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Effects of cooling and freezing on pH of semen extender
CRYOBIOLOGY
19, 16-19
Effects
(1981)
of Cooling
and Freezing
R. S. JEYENDRAN2 Department
of Animal
Science,
AND
of
University
During freezing, ice formation increases the solute concentration of the unfrozen portion of an aqueous solution until it reaches the eutectic temperature of the solution and at this temperature, the solution is completely frozen. However, if a mixture of solutes are present as in semen extenders, then, the solute concentration and the pH of the solution will be determined by the relative eutectic temperature of the individual solutes during freezing. Van den Berg (11, 12) demonstrated this effect on sodium phosphate solution; his analysis revealed the pH of the unfrozen portion of slightly alkaline sodium phosphate solution at room temperature to be 3.6 at-9.9”C. Similar changes in pH were also observed with calcium and magnesium phosphate solutions (13, 14). Moreover, changes in pH during freezing of “muscle juice” (1) and milk (10) have been reported. To date, there is virtually no information available regarding measurement of pH at or below the freezing point for semen extenders. Therefore, the present study was designed to determine the changes in pH of extenders with and without egg yolk and cryoprotecting compounds, and the possible interaction between these during cooling and freezing to a temperature as low as -18°C. MATERIALS
AND
Experimental
St. Paul,
Minnesota
55108
Procedure
The pH of nine buffers and TEST with and without egg yolk and/or cryoprotective compound was determined at room temperature and 5°C using a Corning pH meter Model 12 and a Corning combination triple-purpose electrode. A continuous measurement of pH of a 30 ml sample of TEST yolk was made as it cooled gradually from 37 to 0°C and then to a temperature as low as - 18°C using a Beckman Model 3500 pH meter with a low-temperature combination electrode (Ingold Electrodes, Inc.). These measurements were replicated six times. The sample was stirred continuously to prevent supercooling during freezing.
Extenders
Ten 0.325 M buffers were used in the study. Three were conventional solutions: Received April 22, 1981; accepted July 28, 1981. ’ Scientific Journal Series Paper No. 11, 573, Minnesota Agricultural Experimental Station. * Present address: Institute of Reproductive Medicine, Chicago, 111. 60602. 16 Copyright @ 1982 by Academic F’ress, Inc. AU rights of reproduction in any form reserved.
E. F. GRAHAM Minnesota,
phosphate (KH,PO,-Na,HPO,* H,O), citrate (Na&H,0,.2H20), and skim milk (heated at 90°C for 10 min). Six were buffers described by Good et al. (3): (a) TES, N-Tris(hydroxy-methyl)methyl-2-aminoethanesulfonic acid; (b) TRIS, Tris(hydroxymethylaminomethane); (c) MES, 2-(Nmorpholino)ethanesulfonic acid; (d) MOPS, morpholino-propane sulfonic acid; (e) BES, N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; and (f) PIPES: Piperazine-NN- bis(Zethanesulfonic acid). The last buffer TEST was prepared by titrating TES with TRIS to pH 7.2 (4). Egg yolk (20% v/v) was added to all buffers except skim milk. The cryoprotective compounds: glycerol, dimethylsulfoxide, ethylene glycol, diethylene glycol, and triethylene glycol were added in concentrations of 2 and 6% v/v to TEST buffer, with and without 20% egg yolk, to determine their interactions with pH during cooling (5). TEST yolk buffer was the only extender used in the freezing experiment.
METHODS
001 l-2240/82/010016-04$02.00/O
on pH of Semen Extender’
COOLING/FREEZING
Mean
pH of 10 Buffers
EFFECTS
1
TABLE with
20%
Egg Yolk
Skim Milk) at Room Temperature
(Except
(22°C) and at 5°C
PH
Phosphate yolk Citrate yolk Skim milk TES yolk
TRIS yolk MES yolk BES yolk PIPES yolk MOPS yolk TEST yolk
RESULTS
22°C
5°C
Difference
7.12 7.15 6.62 5.41 9.06 4.78 5.33 6.93 5.31 7.12
7.10 7.10 6.70 5.92 9.72 5.22 5.72 7.18 5.59 7.51
AND
DISCUSSION
-0.02 -0.05 +0.08
+0.51 +0.66 +0.44 +0.39 +0.25 +0.28 +0.39
Table 1 lists the mean pH values of 10 buffer solutions at room temperature (22°C) and 5°C. A very minimal change in pH with temperature was noted for the three conventional buffer solutions. However, a marked increase of pH (about 0.25 to 0.66 units) was observed for all of Good’s buffer solutions. Table 2 lists the mean pH value of the five cryoprotective compounds at concentrations of 2 and 6% in TEST buffers with and without 20% egg yolk. The pH at 5°C (7.57) was significantly higher (P < 0.05)
Mean
TABLE 2 pH of TEST Buffer with and without Egg Yolk and/or 2 or 6% Cryoprotective Compounds (CP)” at Room Temperature (22°C) and 5°C 2% CP
TEST TEST TEST TEST Mean
yolk yolk CP
CP
6% CP
22°C
5°C
22°C
5°C
7.16 7.13 7.14 7.15 7.15
7.64 7.50 7.53 7.60 7.57
7.16 7.12 7.13 7.16 7.14
7.64 7.51 7.50 7.61 7.57
Nore. Mean pH at SC significantly than the mean pH at 22C.
greater
a CP, Glycerol, dimethylsulfoxide, diethylene
glycol,
20%
and triethylene
(P < .05)
ethylene glycol,
glycol.
ON
SEMEN
EXTENDER
pH
17
than at room temperature (7.14). The addition of egg yolk or cryoprotective compound did not alter the pH change significantly, even though a slight drop in pH (0.1 units) was noted with the addition of 20% egg yolk. It is apparent that there is a change in pH using Good’s buffer solutions and that it is primarily due to temperature effect. A similar change in pH has been reported for sodium bicarbonate/carbonate system (6). A continuous measurement of pH was made in a 30 ml solution of TEST yolk to determine if the observed increase of pH continued during freezing. Figure 1 shows the results of this experiment. The pH increased during cooling and freezing to 8.0 5 0.2 from 37 to - 14”C, at which point it dropped abruptly to a pH 6.5 * 0.2. Van den Berg (11) states that at the eutectic temperature of a solution, the solute concentration and pH may be different from the initial concentration and pH, depending on the respective solubilities of the dissolved salts and their ionic state. Van den Berg and Solliman (13) have demonstrated a decrease in pH during freezing for alkaline salts due to their low solubilities at higher concentration. Therefore, the drop in pH could probably be explained on the basis that TRIS salt, which is basic, may be crystallizing out of the solution at - 14°C. However, since the pH of egg yolk is about 6.8, it is also possible that both TES and TRIS (TEST) may be crystallizing at that temperature. Therefore, an attempt was made to determine the eutectic temperature of TES, TRIS, TEST, and TEST yolk. No eutectic freezing was noticed in any of the solutions examined when frozen to -30°C. However, it is known that proteins tend to mask the eutectic freezing (7). It is also possible that TES, TRIS, and TEST may transform into an amorphous form like glycerol (8) rather than a crystalline form and hence, there is no observable eutectic temperature. Nevertheless, these observations do not explain the abrupt drop in pH during freezing.
18
JEYENDRAN
AND
GRAHAM
8.00
7.00
e,
6.00
4oc
oc
-1zc
-18C
TEMPERATURE FIG.
1. pH during
cooling
and freezing
Foote (2), Steinback and Foote (9) have demonstrated the importance of pH on the fertility and longevity of fresh or frozenthawed spermatozoa. The exposure of spermatozoa to these adverse pH changes could be minimized by the proper rate of freezing. Also, it is possible that the spermatozoa may be less sensitive to these changes in pH at low temperatures, but it will be an important factor in cryopreservation of tissues and organs where slow freezing is employed. SUMMARY
The pH change of 10 different buffering systems with temperature ranging from room to 5°C was examined; three were conventional buffers which included phosphate yolk, citrate yolk, and skim milk. Seven were Good’s buffers with egg yolk which included TES, TRIS, BES, MOPS, PIPES, MES, and TEST. The pH of the three conventional buffers did not change with decreasing temperature, but Good’s buffers showed an increase in pH with de-
of TEST-yolk-buffer
solution.
creasing temperature from room to 5°C. The pH change due to temperature was measured for TEST buffer solution with and without 20% egg yolk containing 2 or 6% of five different cryoprotective compounds. The pH at 5°C was significantly higher than at room temperature. The addition of egg yolk and/or cryoprotective compound did not alter the pH significantly during cooling, even though a slight drop in pH was noted with the addition of egg yolk indicating that the change in pH is primarily due to the buffer. The pH of TEST yolk buffer (pH 7.2 at room temperature) was measured continuously from 37°C to below freezing (- 18°C). The pH increased with decreasing temperature to 8.0 + 0.2 from 37 to - 14°C at which point it dropped abruptly to pH 6.5 ? 0.2. REFERENCES 1. Finn, D. B. Denaturation of proteins in muscle juice by freezing. Proc. R. Sot. B 3,396 (1932). 2. Foote, R. H. Influence of pH on survival and fertility of bull sperm. J. Dairy Sci. 47, 807 (1964).
COOLING/FREEZING
EFFECTS
3. Good, N. E., Winget, G. D., Winter, W., Connally, T. N., Izawa, S., and Sign, M. M., Hydrogen ion buffers for biological research. Biachemistry 5, 467 (1966). 4. Graham, E. F., Crabo, B. F., and Bown, K. I. Effects of some zweitter ion buffers on the freezing and storage of spermatozoa. Bull. J. Dairy Sci. 55, 372 (1972). 5. Jeyendran, R. S., and Graham, E. F. An evaluation of cryoprotective compounds on bovine spermatozoa. Cryobiology 17, 458 (1980). 6. Long, C. Chemical data. In “Biochemists’ Handbook” (D. Long, Ed.), p. 19. Van Nostrand Princeton, N.J., 1961. 7. Meryman, H. T. The exceeding of a minimum tolerable cell volume in hypertonic suspension as a cause of freezing injury. In “The Frozen Cell.” G. E. W. Wolstenholme and M. O’Connor, (Eds.), p. 51. Churchill, London, 1970. 8. Rapatz, G., and Luyet, B. On the instability of frozen glycerol solutions stored at various low temperatures. Biod.vnmnica 10, 81 (1967). 9. Steinback, J., and Foote, R. H. Osmotic pressure
ON SEMEN EXTENDER
10. 11.
12.
13.
14.
pH
19
and pH effects on survival of frozen bovine spermatozoa. J. Dairy Sci. 50, 205 (1967). Tessier, H., and Rose, D. Composition of the liquid portion of frozen milk. Canad. J. Technol. 34, 211 (1956). Van den Berg, L. The effect of addition of sodium and potassium chloride to the reciprocal system: KH,PO,-Na,HPO,.H,O on pH and composition during freezing. Arch. Biochern. Biophys. 84, 305 (1959). Van den Berg, L., and Rose, D. Effect of freezing on the pH and composition of sodium and potassium phosphate solutions: The reciprocal system KH,PO,-Na,HP02.H20. Arch. Biothem. Biophys. 81, 319 (1959). Van den Berg, L., and Soliman, F. S. Composition and pH changes during freezing of solutions containing calcium and magnesium phosphate. Cryobiology 6, 10 (1969). Van den Berg, L., and Soliman, F. S. Effect of glycerol and dimethyl sulfoxide on changes in composition and pH of buffer salt solutions during freezing. Cr.vobio/ogy 6, 93 (1969).
19, 16-19
Effects
(1981)
of Cooling
and Freezing
R. S. JEYENDRAN2 Department
of Animal
Science,
AND
of
University
During freezing, ice formation increases the solute concentration of the unfrozen portion of an aqueous solution until it reaches the eutectic temperature of the solution and at this temperature, the solution is completely frozen. However, if a mixture of solutes are present as in semen extenders, then, the solute concentration and the pH of the solution will be determined by the relative eutectic temperature of the individual solutes during freezing. Van den Berg (11, 12) demonstrated this effect on sodium phosphate solution; his analysis revealed the pH of the unfrozen portion of slightly alkaline sodium phosphate solution at room temperature to be 3.6 at-9.9”C. Similar changes in pH were also observed with calcium and magnesium phosphate solutions (13, 14). Moreover, changes in pH during freezing of “muscle juice” (1) and milk (10) have been reported. To date, there is virtually no information available regarding measurement of pH at or below the freezing point for semen extenders. Therefore, the present study was designed to determine the changes in pH of extenders with and without egg yolk and cryoprotecting compounds, and the possible interaction between these during cooling and freezing to a temperature as low as -18°C. MATERIALS
AND
Experimental
St. Paul,
Minnesota
55108
Procedure
The pH of nine buffers and TEST with and without egg yolk and/or cryoprotective compound was determined at room temperature and 5°C using a Corning pH meter Model 12 and a Corning combination triple-purpose electrode. A continuous measurement of pH of a 30 ml sample of TEST yolk was made as it cooled gradually from 37 to 0°C and then to a temperature as low as - 18°C using a Beckman Model 3500 pH meter with a low-temperature combination electrode (Ingold Electrodes, Inc.). These measurements were replicated six times. The sample was stirred continuously to prevent supercooling during freezing.
Extenders
Ten 0.325 M buffers were used in the study. Three were conventional solutions: Received April 22, 1981; accepted July 28, 1981. ’ Scientific Journal Series Paper No. 11, 573, Minnesota Agricultural Experimental Station. * Present address: Institute of Reproductive Medicine, Chicago, 111. 60602. 16 Copyright @ 1982 by Academic F’ress, Inc. AU rights of reproduction in any form reserved.
E. F. GRAHAM Minnesota,
phosphate (KH,PO,-Na,HPO,* H,O), citrate (Na&H,0,.2H20), and skim milk (heated at 90°C for 10 min). Six were buffers described by Good et al. (3): (a) TES, N-Tris(hydroxy-methyl)methyl-2-aminoethanesulfonic acid; (b) TRIS, Tris(hydroxymethylaminomethane); (c) MES, 2-(Nmorpholino)ethanesulfonic acid; (d) MOPS, morpholino-propane sulfonic acid; (e) BES, N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; and (f) PIPES: Piperazine-NN- bis(Zethanesulfonic acid). The last buffer TEST was prepared by titrating TES with TRIS to pH 7.2 (4). Egg yolk (20% v/v) was added to all buffers except skim milk. The cryoprotective compounds: glycerol, dimethylsulfoxide, ethylene glycol, diethylene glycol, and triethylene glycol were added in concentrations of 2 and 6% v/v to TEST buffer, with and without 20% egg yolk, to determine their interactions with pH during cooling (5). TEST yolk buffer was the only extender used in the freezing experiment.
METHODS
001 l-2240/82/010016-04$02.00/O
on pH of Semen Extender’
COOLING/FREEZING
Mean
pH of 10 Buffers
EFFECTS
1
TABLE with
20%
Egg Yolk
Skim Milk) at Room Temperature
(Except
(22°C) and at 5°C
PH
Phosphate yolk Citrate yolk Skim milk TES yolk
TRIS yolk MES yolk BES yolk PIPES yolk MOPS yolk TEST yolk
RESULTS
22°C
5°C
Difference
7.12 7.15 6.62 5.41 9.06 4.78 5.33 6.93 5.31 7.12
7.10 7.10 6.70 5.92 9.72 5.22 5.72 7.18 5.59 7.51
AND
DISCUSSION
-0.02 -0.05 +0.08
+0.51 +0.66 +0.44 +0.39 +0.25 +0.28 +0.39
Table 1 lists the mean pH values of 10 buffer solutions at room temperature (22°C) and 5°C. A very minimal change in pH with temperature was noted for the three conventional buffer solutions. However, a marked increase of pH (about 0.25 to 0.66 units) was observed for all of Good’s buffer solutions. Table 2 lists the mean pH value of the five cryoprotective compounds at concentrations of 2 and 6% in TEST buffers with and without 20% egg yolk. The pH at 5°C (7.57) was significantly higher (P < 0.05)
Mean
TABLE 2 pH of TEST Buffer with and without Egg Yolk and/or 2 or 6% Cryoprotective Compounds (CP)” at Room Temperature (22°C) and 5°C 2% CP
TEST TEST TEST TEST Mean
yolk yolk CP
CP
6% CP
22°C
5°C
22°C
5°C
7.16 7.13 7.14 7.15 7.15
7.64 7.50 7.53 7.60 7.57
7.16 7.12 7.13 7.16 7.14
7.64 7.51 7.50 7.61 7.57
Nore. Mean pH at SC significantly than the mean pH at 22C.
greater
a CP, Glycerol, dimethylsulfoxide, diethylene
glycol,
20%
and triethylene
(P < .05)
ethylene glycol,
glycol.
ON
SEMEN
EXTENDER
pH
17
than at room temperature (7.14). The addition of egg yolk or cryoprotective compound did not alter the pH change significantly, even though a slight drop in pH (0.1 units) was noted with the addition of 20% egg yolk. It is apparent that there is a change in pH using Good’s buffer solutions and that it is primarily due to temperature effect. A similar change in pH has been reported for sodium bicarbonate/carbonate system (6). A continuous measurement of pH was made in a 30 ml solution of TEST yolk to determine if the observed increase of pH continued during freezing. Figure 1 shows the results of this experiment. The pH increased during cooling and freezing to 8.0 5 0.2 from 37 to - 14”C, at which point it dropped abruptly to a pH 6.5 * 0.2. Van den Berg (11) states that at the eutectic temperature of a solution, the solute concentration and pH may be different from the initial concentration and pH, depending on the respective solubilities of the dissolved salts and their ionic state. Van den Berg and Solliman (13) have demonstrated a decrease in pH during freezing for alkaline salts due to their low solubilities at higher concentration. Therefore, the drop in pH could probably be explained on the basis that TRIS salt, which is basic, may be crystallizing out of the solution at - 14°C. However, since the pH of egg yolk is about 6.8, it is also possible that both TES and TRIS (TEST) may be crystallizing at that temperature. Therefore, an attempt was made to determine the eutectic temperature of TES, TRIS, TEST, and TEST yolk. No eutectic freezing was noticed in any of the solutions examined when frozen to -30°C. However, it is known that proteins tend to mask the eutectic freezing (7). It is also possible that TES, TRIS, and TEST may transform into an amorphous form like glycerol (8) rather than a crystalline form and hence, there is no observable eutectic temperature. Nevertheless, these observations do not explain the abrupt drop in pH during freezing.
18
JEYENDRAN
AND
GRAHAM
8.00
7.00
e,
6.00
4oc
oc
-1zc
-18C
TEMPERATURE FIG.
1. pH during
cooling
and freezing
Foote (2), Steinback and Foote (9) have demonstrated the importance of pH on the fertility and longevity of fresh or frozenthawed spermatozoa. The exposure of spermatozoa to these adverse pH changes could be minimized by the proper rate of freezing. Also, it is possible that the spermatozoa may be less sensitive to these changes in pH at low temperatures, but it will be an important factor in cryopreservation of tissues and organs where slow freezing is employed. SUMMARY
The pH change of 10 different buffering systems with temperature ranging from room to 5°C was examined; three were conventional buffers which included phosphate yolk, citrate yolk, and skim milk. Seven were Good’s buffers with egg yolk which included TES, TRIS, BES, MOPS, PIPES, MES, and TEST. The pH of the three conventional buffers did not change with decreasing temperature, but Good’s buffers showed an increase in pH with de-
of TEST-yolk-buffer
solution.
creasing temperature from room to 5°C. The pH change due to temperature was measured for TEST buffer solution with and without 20% egg yolk containing 2 or 6% of five different cryoprotective compounds. The pH at 5°C was significantly higher than at room temperature. The addition of egg yolk and/or cryoprotective compound did not alter the pH significantly during cooling, even though a slight drop in pH was noted with the addition of egg yolk indicating that the change in pH is primarily due to the buffer. The pH of TEST yolk buffer (pH 7.2 at room temperature) was measured continuously from 37°C to below freezing (- 18°C). The pH increased with decreasing temperature to 8.0 + 0.2 from 37 to - 14°C at which point it dropped abruptly to pH 6.5 ? 0.2. REFERENCES 1. Finn, D. B. Denaturation of proteins in muscle juice by freezing. Proc. R. Sot. B 3,396 (1932). 2. Foote, R. H. Influence of pH on survival and fertility of bull sperm. J. Dairy Sci. 47, 807 (1964).
COOLING/FREEZING
EFFECTS
3. Good, N. E., Winget, G. D., Winter, W., Connally, T. N., Izawa, S., and Sign, M. M., Hydrogen ion buffers for biological research. Biachemistry 5, 467 (1966). 4. Graham, E. F., Crabo, B. F., and Bown, K. I. Effects of some zweitter ion buffers on the freezing and storage of spermatozoa. Bull. J. Dairy Sci. 55, 372 (1972). 5. Jeyendran, R. S., and Graham, E. F. An evaluation of cryoprotective compounds on bovine spermatozoa. Cryobiology 17, 458 (1980). 6. Long, C. Chemical data. In “Biochemists’ Handbook” (D. Long, Ed.), p. 19. Van Nostrand Princeton, N.J., 1961. 7. Meryman, H. T. The exceeding of a minimum tolerable cell volume in hypertonic suspension as a cause of freezing injury. In “The Frozen Cell.” G. E. W. Wolstenholme and M. O’Connor, (Eds.), p. 51. Churchill, London, 1970. 8. Rapatz, G., and Luyet, B. On the instability of frozen glycerol solutions stored at various low temperatures. Biod.vnmnica 10, 81 (1967). 9. Steinback, J., and Foote, R. H. Osmotic pressure
ON SEMEN EXTENDER
10. 11.
12.
13.
14.
pH
19
and pH effects on survival of frozen bovine spermatozoa. J. Dairy Sci. 50, 205 (1967). Tessier, H., and Rose, D. Composition of the liquid portion of frozen milk. Canad. J. Technol. 34, 211 (1956). Van den Berg, L. The effect of addition of sodium and potassium chloride to the reciprocal system: KH,PO,-Na,HPO,.H,O on pH and composition during freezing. Arch. Biochern. Biophys. 84, 305 (1959). Van den Berg, L., and Rose, D. Effect of freezing on the pH and composition of sodium and potassium phosphate solutions: The reciprocal system KH,PO,-Na,HP02.H20. Arch. Biothem. Biophys. 81, 319 (1959). Van den Berg, L., and Soliman, F. S. Composition and pH changes during freezing of solutions containing calcium and magnesium phosphate. Cryobiology 6, 10 (1969). Van den Berg, L., and Soliman, F. S. Effect of glycerol and dimethyl sulfoxide on changes in composition and pH of buffer salt solutions during freezing. Cr.vobio/ogy 6, 93 (1969).