Carbon dioxide and pH affect sperm motility of white sturgeon (Acipenser transmontanus)
Department of Biological Sciences and Center for Reproductive Biology, University of Idaho, Moscow, Idaho 83844-3051, USA
* Author for correspondence (e-mail: rolfi{at}uidaho.edu)
Accepted 14 June 2002
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Summary |
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Key words: Acipenser transmontanus, buffering capacity, carbon dioxide, pH, semen, sperm, motility, sturgeon, Acipenser transmontanus
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Introduction |
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As a species, sturgeon are routinely exposed to hypercapnia in their
natural environment (Crocker and Cech,
1998). Environmental hypercapnia is associated with an increase in
arterial PCO2 and a decrease in blood pH in the
white sturgeon A. transmontanus
(Crocker and Cech, 1998
;
Crocker et al., 2000
). We have
previously noted that maintenance of salmonid sperm at low pH or high
CO2 prevents the onset of motility, and reduces fertility, when
sperm are subsequently diluted with water (Bencic et al.,
2000a
,b
,
2001
). Further, the maximal pH
sensitivity of this capacity for motility corresponds to the pH range over
which the buffering capacity of seminal plasma is particularly low
(Ingermann et al., 2002
).
These findings suggest that the functional properties of salmonid sperm may be
especially sensitive to hypercapnia and resulting respiratory acidosis, as
well as metabolic acidosis, prior to release into the environment. As sturgeon
are subjected to environmental hypercapnia, especially in intensive
aquaculture settings (Crocker and Cech,
1996
,
1998
;
Farrell et al., 2001
), and
because fish are occasionally anesthetized with CO2 (e.g.
Fish, 1943
;
Prince et al., 1995
),
including during gamete harvesting, our previous findings with salmonids
suggest that environmental hypercapnia and respiratory and metabolic acidosis
could be associated with reductions in sperm motility and possibly fertility
in the sturgeon. In contrast, Gallis et al.
(1991
) reported that motility
of sperm from the Siberian sturgeon Acipenser baeri is relatively
insensitive to pH changes near physiological semen pH (approximately 8.1).
Williot et al. (2000
) also
found no correlation between sperm motility and pH in A. baeri.
Finally, Gallis et al. (1991
)
reported that semen from A. baeri shows a strong buffering capacity.
As preliminary observations from our laboratory suggested the opposite in the
white sturgeon A. transmontanus, this study was designed to determine
the pH sensitivity of the capacity for motility, the influence of
CO2, and the buffering capacity of semen of the Kootenai River
white sturgeon A. transmontanus.
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Materials and methods |
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The osmolality of sturgeon seminal plasma is low relative to that of
teleosts (Gallis et al., 1991;
Williot et al., 2000
).
Therefore, to determine the effect of pH on sperm motility, 250µl semen
samples were combined with 500µl of low osmolality, sperm immobilizing
buffer (SI buffer; in mmoll-1: NaCl 10; KCl 4; CaCl2
0.0125; Bis-Tris-Propane 50, titrated to various pH values with HCl). These
mixtures were maintained in 2ml sealed microcentrifuge tubes placed on their
sides at 10°C. After 2h, a portion of each sample was centrifuged for 3min
at 12000g (Marathon MicroA microcentrifuge, Fisher Scientific)
and the pH of the supernatant determined at 10°C (Accumet 815 MP pH meter,
Fisher Scientific). Also, the motility of a small portion of each sample was
assessed. The motility of sperm samples was determined using a BH2 Olympic
light microscope. Semen was diluted (>1:1000) using dechlorinated water and
sperm motility was estimated visually at room temperature (approximately
20°C) and expressed as a percentage of motile sperm relative to the total
number of sperm (Terner, 1986
;
Moccia and Munkittrick, 1987
;
Munkittrick and Moccia, 1987
;
Bencic et al., 2000b
.) The
identities of the treatment groups were unknown by the person evaluating
motility.
To examine the relationship between semen PCO2 and pH, 0.7ml semen samples were maintained at 10°C in 600ml metallized polyester bags (Kapak Corp., Minneapolis, MN, USA) filled with humidified air either with or without various amounts of injected CO2. Bags were flaccid, not taut. After 4h, CO2 levels were determined with a Model 920D Dual Trak CO2/O2 analyzer from Quantek Instruments (Northboro, MA, USA). Bags were subsequently cut open and the pH of semen samples determined immediately at 10°C. Similarly, to examine the effect of CO2 on sperm motility, 0.7ml semen samples were maintained for 4h in either the presence or absence of 4-5kPa CO2. After 4h at 10°C, bags were cut open and sperm motility assessed immediately.
The time course of sensitivity to low pH was assessed in the following manner. 250µl semen was added to 500µl SI buffer titrated to high or low pH. Within 1 min of mixing, a small portion of suspension was added to water and motility estimated. The remainder of the suspension was kept on wet ice and motility assessed periodically thereafter. At the end of the experiment, the pH of the centrifuged samples was measured at 10°C.
Buffering capacity analyses were conducted on semen and seminal plasma at
10°C. (Seminal plasma was obtained by centrifuging semen at
12000g for 3 min.) The buffering capacities of the fluid
samples were quantified as described
(Ingermann et al., 2002).
Basically, 10ml of BC saline (in mmoll-1: NaCl 110, KCl 40) was
added to 1ml of semen or seminal plasma. The pH of this solution was then
monitored at 10°C before and upon addition of small volumes of
0.1moll-1 HCl in BC saline. Buffering capacity was quantified as
µmol HCl 1ml-1 semen or seminal plasma pH unit-1
(Slyke; Wolters-Everhardt et al.,
1986
). Buffering capacities were calculated from linear
regressions of plots of pH versus HCl added over the ranges 7.5-8.5
and 6.0-7.0.
14 values of water pH taken from the Kootenai River near Bonners Ferry, ID, USA were obtained from Genevieve Hoyle (Kootenai Tribe of Idaho Fisheries Department) and four additional pH values of Kootenai River water were analyzed in our laboratory. The pH measurements were collected periodically from June 2001 to April 2002.
Chemicals were from Sigma Chemical Co. (St Louis, USA). All data were analyzed by one-way analysis of variance (ANOVA) followed by NewmanKeuls' test, with significance set at P<0.05. Data are presented as mean ± S.E.M.
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Results |
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Inhibition of the capacity for motility could also be achieved by prior maintenance of semen with CO2 (Table 1). With the exception of semen from 2 of 7 males, sperm maintained under 4-5 kPa CO2 for 4h at 10°C showed little, if any, motility upon addition to water (Table 1). Sperm from the single male that showed the lowest CO2 sensitivity in the capacity for motility did show marked pH sensitivity, but over a lower pH range than sperm from other males (Fig. 1; male, filled circle).
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The pH of semen maintained for 4h at 10°C without added CO2 was 8.93±0.06 (N=6, each for two determinations per male; Fig. 3). Addition of CO2 to semen under these conditions resulted in acidification, with the greatest change in semen pH occurring upon CO2 addition at relatively low PCO2 values (Fig. 3A); semen pH appeared less sensitive to changes in PCO2 values above approx. 1kPa. Nonetheless, pH plotted against the logPCO2 demonstrated a linear relationship over a range of 0.01 to 5.3 kPa CO2 that corresponded to a semen pH of approximately 9.0-7.3 (Fig. 3B).
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Addition of HCl to semen and seminal plasma resulted in multiphasic titration curves that demonstrated a lower buffering capacity at high pH than at low pH (Fig. 4A). This can be seen more clearly in Fig. 4B, which shows the buffering capacity relative to the pH of the solution. Quantification of buffering capacities as reciprocals of linear regressions of titration curves indicated that buffering capacities of semen and seminal plasma over the pH range 7.5-8.5 are about 20% of those over the pH range 6.0-7.0 (Fig. 4C). (Statistically indistinguishable results were obtained from four UCD semen samples; data not shown.) The ratio of buffering capacity of semen to that of seminal plasma from the same male was 1.11±0.02 (N=6) and 1.04±0.02 (N=4) over the pH range 7.5-8.5 for KT and UCD samples, respectively. The ratio of buffering capacity of semen to that of seminal plasma was 0.97±0.02 (N=6) and 0.99±0.02 (N=4) over the pH range 6.0-7.0 for KT and UCD samples, respectively.
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Water samples taken from the Kootenai River over a calendar year had pH values of 8.4±0.1 (N=18).
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Discussion |
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Sturgeon sperm maintained for several hours at low pH demonstrated little,
if any, motility when subsequently added to water. In contrast, sperm
maintained at high pH demonstrated appreciable motility when added to water.
Although the specific range of pH that controlled this capacity for motility
varied among males it was, nonetheless, relatively narrow, approximately 1 pH
unit. This is consistent with observations in salmonids
(Ingermann et al., 2002) but
appears to contrast with the results of studies of sperm collected from A.
baeri by Williot et al.
(2000
). They found no
correlation between semen pH and sperm motility. However, the pH values of all
of their semen samples exceeded 7.5 and thus correspond approximately to the
range of pH values in the current study (>7.8) that had little effect on
sperm motility.
Crocker and Cech (1998)
found that maintenance of A. transmontanus under 3.3-4.7 kPa
CO2 resulted in a decrease in arterial blood pH of approximately
0.7 pH units. Gallis et al.
(1991
) reported that the
normal pH of A. baeri seminal plasma is approximately 8.1. Therefore,
if the pH of the semen of A. transmontanus is comparable to that of
A. baeri, the current data suggest that exposure of the animal to
acute hypercapnia will have significant deleterious effects on the functional
properties of A. transmontanus sperm. Whether chronic hypercapnia is
associated with such putative effects is not clear because fish appear to
regulate pH upward under these exposures by adjusting bicarbonate levels
(Lloyd and White, 1967
;
Cameron and Randall, 1972
;
Crocker and Cech, 1998
).
Inhibition of the capacity for motility by exposure to low pH occurred
relatively rapidly. Within 1 min and 5 min, motility had decreased by about
26% and 50%, respectively (Fig.
2). Although it seems likely that the mechanism underlying this
inhibition is associated with intracellular pH change as in salmonids
(Bencic et al., 2000b), the
rapidity of response leaves open the possibility that low pH affects sperm
motility by an extracellular mechanism. A. transmontanus is a
broadcast spawner with gametes released into fast water
(http://refuges.fws.gov/fish/KootenaiRiverSturgeon.html).
Because sturgeon sperm remain motile for several minutes
(Gallis et al., 1991;
Linhart et al., 1995
;
Toth et al., 1997
), it is
possible that the shedding of sperm into acidic waters could be associated
with reduced sperm motility and fertility. Indeed, Gallis et al.
(1991
) reported that although
diluting the sperm of A. baeri 1:100 in a Tris-buffered activating
solution at pH 7.2 had little effect on motility, diluting sperm at pH 6.2
reduced the initial intensity to about 40% of the motility observed in pH 9.2
buffer. However, pH measurements of water samples from the Kootenai River
yielded values above 8.0 suggesting that pH sensitivity of sperm motility
after semen release from the male is not likely to be a concern with the
Kootenai River A. transmontanus.
Sperm maintained under high CO2 levels (4-5 kPa) generally showed poor, if any, motility upon addition to water relative to sperm maintained at low CO2 levels (<0.02 kPa; Table 1). Furthermore, the pH of semen maintained without added CO2 was 8.9 and addition of modest levels of CO2, up to about 1 kPa, resulted in a marked decrease in semen pH (Fig. 3A). Thus, the inhibition of the capacity for motility by CO2 is likely caused, at least in large part, by acidification of semen and probably an accompanying decrease in intracellular pH.
That modest levels of CO2 have a marked effect on semen pH
suggests that semen possesses low buffering capacity. Indeed, within the
approximate normal physiological range of pH7.5, the buffering capacity of
semen was low, and lower than that associated with pH values less than 7.5
(Fig. 4A,B). Furthermore, the
lowest buffering capacity noted in the present study
(Fig. 4B) corresponds to the
seminal plasma pH of 8.1 reported by Gallis et al.
(1991
) for A.
baeri.
Gallis et al. (1991)
reported that the buffering capacity for A. baeri semen was high.
From figure 3 in Gallis et al.
(1991
), it is clear that over
the pH range of approximately 2.5-10.8 there are several pH regions where
buffering capacity is relatively high; however, over the more physiologically
relevant pH range of 7.5-9.0, their results are consistent with our results
and demonstrate a low seminal buffering capacity.
Semen pH was highly sensitive to modest additions of CO2 up to approx. 1 kPa but appeared less sensitive to additions above that amount (Fig. 3A). However, Fig. 3B indicates that over the pH range of approximately 7.3-9.0, pH changed in a consistent and predictable manner in response to changes in PCO2. These results suggest that the low and relatively stable buffering capacity of semen over this pH range was responsible for the direct relationship between pH and logPCO2. The present findings suggest a nonlinear response would be noted if PCO2. values were raised such that the pH was sufficiently shifted into the range where seminal buffer capacity was greater (Fig. 4B).
To determine whether sperm contribute to the buffering capacity of the semen, the ratio of buffering capacity of semen to that of seminal plasma per male was calculated. These data suggest that within the pH range 7.5-8.5, sperm contribute perhaps 5-10% of the buffering capacity of semen whereas they appear to make no contribution over the pH range of 6.0-7.0. The relatively minor contribution of sperm to the semen at the higher pH values seems unlikely to be physiologically significant and may simply be a consequence of the presence of sperm proteins.
That the buffering capacity of sturgeon semen is low within a normal
physiological range is comparable to results obtained with salmonids
(Ingermann et al., 2002).
Morisawa and Morisawa (1988
)
noted that sperm taken directly from the testes of salmonids are immotile when
exposed to water while those taken from the distal portion of the sperm ducts
do become motile when exposed to water. The primary differences between semen
in the testes and in the reproductive ducts are higher pH values and higher
bicarbonate levels in semen in the reproductive ducts (Morisawa and Morisawa,
1986
,
1988
). We have suggested that
a low semen buffering capacity represents a physiological adaptation allowing
the epithelial cells of the reproductive tract to exert control over the
capacity for sperm motility by regulating semen pH via bicarbonate
secretion (Ingermann et al.,
2002
). A comparable situation may exist in the sturgeon as well:
the low semen buffer capacity may permit control of the potential for sperm
motility via pH regulation mediated by acid/base secretions into the
semen. However, the low buffering capacity coupled with the high pH
sensitivity of sperm motility within the physiological pH range may also make
the sturgeon sperm vulnerable to acute environmental hypercapnia and
respiratory and metabolic acidosis.
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Acknowledgments |
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References |
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Bencic, D. C., Krisfalusi, M., Cloud, J. G. and Ingermann, R. L. (2000a). Short-term storage of salmonid sperm in air versus oxygen. N. Am. J. Aquacult. 62, 19-25.
Bencic, D. C., Cloud, J. G. and Ingermann, R. L. (2000b). Carbon dioxide reversibly inhibits sperm motility and fertilizing ability in steelhead (Oncorhynchus mykiss). Fish Physiol. Biochem. 23,275 -281.
Bencic, D. C., Ingermann, J. G. and Cloud, J. G. (2001). Does CO2 enhance short-term storage success of chinook salmon (Oncorhynchus tshawytscha) milt? Theriogenology 56,157 -166.[Medline]
Billard, R. (1978). Changes in structure and fertilizing ability of marine and freshwater fish spermatozoa diluted in media of various salinities. Aquaculture 14,187 -198.
Cameron, J. N. and Randall, D. J. (1972). The effect of increased ambient CO2 on arterial CO2 tension, CO2 content and pH in rainbow trout. J. Exp. Biol. 57,673 -680.[Medline]
Ciereszko, A. and Dabrowski, K. (1994). Relationship between biochemical constituents of fish semen and fertility: the effect of short-term storage. Fish Biochem. Physiol. 12,357 -367.
Crocker, C. E. and Cech, J. J., Jr (1996). The effects of hypercapnia on the growth of juvenile white sturgeon, Acipenser transmontanus. Aquaculture 147,293 -299.
Crocker, C. E. and Cech, J. J., Jr (1998). Effects of hypercapnia on bloodgas and acidbase status in the white sturgeon, Acipenser transmontanus. J. Comp. Physiol. B 168,50 -60.
Crocker, C. E., Farrell, A. P., Gamperl, A. K. and Cech, J. J.,
Jr (2000). Cardiovascular responses of white sturgeon to
environmental hypercapnia. Am. J. Physiol.
279,R617
-R628.
Farrell, A. P., Thorarensen, H., Axelsson, M., Crocker, C. E., Gamperl, A. K. and Cech, J. J. (2001). Gut blood flow in fish during exercise and severe hypercapnia. Comp. Biochem. Physiol. A 128,549 -561.
Fish, F. F. (1943). The anesthesia of fish by high carbon dioxide concentrations. Trans. Am. Fish. Soc. 72,25 -29.
Gallis, J. L., Fedrigo, E., Jatteau, P., Bonpunt, E. and Billard, R. (1991). Siberian sturgeon, Acipenser baeri, spermatozoa: effects of dilution, pH, osmotic pressure, sodium and potassium ions on motility. In Acipenser (ed. P. Williot), pp. 143-151. Antony: Cemagref Publ.
Ingermann, R. L., Bencic, D. C. and Cloud, J. C. (2002). Low seminal plasma buffering capacity corresponds to high pH sensitivity of sperm motility in salmonids. Fish Physiol. Biochem. 24,299 -307.
Lahnsteiner, F., Berger, B., Weismann, T. and Patzner, R. A. (1998). Determination of semen quality of the rainbow trout, Oncorhynchus mykiss, by sperm motility, seminal plasma parameters, and spermatozoal metabolism. Acquaculture 163,163 -181.
Linhart, O., Mims, S. D. and Shelton, W. L. (1995). Motility of spermatozoa from shovelnose sturgeon and paddlefish. J. Fish Biol. 47,902 -909.
Lloyd, R. and White, W. R. (1967). Effect of high concentration of carbon dioxide on the ionic composition of rainbow trout blood. Nature 216,1341 -1342.
Moccia, R. D. and Munkittrick, K. R. (1987). Relationship between the fertilization of rainbow trout (Salmo gairdneri) eggs and the motility of spermatozoa. Theriogenology 27,679 -688.
Morisawa, S. and Morisawa, M. (1986). Acquisition of potential for sperm motility in rainbow trout and chum salmon. J. Exp. Biol. 126,89 -96.[Abstract]
Morisawa, S. and Morisawa, M. (1988). Induction of potential for sperm motility by bicarbonate and pH in rainbow trout and chum salmon. J. Exp. Biol. 136, 13-22.[Abstract]
Munkittrick, K. R. and Moccia, R. D. (1987). Seasonal changes in the quality of rainbow trout (Salmo gairdneri) semen: effect of a delay in stripping on spermatocrit, motility, volume and seminal plasma constituents. Aquaculture 64,147 -156.
Prince, A. M., Low, S. E. and Lissimore, T. J. (1995). Sodium bicarbonate and acetic acid: an effective anaesthetic for field use. N. Am. J. Fish. Management 15,170 -172.
Rurangwa, E., Volckaert, F. A., Huyskens, G., Kime, D. E. and Ollevier, E. (2001). Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standard fertilization in African catfish (Clarias gariepinus). Theriogenology 55,751 -769.[Medline]
Terner, C. (1986). Evaluation of salmonid sperm motility for cryopreservation. Prog. Fish Cult. 48,230 -232.
Toth, G. P., Ciereszko, A., Christ, S. A. and Dabrowski, K. (1997). Objective analysis of sperm motility in the lake sturgeon, Acipenser fulvescens: activation and inhibition conditions. Aquaculture 154,337 -348.
Williot, P., Kopeika, E. F. and Goncharov, B. F. (2000). Influence of testis state, temperature and delay in semen collection on spermatozoa motility in the cultured Siberian sturgeon (Acipenser baeri Brandt). Aquaculture 189, 53-61.
Wolters-Everhardt, M., Dony, J. M. J., Lemmens, W. A. J. G., Doesburg, W. H. and DePont, J.-J. H. H. M. (1986). Buffering capacity of human semen. Fert. Steril. 46,114 -119.[Medline]