~ERE IS considerable information regarding viability of bovine sperm

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1 Freezing-Point Depression and Viability of Bovine Sperm During Freezing and Storage P. S. RAO,* PH.D., J. D. SIKES, PH.D., and C. P. MERILAN, PH.D. ~ERE IS considerable information regarding viability of bovine sperm during freezing and storage. Factors studied include: (1) the influence of freezing methods,2. 6 (2) storage at low temperatures,4, 8 (3) variations in the survival of sperm frozen and stored at subze,ro temperature according to breeds, bulls, and ejaculates,3, 9 (4) the effects of hypertonic and hypotonic solutions on viability,lo and (5) the freezing-point depression of bovine semen, seminal plasma, and the expanders , 16 In spite of such exhaustive studies, there is no evidence to support the view that research workers have correlated the freezing-point depression of bovine semen, seminal plasma, and the expanders with freezing and storage. The aims of the study reported here were as follows: (1) to observe the activity of spermatozoa when expanded, frozen, and stored in hypotonic, isotonic, and hypertonic expanders, and (2) to determine the difference between the freezing-point depression of bovine semen and seminal plasma of the same sample and the possible correlation with the sperm concentration. EXPERIMENTAL PROCEDURE Bovine semen was collected by means of an artificial vagina, and the total number of live cells in each sample was estimated microscopically.7 Semen was expanded to a final expansion rate of 1:20. The addition of 8% glycerol to the expanders was carried out according to the method described by Sikes and Merilan. The percentage of egg yolk (20%) was constant in all expanders, and the change from hypotonicity to hyper- From the Department of Dairy Husbandry, University of Missouri, Columbia, Mo. Presented at the 23rd Annual Meeting of The American Fertility Society, Washington, D. C., Apr , The authors are indebted to Mr. K. W. Bower for his assistance: *Present address: Department of Gynecology and Obstetrics, Saint Louis University School of Medicine. Saint Louis, Mo. 129

2 130 RAO E'I' AL. FERTILITY & STElULITY tonicity was obtained by using different levels of sodium citrate dihydrate. Two hypotonic, one approximately isotonic, and two hypertonic expanders were used. A Fiske Model-J* milk cryoscope was used to determine the freezingpoint depression values. The standard procedure was not applicable in freezing semen with any certainty. Certain changes from the standard procedure accordingly, became imperative, and the necessary modifications were adopted. The freezing-point depression values were obtained within 30 min. after semen collection. Two consecutive readings of the same sample were taken to serve as a double check. Seminal plasma was obtained by centrifuging semen between 25,032 and 28,735 X gravity for 20 min. Calculation of osmotic pressure from freezing-point depression values was done as described by West and Todd. Sperm concentrations were estimated by use of a hemocytometer. 7 Two different freezing methods were studied to evaluate the possible damage to the cells at a rapid cooling rate. An alcohol bath containing ethyl alcohol at +5 C. and liquid nitrogen was used for freezing. A temperature drop of 1 0 C./min. from +5 to C. and 2 C. from -15 to 0-79 C. was obtained by adding dry ice to the alcohol bath. Three aliquot samples of four from each expander were frozen in this manner. One was thawed immediately, and the rate of motility was observed. The second and third samples were stored in the dry ice chamber at C. for 7 and 30 days, respectively, and the motility ratings were made upon removal from storage. The fourth sample, after reaching a temperature of C. in the alcohol bath, was transferred directly into liquid nitrogen. Samples were held at that temperature for 10 min., then removed, thawed, and checked for motility. Semen RESULTS AND DISCUSSION A total of 71 samples from 9 bulls was studied to determine freezingpoint depression values. The mean values of semen and seminal plasma, with corresponding standard-deviation values for each bull, are shown in Table 1. The mean freezing-point depression, C. (range, to C.), obtained for semen is in relative agreement with the reports of Bernstein and Sergin, and of Roemmele as noted by Anderson and by Salisbury et al. The mean and standard deviation of the calculated osmotic pressure values was ± atm. Statistical analysis showed a sig- *Fiske Associates, Inc., Bethel, Conn.

3 VOL. 19, No.1, 1968 BOVINE SPERMATOZOA 131 TABLE 1. Variation of Freezing-Point Depression of Bovine Semen and Seminal Plasma Booine semen freezing-point Seminal plasma freezing-point Bull depression, mean value (_oc.) depression, mean value (_oc.) ~ = ±* ± ± ± ± ± ± ± ± OVER-ALL MEAN ± '" ± = standard deviation ± ± ± ± ± ± ± ± ±0.OO ± nificant difference between bulls and between samples within bulls at the 0.5% level. This would indicate that the freezing-point depression of semen differs between bulls and also that the individual samples collected over a period of time from a single bull differ significantly. Seminal Plasma The over-all mean value for seminal plasma was C. (range, to C.). The mean and standard deviation of the calculated osmotic pressure values was atm. In comparison, results obtained in this experiment were slightly higher than those of Rothschild and BamesP 13 Analysis of variance showed a significant difference between samples within bulls at the 0.5% level, but no significant difference was observed among bulls. It is difficult to explain the findings of the seminal-plasma analysis. The accessory sex glands cannot be considered independently,13 and some form of overriding "control" is exercised on these secretions. Since the bulls are not uniform in responding to sexual stimulation prior to semen collection, the response of accessory sex glands would differ. This would result in seminal plasma of varied freezing-point-depression values within the same bull. Expanders The mean and standard deviation for the freezing-point-depression and osmotic-pressure values for the five different expanders are presented in Table 2. The determinations were in concurrence with the findings of

4 TABLE 2. Freezing-Point Depression and Osmotic Pressure of Expanders Sodium Sodium citrate citrate dihydrate dihydrate Egg yolk Freezing-point depression Osmotic pressure (calculated) Expander (gm./loo ml.) (%) (%) mean value (- C.) mean value (atm.) Hypotonic! ~*0.OO ~ Hypotonic ~0.OO ~ Approximately isotonic ~ ~ Hypertonic! ~0.OO ~ Hypertonic ~0.OO ~ * ~ = standard deviation.

5 VOL. 19, No. I, 1968 BOVINE SPERMATOZOA 133 Swanson and of Pursley and Herman. An analysis of variance showed a significant difference among the expanders, trials, and expanders versus trials at the 0.5% level. Sperm Concentration as Related To Freezing.Point Depression The regression analysis for 22 semen samples was studied to determine the corre!ation between the difference in freezing-point depression of semen and seminal plasma of the same sample and that of sperm concentration (Fig. 1). The values indicated a positive and significant correla- Fig. 1. Difference in freezingpoint depression of semen and plasma of sample and its relationship to sperm concen tration.., 200 E E... ISO.11) o g ISO Q «o N o i ~ ~ SO. a:: ~ SO :E ~ 40 Y=5S.29S+17SS.S33X Sb= '--"":0~.0::;'3~"""-"":O::-;;.0=:;:5~""-"":Q;:;;O!;:;7""""""-"":0;;1.09 FREEZING POINT DEPRESSION, _OC tion between sperm count and the difference in freezing-point depression at 0.1% level, showing that as the sperm count increased, the magnitude of the freezing-point depression increased correspondingly. Since seminal plasma constihltes a large portion of semen and influences the freezing-point depression considerably, the sperm may be acting as large colloidal particles even though their size differs, thus increasing the freezing-point depression of semen over seminal plasma. Another possible explanation would be that during the process of centrifugation larger colloidal particles along with spermatozoa may settle to the bottom, thus leaving seminal plasma with a freezing-point depression value less than that of semen. It is difficult to determine what effect the outward diffusion of intracellular electrolytes and inward diffusion of extracellular electrolytes would have on the freezing-point depression of the sample.

6 134 RAO ET AL. FERTILITY & STERILITY Spenn Viability The statistical analysis presented in Table 3 shows a significant difference among bulls, viability, and expanders at the 0.5% level. Interaction terms such as bulls versus viability and expanders versus bulls did not show statistical significance; however, expanders versus viability was significant at the 10% level. This showed a marked difference among bulls in the ability of their sperm to withstand freezing and in the ability to survive when stored at C. in expanders of different osmotic-pressure values. The significant difference of the interaction among expanders versus viability indicated that the influence of expanders on sperm in freezing and storage did not follow a specific pattern but varied with different expanders. Figure 2 shows the mean percentage of live cells per sample for each expander in each viability factor. It is obvious that the 2.75% and 3% are superior in comparison to others. To test this statistically, Duncan's new multiple-range test was applied at the 5% level. The Duncan's test showed that the 3% sodium citrate expander (approximately isotonic) and 2.75% sodium citrate expander (hypotonic 2 ) were not significantly different in affording protective action for sperm. However, the mean values would show a slight advantage in the approximately isotonic expander. In freezing and storage the 3.25% sodium citrate expander (hypertonic L) showed a significant loss of live cells in comparison with hypotonic and approximately isotonic expanders, even though there was no marked difference in prefreezing. The 4% sodium citrate expander ( hypertonic 2 ) was detrimental to the sperm, and adverse effects could be seen even in prefreezing. In hypotonic and approximately isotonic ex- TABLE 3. Analysis of Variance for Total Number of Live Cells when Frozen and Stored Degrees of Sum of Source freedom squares jl,f ean squares F Rat1:o F.005 Bulls 8 63,665 7, * 3.13 Viability 4 153,494 38, * 4.62 Expanders 4 107,920 26, * 4.66 Bulls X viability 32 2, Expanders X bulls 32 1, Expanders X viability 16 2, t Error , TOTALS ,946 * Significant at 0.5% level. t Significant at 10% level F.l

7 VOL. 19, No.1, 1965 BOVINE SPERMATOZOA 135 panders no significant loss in motility was observed during the first 7 days of storage (significant at 30 days); however, the loss in motility was significant in the 3.25% sodium citrate expander (hypertonicl).."":~:rhe beneficial effect of 3% sodium citrate dihydrate in the liquid storage I7 and of 2.9% in W...J a. ~ «en Fig. 2. Effect of freezing ;;; and storage time on semen :J samples with a mean freezing- ~ point depression of C. w > :J PRE-FREEZING POST FREEZING (LIQUID N2) FiJ POST FREEZING (DRY ICE) ~ 7 DAYS STORAGE AT-7geC III 30 DAYS STORAGE AT -79"C mi CONCENTRATION OF SODIUM CITRATE. gm.llooml. freezing! have been reported. When different freezing methods (dry ice and alcohol versus liquid nitrogen) were tested, a significant difference at the 5% level was observed in favor of the dry-ice and alcohol-freezing technic. This would suggest either a critical temperature range and/or an optimal freezing rate. Therefore, it can be concluded that the best choice among the expanders would be an isotonic expander. It could be further stated that it would be far safer to use a hypotonic expander than a hypertonic expander, however slight the hypertonicity might be. SUMMARY The mean and the standard deviation of the freezing-point depression for semen and seminal plasma were ± C. and ± C., respectively. For the expanders the range was C. to C., but in terms of sodium citrate dihydrate concentration the range was 2.5 to 4%. Statistical analysis showed a significant difference at the 0.5% level for semen among the 9 bulls sampled. A significant difference at the 0.5% level was also noted for both semen and seminal plasma among all the samples (71) for the 9 bulls. A correlation between the difference in freezing-point depression of

8 136 BAo ET AL. FERTILITY & STERILITY semen and seminal plasma of the same sample and that of sperm concentration was observed. The regression coefficient and the sample standard deviation were ± (1O,OOO's per cu. mm.). It was concluded that the best choice among the expanders was the approximately isotonic expander (3% sodium citrate). It is far safer to use a hypotonic expander than a hypertonic expander, however slight the hypertonicity might be S. Grand Blvd. St. Louis, Mo REFERENCES 1. ANDERSON, J. The semen of animals and its use for artificial insemination. Imperial Bureau of Animal Breeding and Genetics, Technical Communication, Edinburgh, BEAN, B. H., PICKETT, B. W., and MARTIG, R C. Influence of freezing methods, extenders and storage temperatures on motility and ph of frozen bovine semen. I Dairy Sc 46:145, BUCH, N. c., SMITH, V. R, and TYLER, W. J. Bull and line differences in the survival of spermatozoa after freezing and thawing. I Dairy Sci 39:1712, CRAGLE, R G., MEYERS, R M., 'VAUGH, R K., HUNTER, J. S., and ANDERSON, R L. The effects of various levels of sodium citrate, glycerol and equilibration time in survival of bovine spermatozoa after storage at C. I Dairy Sc 38:508, Fiske Milk Cryoscope Manual. Fiske Associates, Inc., Bethel, Conn., FORGASON, J. L., BERRY, 'vv. T., JR., and GOODWIN, D. E. Freezing bull semen in liquid nitrogen vapor without instrumentation. I Animal Sc 20:970, HERMAN, H. A., and MADDEN, F. W. The Artificial Insemination of Dairy and Beef Cattle. Lucas Bros., Columbia, Mo., POLZE, C. The storage of bull semen at low temperatures. Vet Rec 65:557, POORE, M. E., and BRUGMAN, H. H. Variation in survival of bovine spermatozoa when stored at sub-zero temperatures as affected by differences in breeds, bulls and ejaculates. I Dairy Sc 38:62.3, PURSLEY, G. R, and HERMAN, H. A. Some effects of hypertonic and hypotonic solutions on the livability and morphology of bovine spermatozoa. I Dairy Sc 33: 220, RAO, P. S. The freezing point depressioll and livability of spermatozoa as affected by different extenders. Ph.D. Thesis, University of Missouri, ROTHSCHILD, L., and BARNES, H. Osmotic pressure of bull semen diluents. Nature 173:636, ROTHSCHILD, L., and BARNES, H. Constituents of bull semen plasma. I Exper Biol 31:561, SALISBURY, G. W., KNODT, C. B., and BRATTON, R \V. The freezing point depression of bull semen and its relation to the diluter problem. I Animal Sc 7:283, SIKES, J. D., and MERILAN, C. P. Preliminary results on the preservation of bovine. semen in a milk-egg yolk-glycerol extender. I Dairy Sc 41:205, SMITH, J. T., MAYER, D. T., and HERMAN, H. A. Osmotic pressure of extended bovine semen during storage. Missouri Agricultural Experiment Station Research Bulletin 583, Univ. Missouri, Columbia, Mo., SWANSON, E. W. The effect of varying proportions of egg yolk and sodium citrate buffer in bull semen diluents upon sperm motility. I Dairy Sc 32:345, WEST, E. S., and TODD, W. R Textbook of Biochemistry (ed. 3). Macmillan, New York, 1961.