on glycerol additions beginning on page 17.

=Effect of further dilution and refreezing after the initial freezing.= Under some circumstances it might be advantageous to freeze semen with 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 deg. C. and dilution and refreezing following an initial freezing of concentrated samples.

Table 6.--Effect of Sperm Numbers and Glycerol Level in Final Mixture on Freezability of Bull Sperm at -79 deg. C. (Average of 10 ejaculates)

================================================================ Post-thawing motility (percent)[F] --------------------------------------------- Glycerol level Number of sperm (millions/ml.) (percent) -------------------------------- 90 30 10 Average ---------------------------------------------------------------- 5 36.0 34.0 36.0 35.0 10 22.0 24.0 23.0 23.0 15 3.2 0.9 0.2 1.4 Average 20.3 19.8 19.9 20.0 ----------------------------------------------------------------

[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 deg. C. for 3 to 7 hours. The other two portions were refrozen.

Table 7.--Effect of Further Dilution and Refreezing on Sperm Motility After the Initial Freezing of Bull Semen

======================================================================== Post-thawing motility Dilution Pre- --------------------------------------------------- of freezing After After storage[G] After refreezing[H] semen motility first ------------------- -------------------- (semen: (percent) freezing No Diluted No Diluted extender) further to 15 further to 15 dilution million/ml dilution million/ml ------------------------------------------------------------------------ First trial: 4 samples 1:1 60 49 46 34 31 6 1:10 53 45 36 30 25 5

Second trial: 7 samples 1:9 67 47 41 35 28 11 15 million/ml 67 30 32 .. 18 .. ------------------------------------------------------------------------

[G] Stored at 5 deg. C. for 3 to 7 hours after first thawing.

[H] Refrozen following first thawing.

Table 8.--Effect of Glycerol Level and Storage at 5 deg. C. on Motility of Sperm in Yolk-Citrate Extender

==================================================================== Sperm motility -------------------------------------------------------- Post- After storage at 5 deg. C. Glycerol thawing ---------------------------- Average level 1 day 3 days 7 days (percent) --------- --------- --------- --------- --------- per- rate per- rate per- rate per- rate per- rate cent cent cent cent cent -------------------------------------------------------------------- Control[I] 56 2.5 55 1.9 46 1.8 38 1.4 48 1.90 0 54 2.4 44 1.9 46 1.8 36 1.4 45 1.87 5 52 2.2 50 1.9 46 1.7 32 1.4 45 1.80 10 52 2.3 46 1.8 42 1.7 28 1.6 42 1.85 20 52 2.1 50 1.7 44 1.6 38 1.1 46 1.62 30 50 0.7 44 0.5 42 0.4 30 0.4 42 0.51 Average 53 2.03 47 1.62 44 1.50 34 1.22 .. .... --------------------------------------------------------------------

[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 in Table 7.

From Table 7 it 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 deg. C. However, in all cases, survival was best in the samples at the lower dilution levels.

GLYCEROL ADDITIONS

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 deg. 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 deg. 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 deg. 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 deg. 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 in Table 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 to rotate 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 in Table 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. From Table 9 it 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 deg. C. storage for 3 days following freezing and thawing with varying glycerol levels (see Table 10).

The results shown in Tables 9 and 10 were 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]

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

(Average of 13 ejaculates)

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

=================================================================== Temperature Post-thawing motility (percent) during Equilibration ---------------------------------- addition time Glycerol additions of glycerol (hours) ---------------------------------- ( deg. 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 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 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 semen was cooled to 4.5 deg. C. before the glycerol was added. The survival at 10 deg. and 15.5 deg. C. was reduced with each rise in temperature. Thus, it appears that cooling to refrigerator temperature (4-5 deg. 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 deg. C.). Results of attempting to reduce equilibration time are given in Table 11. At 4.5 deg. 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 deg. and 15.5 deg. C., the shortest equilibration time--2 hours--was slightly more detrimental with the differences significant at the 5-percent level at 15.5 deg. 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 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 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 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 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 ------------------------------------------------------------

[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.

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

(Average of 10 ejaculates)