Part 3

In the first efforts to find the optimum level of egg yolk, the level of yolk in the final frozen mixture was varied from about 6 to 46 percent. These levels were obtained by varying the proportion of yolk to 2.9 percent citrate solution in the original extending media and also in the media added in glycerolating the samples.

Split portions of 20 semen samples were frozen in each of the extender combinations indicated in Table 4. The mean percentages of motile sperm found before and after freezing and thawing are shown also. The highest percentages were found with extenders containing 23 and 24 percent yolk. The highest percentage of yolk, resulting when a 1:1 (yolk to citrate) extender was used for both extending and glycerolating, proved to be most detrimental to sperm survival during freezing. The lowest percentage of yolk used (6 percent) was not as effective in protecting sperm during freezing as the intermediate levels tested (Fig. 2).

Table 4.--Effect of Egg-Yolk Level in Diluent on Freezability of Semen (Average of 20 semen samples)

========================================================================= Medium Yolk in Pre- Post- Sur- ---------------------------- final freezing thawing vival Diluent Extending Glycerolating mix- motility motility (per- ------------ ---------------- ture (percent) (percent) cent) yolk:citrate yolk:citrate (percent) ------------------------------------------------------------------------- 1 1:1 1:1 45.7 65 5 8 2 1:1 1:3 34.9 64 27 42 3 1:3 1:1 33.6 63 33 52 4 1:1 0:1 24.2 64 39 61 5 1:3 1:3 22.8 63 37 59 6 1:3 0:1 12.1 59 33 56 7 1:7 1:7 11.4 56 35 62 8 1:7 0:1 6.0 52 26 50 9 1:15 1:15 5.7 49 25 51 -------------------------------------------------------------------------

This mixture included 14 percent glycerol.

The average initial sperm concentration was 900 x 10^{6}/ml. Sufficient extender was added to give 30 x 10^{6}/ml. at the first extension. Thus the final concentration was 15 x 10^{6} sperm/ml. after glycerolization.

Since rather large changes in the percentages of yolk were used in this experiment, two further trials were conducted in which 16, 24, and 32 percent yolk in the final mixture were compared, with the final citrate percentages held constant. In these tests, 16 and 24 percent yolk maintained sperm better at all citrate levels tried than 32 percent yolk. The 16 percent level was slightly better at most of the levels of citrate tested (Fig. 3).

=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,,,,, 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.,

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 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 ----------------------------------------------------------------------

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.,

During the early studies in the Illinois laboratory, the effects of glycerol level were also tested. 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) --------------------------------------------- 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 ----------------------------------------------------------------

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 After refreezing 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 .. ------------------------------------------------------------------------