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° 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 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]
| Stage when observed | Sperm motility (percent) | ||||
| Glycerol only | Glycerol and glucose | Glycerol 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.