Freezability of Bovine Spermatozoa in Tris-Buffered Yolk Extenders Containing Different Levels of Tris, Sodium, Potassium and Calcium Ions

Freezability of Bovine.Spermatozoa in Tris-Buffered Yolk Extenders Containing Different Levels of Tris, Sodium, Potassium and Calcium Ions A. M. YASSEN ~'2 and R. H. FOOTE

Department of Animal Science, Cornell University, Ithaca, New York Abstract

This study was designed to investigate the effect of different species of ions and osmotic pressure on survival of bull spermatozoa during freezing to - - 1 9 6 C. Replacing 20 to 80% of the Tris-buffered 20% yolk extender with isosmotic NaC1, KC1, or CaCI~ solutions was found to be harmful. Potassium was more harmful than Na or Ca at the highest level of replacement. When either 10 or 20% by volume of a 0.25 ~ Tris-buffered 1% yolk extender was replaced with isosmotic Na and K chloride and citrate salt solutions, survival of spermatozoa during freezing was not affected. Replacement of 50% or more of the Na citrate with K citrate in 1 and 20% yolk-citrate extenders reduced sperm cell survival before and after freezing. These results indicate that the optimmn levels of Na and K in extenders for freezing spermatozoa are less than the normal intracellular concentrations of these ions. Osmotic pressure of a 0.25 ~ Tris-buffered 20% egg yolk extender (freezing point depression, 0.55 C) was increased by adding NaC1, KC1, and CaCl: singly and in all combinations. As the osmotic pressure increased, motility of the spermatozoa after freezing declined in a linear manner, regardless of the tonicity contributed by each cation. Living cells require optimal external and internal Na, K, and Ca ion concentrations to maintain normal function. Cragle et al. (4) reported that t ( concentration was higher than Na in spermatozoa and lower in seminal plasma. Much less Ca was found than either Na or K in spermatozoa or in seminal plasma. Extending semen at high rates is expected to alter

the internal and external ion relationships and, similarly to washing sperm cells, may alter sperm viability (16). A number of studies (1, 3, 5, 7, 10, 15-17) have indicated that low levels of K (less than 0.01 ~ ) are beneficial to washed or fresh bull or ram sperm cells, but high levels of K are toxic. Most of these studies have been conducted with unfrozen cells. Likewise, studies with Ca additions to unfrozen bull or ram sperm have shown that high levels of Ca are toxic to these cells and that the effect appears to be independent of K (1, 15). Tris (hydroxymethyl) aminometh ane ( T r i s ) , used as a buffer for spermatozoa, provides no inorganic cations, and Bomstein and Steberl (2) found that replacing 40% of a synthetic medium containing 0.132 ~ Tris with a Ca-free Kreb'sRinger phosphate and a sodium citrate buffer improved sperm cell survival at room temperature. Tris penetrates some cells and competitively inhibits K uptake (9). I t has been postulated that Tris decreases the adverse effect of high K on sperm cells by this mechanism (10). Van den Berg (13) and Van den Berg and Rose (14) have reported that the p H of the medium may change during freezing. The salt composition of the medium was found to play an important role in the magnitude and direction of shifts in pH. These changes may alter the molecular structure of cell membranes (8). The present study was planned to investigate the influence of Na +, K ÷, and Ca ++ (hereafter identified as Na, K, and Ca), when added at various levels separately and in combination, on survival of bovine spermatozoa in a Trisbuffered egg yolk extender (TY). Experimental Procedures General. Semen used throughout the study was provided by Eastern Artificial Insemination Cooperative, Inc., from bulls in the regular stud. I t was extended with the appropriate extender to give 30 million motile sperm per milliliter in the nonglycerolated fraction and 15 million motile sperm per milliliter in the final extender following glycerolation. Extended semen in the nonglycerolated fraction was cooled gradually and then glycerolated with an equal

Received for publication December 14, 1966. 1 Supported by a goverment fellowship from the United Arab Republic. 2Present address : Animal Production Departmerit, College of Agriculture, Alexandria University, Alexandria, U. A. R. 887

888

YASSEN AND FOOTE

volume of the precooled glycerol fraction at 5 C in four proportions at 20-rain intervals, as previously described (6, 12). The four portions added were 10, 20, 30, and 40% of the initial volume. The glycerolated semen was equilibrated for 3 hr and then 0.8 ml was transferred per ampule for freezing. Ampules were frozen in a dry ice-alcohol bath or a Linde BF-3 freezing unit. Semen was frozen at a rate of 3 C/min from 5 to -- ]5, 5 C/min from - - 1 5 to - - 4 0 C, and 10 C/rain from - - 4 0 to - - 7 0 C. Then ampules were stored in liquid nitrogen. The Na, K, and Ca were added to the TY extenders as chloride or citrate salts, as described under each experiment. All solutions were made with water redistilled through glass. The final extenders contained 20% egg yolk by volume, unless otherwise indicated, and 1,000 units of penicillin and one mg of dihydrostreptomycin per ml. The osmotic pressure of the extender was determined as the freezing-point depression ( F P D ) with a Fiske cryoscope. Freezability of the semen refers to the percentage of progressively motile cells after freezing and thawing. This was estimated in duplicate and results analyzed statistically, according to procedures previously described by Steinbach and Foote (12) and Steel and Torrie (11). All data were processed electronically and tests of significance were computed for all main effects and interactions. Only the major sources of variance have been presented. Experiment 1. This experiment consisted of five levels of Tris (Table 1), each replaced TABLE 1 Composition of the initial Tris extenders in Experiment One Composition of 100 ml of extender ~ Tris molarity .10 .15 .20 .25 .30

Tris

(g)

].2112 1.8170 2.4224 3.0282 3.6340

Citric acid monohydrate

(g)

0.6698 1.0048 1.3396 1.6747 2.0098

pit

Freezing point depression of the nonglycerol extender b

6.67 6.64 6.64 6.64 6.64

.250 .351 .454 .551 .649

(C)

The extenders included 20 ml of egg yolk per 100 ml. Varying levels of Na, K, and Ca were achieved by replacing equivalent amounts of Triscitric acid buffer as indicated in the text. b Final extenders contained equal volumes of nonglycerol and 14% by volume of glycerol-containing extenders, so that 7% glycerol was present in the media used for freezing. J. DAIRY SCIENCE ~OL. 50, NO. 6

with five levels of Na, K, and Ca in a 5 × 5 X 3 factorial arrangement. The five levels of replacement with NaC1 and KC1 were 0, 20, 40, 60, and 80%. Because of gelation of egg yolk, CaCl~ was used at 0, 10, 20, 30, and 40% levels. The osmotic pressure ( F P D ) was kept constant within each Tris molarity, regardless of the concentration of NaC1, KC1, or CaC12 in the extender. The F P D of all 75 solutions was determined and means for the five different molarities are shown in Table 1. The p H decreased as much as 0.2 unit, with the highest salt replacements due to the decreased buffering capacity of the solution and the lower p H of egg yolk. Because of the large number of treatments, the semen was frozen in three batches, handling each ion series as a separate batch. The semen was stored in liquid nitrogen for one hr and for one week. Experiment 2. Because of the large contribution of Na and K ions by the egg yolk in the previous experiment, the level of egg yolk was reduced in the extender to 1%. This was added to 0.25 ~ Tris buffer of the composition shown in Table 1. Tris buffer was replaced with isosmotic solutions of NaC1 and KC1 singly and in combination, in graded levels up to a maximum replacement of 10%. Other conditions of the experiment were the same as described under the general procedure. Experiment 3. This study was designed to compare sperm survival when Na, K, and Ca were used singly and in combination to increase the osmotic pressure of the medium. Five levels of cations were chosen and these represented 0, 1/3, 2/3, 3/3, and 4/3 of the concentrations of the Na, K, and Ca reported to be in bull spermatozoa by Cragle et al. (4). The five levels chosen are shown in Table 2. These were combined in a 5 X 5 × 5 factorial arrangement added to 0.25 ~t Tris buffer. The final extender contained 1% egg yolk and 9% glycerol by volume, p H and freezing-point depression were determined for the 125 extenders. The p H of all extenders was approximately 6.9 and the freezing-point depression ranged from TABLE 2 Composition of extenders used in Experiment Three Coded levels" Ingredients

0

1

--(g/lOOml Nail XCI CaCle

O 0 0

2

3

4

extender)---

. 1 4 6 6 . 2 9 3 2 . 4 3 9 8 .5864 . 1 5 3 2 . 3 0 6 4 . 4 5 9 6 .6128 . 0 2 0 8 . 0 4 1 6 . 0 6 2 4 .0832

a These levels were combined singly and in combination and added to a 0.25 ~ Trls buffer.

FREEZABILITY OF BOVINE SPERMATOZOA 0.45 C to values in excess of 1.0 C. The latter values could not be determined accurately and the solutions were highly spcrmicidal; therefore, as finally conducted, the experiment contained only four levels of NaC1. Thus, it became a 4 × 5 × 5 factorial arrangement. The experiment was replicated with 10 bulls. Extended semen was examined in duplicate before freezing and after storage for 1 wk in liquid nitrogen, making a total of 4,000 motility estimations. Experiment 4. I n the previous experiments the effects of increasing the level of cations also might have been attributed to chloride ions, which increased simultaneously. Citrate is the anion present in highest concentration in the most commonly used yolk extender for freezing semen. The permeability of the cell to the citrate ion may be considerably different from the chloride ion. Experiment 4 was planned to compare the freezability of bull semen in Tris extender containing the chloride and citrate salts of Na and K. Again, the 0.25 ~ Tris provided the basic medium. NaC1 and KC1 solutions equal in osmotic pressure to the Tris buffer were used to replace 10 and 20% of this buffer. Isosmotic Na citrate and K citrate solutions replaced portions of Tris buffer, to give Na and K concentrations equal to the concentrations provided by the NaC1 and KC1 solutions. One per cent egg yolk was included and 19% water was added, to keep the ions contributed by the buffer equal to those when 20% egg yolk was used; this reduced the F P D equivalent to the 0.20 ~ Tris shown in Table 1. Semen was processed for freezing and storage in the usual manner. Experiment 5. Since high levels of K generally are considered to be more toxic to cells than Na, it was of interest to determine what effect complete replacement of Na by K in buffer maintained at isosmotic levels had on sperm survival, and to measure possible protective influences of different levels of egg yolk. A 2 × 5 factorial arrangement consisting of two levels of egg yolk (1 and 20%), and five levels of sodium and potassium citrate was planned. Separate solutions containing 2.90 g of sodium citrate dihydrate and 3.17 g of potassium citrate monohydrate per 100 ml of water were prepared. These solutions were used separately and in 1:3, 1:1, and 3:1 combinations, giving five concentrations of each cation. Semen from ten bulls was processed as described for the previous experiments.

before freezing and 1 hr and 1 wk after freezing. Analysis of variance revealed that the main factors, bulls, freezing, concentration of Tris, and level of salt added gave highly significant differences (P < .01), and these factors contributed most of the variance. There was little difference between storage at - - 1 9 6 C for 1 hr or I wk and these data are combined in Figures I and 2. Results shown in Figure 1 indicate that sperm cell survival during freezing was highest in the 0.20 to 0.25 ~ extenders, which were isosmotic or slightly hypo-osnmtic to semen. Motility was drastically reduced in 55 NoCl KCI

50

Experiment 1. The mean percentages of progressively motile sperm cells were estimated

.....o-...'~'" ... +5C

.....

,.."

40

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i

20

10

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-196C

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i.Y

;"

-

~:."

0,/, 0 .10

, .15

; . 0

MOLARITY

; . 5

OF

, .30

TRIS

Fro. 1. Freezability of bull semen extended with TY extenders having different levels of Tris in Experiment One. 45 ~. . . . . . . o,, " ...... o....... "'~".,,. 40' - ~ . , . , ~ q . .,,.

NaCl ., KCI ..... CaCl 2

. . . . . . . . .

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-196C

~1'~"- ~ ' ~ I

0

' 10

% Results and Discussion

889

2'o

3o

40'

' 50

60'

70

80

TRIS BUFFER REPLACED W I T H SALT

FZG. 2. Freezability of bull semen in TY extenders having different levels of NaC1, KC1, or CaC12 in Experiment One. J. D A I R Y S C I E N C E V O L . 5 0 , l~O. 6

890

Y A S S E N AND F O O T E

the 0.10 and 0.15 ~ solutions, even before freezing. This effect has been reported previously (12), but the depression was less pronounced at corresponding levels of Tris, due to inclusion of one percent fructose in the earlier studies. Data showing the interactions of molarity with other components are not given, because most of the interactions were relatively small, although sometimes statistically significant. The molarity times level of salt replacement interaction was large (P < .01), due to an increasing spermicidal effect of high cation replacement in the Tris extenders of high molarity. Figure 2 shows that the addition of all cations was harmful when 20% or more of the Tris was replaced. The effect is present both before and after freezing. The 20% egg yolk was found to furnish a considerable amount of Na, K, and Ca to the extender (19). However, sperm cells contain about 4 times the Na and l0 times the K concentration (4) found in the control TY extender. Assuming that high intracellular ion concentrations are harmful, this suggests that one of the beneficial effects of Tris without salt replacement is that it may permit the outflux of the intracellular Na and K to the extracellular media. From these results it was concluded that 20% yolk in the extender furnishes enough Na, K, and Ca, and that additional amounts are detrimental to spermatozoa. The experiment did not establish a minimum requirement, since the ion concentration in a 20% yolk medium may have been more than was necessary. Furthermore, the proportion of free ions in the egg yolk was not determined. Experiment 2. Tabular presentation of the results has been omitted, since the analysis of variance revealed that there was no sig'nificant effect (P > .05) of adding low levels of NaC1 or KC1 to the TY medium containing 1% egg yolk. The mean percentages of motile sperm cells in extenders containing 0, 5, and 10% NaC1 replacement and 0, 5, and 10% I~iC1 replacement were 50, 48, 50, 50, 50, and 55 before freezing and 28, 28, 23, 28, 30, and 30 after freezing, respectively. Since only 1% egg yolk was used, these results suggest that Na and K in the extender at levels much lower than found in sperm cells were as effective as higher levels in maintaining sperm viability. Experiment 3. The cations added to increase the tonicity of the media decreased sperm survival as osmotic pressure of the media increased (P < .01). This relationship is shown by the data in Table 3 and the regression equation in Figure 3. Orthogonal polynomial comparisons revealed that Na, K, and Ca each affected J . ])AIRY SeImNC~ VOL. 50, No. 6

TABLE 3 Overall effect of salt level on sperm cell survival in Experiment Three Temperature

Level of salt added* Salt

0

:1

2

3

4

--(% Motile spermatozoa)-5C -- 196 C

NaC1 KC1 CaC12 NaC1 KC1 CaC12

54 50 36 21 19 12

45 47 36 15 16 12

30 38 35 8 12 12

ll 25 35 2 7 11

.... b 14 33 .... b 3 11

See Table 2 for composition. b Spermicidal, due to the high tonicity of the media, and this level was discontinued. 40

=62.49-67X

A >30

Z

•

|%

•

~f/

O 0

I .45

FREEZING

I .S5

POINT

°~o°°

°

i

.65

.75

DEPRESSION,

.85

.95

C (X)

FIG. 3. Regression of the percentage of motile cells following freezing and thawing on the FPD of the extenders in Experiment Three. sperm motility before and after freezing (P < .01). However, the amount of Ca added was much less than for the Na and K (Table 2), because of the lower level of Ca ion in spermatozoa. Therefore, the increasing amounts of Ca shown in Table 3 had little effect on sperm survival. The analysis of variance revealed that 54% of the variance associated with cation effects was due to K, 44% to Na, and 2% to Ca. The generally low motility values for frozen sperm cells given in Table 3 is due to the fact that for each level of cation shown the motilities have been averaged over all levels of the other cations. The relationship depicted in Figure 3 shows that the total ion concentration present was more important than the proportions of the

891

F R E E Z A B I L I T Y OF B O V I N E S P E R I ~ A T O Z O A

tonicity contributed by each cation studied. The a p p r o x i m a t e relationship is shown by the linear regression equation given in F i g u r e 3. Although inspection of the data suggests a slight dep a r t u r e f r o m linearity, this was not significant statistically (P > .10). Experiment 4. Results of this experiment, summarized in Table 4, show that replacing the TABLE 4 Percentage of progressively motile spermatozoa in Experiment Four %Replacement of Tris by volume *

% Motile spermatozoa

Replacement salt

Before freezing, 5 C

After storage for 1 wk at --196 C

O 10 10 10 10

NaC1 Na citrate KC1 K citrate

54 52 53 52 54

30 27 31 30 28

20 20 20 20

NaC1 Na citrate KC1 K citrate

53 52 52 52

28 32 27 28

a The Tris buffer was replaced with isosmotic amounts of the salt solutions shown. After addition of 1% egg yolk the pH was approximately 7.0 in all extenders. Tris buffer with either 10 or 20% NaC1, KC1, N a citrate, or K citrate had no significant effect on the viability of bull sperm cells ( P > .10). Most of the variance was associated with bulls and t e m p e r a t u r e of storage ( P < .01). These results indicate that considerable variation in the level of N a and K and associated anions is possible under the conditions tested without appreciable effect on sperm survival following freezing. This tends to confirm the previous results, which indicated that total osmotic pressure exerted a greater effect on sperm cell survival than the p a r t i c u l a r species of ion used. Experiment 5. Results of this experiment, presented in Table 5, show that replacing Na by K reduced the viability of sperm cells significantly ( P < .01), both before and a f t e r freezing. The effect was particularly pronounced when 50% or more of the cation was present as K. The f a c t that sperm cells survived better at 5 C in 1 % egg yolk with high K levels than in 20% egg yolk may be due to a lower total K content of the extender, since the egg yolk was replaced on a volume basis by water. This i n j u r y of the sperm cells before and a f t e r freezing m a y result f r o m an accumulation of K inside the cells (18). The motility of spermatozoa in 1 % egg yolk

TABLE 5 Percentage of progressively motile spermatozoa in Experiment Five % Motile spermatozoa Proportions in the buffer Egg yolk - (% by Na K volume) citrate citrate

After storage Before for 1 freezing, wk at 5C --196 C

1 1 1 1 1

100 75 50 25 0

0 25 50 75 100

58 57 54 46" 38 b

28 26 19 b 12 b 12 b

20 20 20 20 20

1O0 75 50 25 0

0 25 50 75 ]00

56 50 46 b 38 b 21 b

31 29 22 b 16 b 8b

51 42 b

20 21

Overall average of 1% egg yolk Overall average of 20% egg yolk

* P ~ .05. b p ~ .01 when comparing the level of K citrate with no K citrate or 1 vs. 20% egg yolk. a f t e r freezing was essentially the same as in 20% egg yolk. Based u p o n the motility estimates at 5 C, these data suggest that 1 % egg yolk is slightly less effective than 20% egg yolk in protecting sperm cells during freezing. Nevertheless, the final motility is similar, indicating that the concentration of the protective agents provided by egg yolk and required during freezing and thawing is low.

Acknowledgments This work was supported in part by grants from the Eastern Artificial Insemination Cooperative, Inc., and the Northeastern Council of Artificial Breeding Cooperatives. The authors are grateful to Professor L. D. VanVleck for assistance with the statistical analyses and to Professor F. W. Shipe for use of the cryoscope.

References (1) Bl~kshaw, A. W.

(2)

(3)

(4)

(5)

1953.

The Effects of

Potassium and Calcium Salts on the Motility of Ram, Rabbit and Bull Spermatozoa. J. Physiol., 120: 465. Bomsteln, R, A., and Steberl, E. A. 1959. Preservation of Washed Bovine Spermatozoa in Synthetic Medium at Room Temperature. Exptl. Cell Research, 18: 217. Choong, C. H., and Wales, R. G. 1963. The Use of Vaxious Diluents for Deep-Freezing Bull Spermatozoa. Australian J. Biol. Sci., 16 : 896. Cragle, R. G., Salisbury, G. W., and VanDemark, N. L. 1958. Sodium, Potassium, Calcium, and Chloride Distribution in Bovine Semen. J. Dairy Sci., 41: 1267. Dott, H. M., and White, I. G. 1964. Effect of Potassium on Ram Spermatozoa Studied J. DAIRy SCIENCE VOL 50, No. 6

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(13)

YASSEN AND FOOTE by a Flow Dialysis Technique. J. Reprod. Fertil., 7 : 127. Foote, R. H., and Dunn, H. O. 1955. Buffers, Extenders and Methods for Freezing Semen. Routine Lab. Procedure no. 11. Corne]l University, Ithaca, N. Y. Lardy, H. A., and Phillips, P. H. 1943. Effect of p H and Certain Electrolytes on the Metabolism of Ejaculated Spermatozoa. Am. J. Physiol., 138: 741. Lovelock, J. E. 1954. Physical Instability and Thermal Shock in Red Cells. Nature, 173 : 659. MacLeod, R. A., and Onofrey, E. 1954. Cation Antagonism of the Antibacterial Action of Amines. J. Biol. Chem., 2]0: 193. O'Shea, T., and Wales, R. G. 1964. Effects of Potassium on Ram Spermatozoa, During Chilling to and Storage at 5°C. J. Reprod. Fertil., 8: 121. Steel, R. G. D., and Torrie, J. H. 1960. Principles and Procedures of Statistics. McGraw-Hall Book Co. Inc., New York. Steinbach, J., and Foote, R. H. 1967. Osmotic Pressure and p H Effects on Survival of Frozen Bovine Spermatozoa. J. Dairy Sei., 50 : 205. Van den Berg, L. 1959. The Effect of Addition of Sodium and Potassium Chloride to

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(14)

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(18)

(19)

the Reciprocal System : KH~PO~ -- Na~_HPO~ --H20 on pH and Composition During Freezing. Arch. Biochem. Biophys., 84: 305. Van den Berg, L., and Rose, D. 1959. Effect of Freezing on the pH and Composition of Sodium and Potassium Phosphate Solutions: The Reciprocal System KH~PO~ - - N a 2 H P O 4 - H20. Arch. Biochem. Biophys., 81 : 319. Wales, R. G., and White, I. G. 1958. The Effect of the Ions of the Alkali Metals Magnesium and Calcium on Dog Spermatozoa. J. Physiol., 142: 494. White, I. G. 1953. The Effect of Potassium on the Washing and Dilution of Mammalian Spermatozoa. Australian J. Exptl. Biol. Med. Sci., 31: 193. White, I. G. 1953. Studies on the Alkali Metal Requirements of Ram and Bull Spermatozoa. Australian J. Biol. Sci., 6: 716. Yassen, A. M. 1966. Effect of Electrolyte Concentration, Method of Glycerolation and Aging Period on the Freezabllity of Bull Semen Extended with Tris-Buffercd EggYolk Extender. Ph.D. thesis, Cornell University, Ithaca, New York. Yassen, A. M., and Foote, R. H. 1966. Na* and K + Concentration of Extended Bull Semen. J. Animal Sci., 25: 933.