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Objective analysis of sperm motility in the lake sturgeon, Acipenser fulvescens: activation and inhibition conditions
Aquaculture ELSEVIER
Aquaculture
154 (1997) 337-348
Objective analysis of sperm motility in the lake sturgeon, Acipenser fulvescens: activation and inhibition conditions Gregory P. Toth a,*, Andrzej Ciereszko b,c, Suzanne A. Christ a, Konrad Dabrowski b ’ MolecularEcology Research Branch, Ecological Exposure Research Division, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268, USA b School of Natural Resources, The Ohio State Uniuersity, 210 Kottman Hall, 2021 Coffey Road, Columbus, OH 43210, USA ’ Centre for Agrotechnology and Veterinary Sciences, Polish Academy of Sciences, IO-718 Olsztyn-Kortowo, Poland Received
10 February 1997; accepted 10 February 1997
Abstract An objective analysis of the duration of motility of sperm from the lake sturgeon, Acipenser has been performed using computer-assisted sperm motion analysis at 200 frames/s. Motility was measured in both 1993 and 1994. The percentage of activated motile sperm and their velocities ([ 19931 curvilinear velocity: 313 pm/s; straight-line velocity: 129 pm/s) were unchanged after 5 min in Tris/glycine buffer, pH 9.0, with 10 mM added Na+. Qualitative, visual measurements revealed motility lasting for 30 min in this medium. The percentage of motile cells, but not the velocities of the motile cells, was reduced by the addition of potassium to the Tris/glycine buffer. The percentage of motile sperm was inhibited by 50% at 0.5 mM added K+. Elemental analysis of seminal plasma revealed the following concentrations: P (3.02 mM); K (5.78 mM); Ca (0.16 mM); Mg (0.21 mM); Na (25.6 mM); Zn (0.76 FM); and Cl (5.41 mM). Differences between two years of milt collection were not significant. 0 1997 Elsevier Science B.V.
fulcescens,
Keywords:
Sturgeon;
Sperm motility; CASA
* Corresponding author. U.S. EPA, MD-642,
National
Exposure
Research
45268. 0044-8486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SOO44-8486(97)00066-5
Laboratory,
Cincinnati,
OH
338
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1. Introduction Both the basic study of sturgeon reproductive biology and the production of domesticated fish would be aided by a quantitative evaluation of the motility of sturgeon sperm. In contrast to the spermatozoa of teleosts that spawn in fresh water (common carp, Cyprinus carpio; rainbow trout, Oncorhynchus mykiss; yellow perch, Perca jlaL;escens), which remain motile for less than I min post-activation, the spermatozoa of Chondrostei such as sturgeon retain their motility and their fertilizing capacity for several hours after activation (Ginsburg, 1972; Detlaff et al., 1993). Gallis et al. (199 1) reported on the effects of dilution, pH, osmotic pressure, and sodium and potassium ions on the activation of sperm from the Siberian sturgeon, Acipenser baeri. Under physiological conditions, K+ concentration, but not osmolality, seemed to be the principal cause of sperm immotility. Until now, the evaluation of sturgeon sperm motility has relied on subjective, qualitative assessments of activated sperm movement (Persov, 1941 [as reported in Detlaff et al., 19931; Gallis et al., 1991). Previously computer-assisted sperm motion analysis technology has been used to evaluate optimal activation conditions and seasonal variation for sperm from the common carp (Toth et al., 1995; Christ et al., 1996). Computer digitization of the sperm head after videotaping under phase contrast microscopy allowed accurate and reliable measurements of the physical parameters of sperm motion, curvilinear and straight-line velocity. The percentage of motile sperm was measured using software criteria (minimum straight-line velocity) to define movement apart from drift and shaking with no forward progression of the sperm head. The present study describes the application of this technology to the evaluation of semen from the lake sturgeon, Acipenser fuluescens, captured in the Wolf River, Wisconsin, in May, 1993 and April, 1994. Our objectives were to (1) visually assess the (given the report of Gallis et al. duration of motility across Na ’ ion concentrations (1991) on the lack of an effect of Na+ concentration on intensity but no report on duration), (2) objectively measure the percentage of motile sperm and their velocities after videomicroscopy at 200 frames/s between 5 and 12 s after activation in media of and (3) measure the concentration of ions in varying Na+ and Kf concentrations, seminal plasma in relation to their regulatory role in sperm activation. Given that the dependence of motility activation on ionic conditions and the seminal plasma ion concentrations were examined over two consecutive years, variation in motion endpoints across years could be explained in terms of seasonal temperature effects. Although the 1993 and 1994 studies differed with respect to sperm storage period, recognition of these annual changes was possible.
2. Materials and methods Lake sturgeons (A. fuluescens) were obtained from the Wolf River (Wisconsin) near Shawano dam (May 1993, April 1994). Semen was collected within 5 min after capture from nine males, 121-140 cm length, 15-22 yrs old (R. Bruch, Wisconsin Department of Natural Resources). Fish capture and semen collection were as described in Folz et al.
G.P. Toth et al./Aquaculture
154 (19971 337-348
339
r
Sturgeon #930000 1
/ VCL Histogram
- 1994
1
0 0
100
300 200 Curvilinear Velocity
400
Sturgeon #940081 \ Fig 1. Distributions year.
of curvilinear
velocities
for individual
sperm from each of two fish from each sampling
(1983). Rather small volumes (l-5 ml) of milt were obtained from individual fish in 1993. However, in 1994, more semen was obtained (4-32 ml). Extreme care was used in collection to avoid contamination with urine. Perchec et al. (199.5) have shown that the ratio of high velocity to low velocity sperm changes significantly immediately with the addition of urine to catheter-collected semen. Fig. 1 shows that the distributions of curvilinear velocities from one fish from each sampling year were unimodal and contained no sperm of low velocity.
G.P. Toth et al./Aquaculture
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I54 (1997) 337-348
Semen was stored on melting ice (0°C) and transported within one day to the laboratory where it was stored for an additional day, under an atmosphere of oxygen (Ciereszko et al., 1994). Analyses of sperm motility were conducted 3 days post collection in 1993, while the 1994 studies were conducted on sperm stored for 9 days. 2.1. Preparation
of seminal plasma and analysis
The subsamples of milt (0.5 ml) from nine males were centrifuged (4°C) at 7000 g for 10 min. Three samples of pooled seminal plasma were formed, each consisting of three individual seminal plasma samples. Ionic composition of seminal plasma was measured by the inductively couple plasma (ICP) emission spectrophotometry method with the use of a ARL-3560 spectrometer (Applied Research Labs, Valencia, CA). 2.2. Manual assessment
of sperm motility
The milt from 1993 (n = 3) was diluted 100 times with 20 mM Tris, 30 mM glycine, pH 9.0, supplemented with NaCl (O-50 mM). The duration of motility (time from activation to less than 1% motility) was recorded at 12°C. 2.3. Dilution and actiuation of semen All solutions and microscopic equipment were kept in an environmental chamber maintained at 10°C. Semen was diluted 1:500 (1993) to 1:750 (1994) for activation in 50 mM Tris/glycine buffer, pH 9.0, with either 0, 10, or 25 mM Na+ added. For the inhibition studies 0, 0.1, 0.3, 0.6, or 1.0 mM K+ was added to the Tris/glycine buffer. 2.4. Videomicroscopy Less than 5 ~1 of the activated sperm suspension was loaded into a 20-pm deep P-Cell semen analysis chamber (Fertility Assoc., MA) and was videotaped on an Olympus BH2 microscope (phase contrast; 50 X total magnification) using a 200 Hz video camera (Motion Analysis, CA) and a high speed (200 Hz) video cassette recorder (V-32 VTR, Nat, Japan). An internal stopclock in the video tape recorder was started when the milt was mixed with an activating medium to record the time elapsed following initiation of motility. Videotaping commenced by 5 s post activation and was carried out to 30 s. Activated suspension of each sample was added to new analysis chambers, 2 and then 5 min post-activation to assess the time course of motility. Two repetitions of each milt sample were performed for each activation medium. 2.5. Computer-assisted
sperm motion analysis
The CellTrak/S computer-assisted sperm analysis system (CellTrak, Version 3.20) from Motion Analysis was used for measurement of sturgeon sperm motion endpoints. A detailed description of the derivation of the motion measurements can be found in Boyers et al. (1989). Table 1 lists the user-adjusted calibration settings. The VP110
G.P. Toth et al./Aquaculture Table 1 Cell trak research
341
154 (1997) 337-348
system settings
Frame rate Duration of data capture Minimum path length Minimum motile speed Maximum burst speed Distance scale factor Camera aspect ratio ALH path smoothing factor Cent. X search neighborhood Cent. Y search neighborhood Cent. cell size minimum Cent. cell size maximum Path max. interpolation Path prediction percentage Depth of sample Video processor model
200 frames/s 25 frames 25 frames 25 microns/s 1000 microns/s 1.2769 microns/pixel 1.0220 23 frames 2 pixels 2 pixels 4 pixels 16 pixels 2 frame 0% 20 microns VP1 10
video processing unit was operated in the quad edge mode since digitized sperm heads were elliptical (the horizontal edges contained more pixels than the vertical edges for sperm in some orientations). Output from the CellTrak analysis includes (1) the percentage of motile cells (those that have straight-line velocities over the minimum threshold velocity [25 pm/s]), (2) the curvilinear velocity (VCL) (the time-average velocity of the sperm head along its actual trajectory), and (3) the straight-line velocity (VSL) (the time-average velocity of the sperm head along a straight line from its first detected position to its last detected position), and (4) the amplitude of lateral head
0 Plot showing Actual sperm head motion (dotted) 0 Smoothed
head motion for ALH computations
(line)
238.73 1 Fig. 2. Diagram of an actual track from a sturgeon computerized motion endpoints.
sperm analysis
illustrating
the various sperm
342
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154 (1997) 337-348
displacement (ALH), the displacement of the actual track from a calculated path. Fig. 2 shows an actual sample path with the associated velocity vectors. 2.6. Statistical
average
analysis
Data were sufficiently normal 197 1). Effects of time, added computer-assisted sperm motion variance. Multiple comparisons
and the variance homogeneous (Levene’s test) (Wirier, Na+ concentration, and added K+ concentration on endpoints were each analyzed by a one way analysis of were done according to the Tukey procedure.
3. Results 3.1. Sperm motility - 1993 -
-----.-.-
3x0
25(x)-
3
2m-
I
2 J
% “E
1500-
: ‘i s
1m-
0 0
I 10
I Xl
I 30
I 40
-47 so
60 Na+[mMl
Fig. 3. Effect of sodium ion on duration of sperm motility - subjective observations.
G.P. Toth et al./Aquaculture Table 2 1993 Sturgeon
sperm motility endpoints
1.54 (1997) 3.37-348
343
(n = 2)
Time (min)
0 mM Na+
lOmMNa+
2.5 mM Na,!
% Motile sperm
0 2 5
,s7.0+ 1.9” ,56.5 f 11.3” ,36.0k3.0”b
,58.1 +6.6h ,65.7 f 3.3” ,55.5 * 10.8’
78.5 + 9.3”b \ 14.G 10.9b ,13.3+9.@
VCL (pm/s)
0 2 5
,312.9*42.3” ,318.3i5.9” ,306.5 * 24.3”
,315.7+6.4” ,313.3*6X’ ,304.3 + 11.5”
,308.3f 13.1” ,320.6 + 15.4” ,251.0*31.3“
VSL (km/s)
0
r 128.8 + 26.0“
5
,127.9+ 13.0“ ,73.6 f 5.0ab
r120.0i2.8” ,98.0+3.0.‘h ,93.7 f 13.8”
rl 15.6k 12.8” ,50.7 f 27.0h p3.9 + 13Xb
Linearity
0 2 5
,41.2*2.7” ,40.5 i 3.4” ,23.9 f 3.3”
,38.2?0.8” ,32.9f3.3”h ,31.6*3.8”
(37.7 f 5.8 d ,15.8f7.2b r17.4+3.5”
ALH
0 2 5
,4.05 * 0.37” ,4.29 i 0.06” ,4.42 i 0.40”
,4.38 f0.23” ,4.70+0.2Fh ,4.50*0.17”
,4.25 + 0.03“ ,5.18*0.29b ,4.81 +0.55”
“Superscripts with the same letters within a time (row) are not significantly different ( p I 0.05). ,Subscripts with the same letters within a treatment (column) are not significantly different ( p 5 0.05) Values are means f population standard deviations.
observed sperm in 10 mM Na+ described above (i.e., the longest subjectively-observed duration was observed with 10 mM Na+). The curvilinear velocity remained essentially unchanged over time and concentration of added Naf, while the straight-line velocity decreased steadily over time with no added Na+ or 25 mM Na’ (changes were insignificant based on variation between the two samples at 2 min). Two minutes after activation, addition of 25 mM Na+ brought about significant changes in linearity and ALH relative to those samples with no added Na+. 3.2. Sperm motility - 1994 - Qect
qf added sodium
Although velocities lower than those of 1993 were evident in the 1994 data set (Table 3), the percentages of motile sperm ranged within the 1993 data set. The addition of 25 mM Na+ to the Tris/glycine buffer resulted in no loss of the percentage of motile sperm during the first 5 min. A significant reduction in motile sperm in relation with time was seen with no added Na+, while 10 mM added Na+ reduced motility means by more than half. The lack of statistical significance of the latter observation should be viewed in the context of the high variability. Velocities and sperm trajectory pattern indices (linearity and ALH) were unchanged across media Na+ concentrations or across time. 3.3. Sperm motility - 1993 - effect
qf added potassium
The effect of added K+ on sturgeon sperm motility is shown in Fig. 4. A clear dose response is seen with a 50% reduction in the percentage of motile sperm at approxi-
344 Table 3 1994 Sturgeon
G.P. Toth et al./Aquaculture
sperm motility endpoints
154 (1997) 337-348
(n = 5)
Time (min)
OmMNa+
10 mM Na+
25 mM Na:
0 2 5
,564 + 22.1” ,,26.4+23.5 ,12.2+8.61”
,51.8+25.5” ,45.8 f 25.5 ,21.0+27.7”
49.8 + 24.6” ,34.9* 18.5 ,42.4+ 18.0a
VCL (pm/s)
0 2 5
217.2*68.5# 207.6 f 63.5 201.5+62.0
210.7i61.0 214.3k53.6 199.3rt41.3
218.lk44.2 228.1 f 56.3 213.8552.7
VSL (I*.m/s)
0 2 5
61.9+ 14.5 55.8 + 24.9 46.7 f 18.5
67.4+ 11.1 47.8 + 14.6 53.4+ 19.7
57.4* 12.0 48.9 + 12.6 41.3k7.4
Linearity
0 2 5
32.6 + 14.9 30.6+ 18.1 25.8 + 14.1
35.4+ 13.9 25.0* 12.2 28.7 + 13.6
28.3 + 8.6 23.9+ 10.7 21.7+8.8
ALH km
0 2 5
3.68 kO.35 3.84kO.30 4.19f 1.14
3.51 kO.20 3.5orto.30 3.44 f 0.34
3.82+0.26 3.84 + 0.3 1 3.54+0.44
% Motile sperm
‘Superscripts with the same letters within a time (row) are not significantly different ( p I 0.05). &Subscripts with the same letters within a treatment (column) are not significantly different ( p < 0.05). Comparisons within and across time not shown were not significant. Values are means + SD.
STURGEON
SPERM
MOTILITY
Effect of Added
K’
60 -
- 240
40 -i
- 160
________-------.o-_,,
0
-M-
0.3 K’ Concentration Percent Motile
.. *...
“C.
0.6 (mM) --0--
“SL
Fig. 4. Effect of potassium ion on CASA-measured sperm motility. Motility means with the same letters are not significantly different ( p I 0.05) (n = 2). Error bars denote standard deviations.
G.P. Totit rt al. /Aquculture
154 flYY7J 337-34X
345
Table 3 Seminal plasma ion concentrations
Na
K P Cl Ca Mg Zn
I993 Mean (mM) f SD
I994 Mean (mM) f SD
25.6 * 2.8 5.78 i 0.49 3.02 + 0.37 5.41 * 2.79 0.16+0.05 0.2 I * 0.02 0.00076 f 0.000 I5
x1.8+7.0 6.97 + I .42 3.665 1.37 2.3 I k I .28 0.13kO.02 0.22 f 0.07 0.00076 + 0.00030
mately 0.5 mM added Kf (Tris/glycine)(n = 2). Curvilinear velocity and straight-line velocity were unchanged for the remaining motile cells across all concentrations of potassium. 3.4. Seminal plasma inn corlcentrutions Table 4 shows the ion concentrations determined for seminal plasma from the lake sturgeon in two spawning seasons. There were no significant differences between ion concentrations, although chloride levels were twice as high in 1993 as in 1994.
4. Discussion The present study of sperm motility in the lake sturgeon, A. ,jidce.scer~~, provides for the first time an objective demonstration of the extended duration of motility, previously reported by Gallis et al. (1991). Gallis et al. (1991) described the sperm from the Siberian sturgeon, A. baeri, showing subjective sperm motility evaluations in domesticated fish. The activation medium consisting of the Tris/glycine buffer with 10 mM added Na+ provided no loss in the percentage of motile sperm after 5 min and no decrease in the velocities of the sperm. Visual microscopic observation revealed maintenance of motility over 30 min. Duration of motility in our studies exceeded that described by Gallis et al. (1991), where ‘intensity’ was decreased within the first minutes after activation. Gallis et al. (1991) examined motility at room temperature while our subjective study was conducted at 12°C. Billard and Cosson (1992) reported that increased temperature (range: 5” to 26°C) increased the beat frequency of trout sperm and decreased the duration of forward movement. A temperature of 12°C is more relevant to natural spawning activities. Several years of observations with Wolf River lake sturgeon indicate spawning being associated with a relatively narrow range of water temperature (9- 11“C, R. Bruch, personal communication). Nevertheless, the duration of sperm motility at either temperature exceeds that of sperm from teleosts that spawn in fresh water (trout < 30 s; carp < 120 s). As described by Detlaff et al. (1993). the increased duration of sturgeon sperm motility and fertility increases the effectiveness of natural spawning which takes place in strong river currents. Our results show swimming
346
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154 (1997) 337-348
velocities (straight-line velocities of 120-130 pm/s and curvilinear velocities of 310-320 pm/s> similar to those described for other fish sperm, both freshwater and marine. Redondo-Miiller et al. (1991) reported trout sperm curvilinear velocities of approximately 250 km/s. There were two objectives in studying the motility characteristics of sturgeon sperm. One was the characterization of the factors influencing activation of the sperm and the second was the setting of reference values for fresh sperm for a given location and date of spawning. Given the inability to conduct on site experimentation with electronic equipment and microscopes that were not field-ready, attainment of the first objective depended upon transport of sperm samples to the distant laboratory and attainment of the second goal was left for another time. The assumption was made that given moderate motility (percentage of motile sperm > 50%), long-term storage (9 days) would not influence the nature of the activation and inhibition of sturgeon sperm. Sample size was limiting and no argument is made that this is a weakness of the study. However, the present studies have adequate sample size to illuminate factors influencing activation and inhibition of sturgeon sperm as previously described subjectively by Gallis et al. (1991). In the present work, a half-maxima1 inhibition of motility activation has been reported following the addition of 0.5 mM K+ to the Tris/glycine buffer. This observation confirms and extends those of Gallis et al. (1991) who reported complete inhibition in the presence of 0.1 mM potassium ion. Confirmation that the osmotic pressure of seminal plasma (38 mosM/kg, see below) is not limiting to sperm activation, the present study shows that activation of sturgeon sperm occurs over a theoretical range of 50 to 100 mosM/kg. Gallis et al. (1991) reported a mean osmotic pressure for seminal plasma of the Siberian sturgeon of 38 mosM/kg. This value is far below those of the freshwater, amphihaline, and marine species of teleosts (range: 232-400 mosM/kg) as listed by Suquet et al. (1994). The major elemental seminal plasma constituents are described for the lake sturgeon. These observations extend those of Gallis et al. (1991) who reported mean sodium and potassium concentrations of 28 mM and 2.5 mM, respectively, in Siberian sturgeon seminal plasma. The sodium and chloride concentrations in the lake sturgeon (25-30 mM and 2-5 mM, respectively) mark the major contributions to the differences between the chondrosts and teleosts - Na: 140 mM and Cl: 150 mM in rainbow trout (Morisawa and Morisawa, 1988) - Na: 133 mM and Cl: 129 mM in turbot (Suquet et al., 1993). The potassium concentration (6 mM) of lake sturgeon seminal plasma resembles those of marine teleosts, such as turbot (3.8 mM) (Suquet et al., 1994). It is of interest that the added Na+ concentrations eliciting no loss of motility 5 min post-activation differed between the study years (1993, 10 mM Na+; 1994, 25 mM Na+). Given that the seminal plasma ions contributing most to osmotic pressure (Na+ and Kf) appeared to be higher in 1994, it is suspected that the sperm from the 1994 sampling could be activated with a higher osmolality medium - i.e., a 25 mM Na+ addition to Tris/glycine - since the osmotic change would remain constant. Clemens and Grant (1965) reported on a seminal thinning response (or hydration) in carp and rainbow trout after injections of pituitary extracts. Changes in the water and salt content
C.P. Toth et ul./Ayuaculture
154 IIYY7) 337-348
347
and sperm cell density of semen occurred in direct proportion to doses of these extracts. It could be hypothesized that since the sturgeon spawned early in 1994 that the hydration of their testes would not have been as substantial as at a later time. While the ion concentrations in the present study were not significantly different across years, it is felt that the reported trends are not inconsistent with the hypothesis. Several advantages are gained by the application of computer-assisted sperm motion analysis (CASA) to the measurement of sturgeon sperm motility endpoints. For adequate measurement of oscillating biologic systems, the sampling rate should be two to four times the frequency of the event (Boyers et al., 1989; Davis and Katz, 1993). The high speed videomicrography (200 frames/s) used in the present study allowed adequate sampling of trajectories of fish sperm with an initial flagellar beat frequency between 40 and 60 Hz. Previous references to fish sperm motility have used only straight-line velocities and beat frequencies (Billard and Cosson, 1992; Oda and Morisawa, 1993). The latter employed a computer-assisted analysis system (CellSoft@), but was not working at video speed above 30 Hz (which are not required for straight-line velocity). Measurement of physical components of the trajectory of the sperm such as curvilinear velocity and linearity provides indices of pattern which may be more robust indicators of the fertilizing capacity. In the present study, automated tracking of the phase-contrast-illuminated sperm head required digitization of the entire sperm head (4 edge analysis in contrast to the 1 edge analysis used for carp sperm [Toth et al., 199.51) since the sturgeon sperm head is elliptical. Tracking of one permanent XY quadrant (in the 1 edge mode) could allow biased tracking of the center of the sperm head, allowing oscillation between ellipse axis maxima and minima.
5. Conclusions Computer-assisted sperm motion analysis of activated lake sturgeon sperm revealed peak durations of sperm motility with unchanging velocity parameters for up to 5 min after addition of various concentrations of Na ion to Tris/glycine buffer. Added potassium ion inhibited activation with an apparent EC,, of 0.5 mM K+. This ability to objectively analyze sturgeon sperm is already being used in other studies of cryopreservation in the lake sturgeon.
Acknowledgements We thank Ron Bruch, Steven Fajfer, and the personnel of the Wisconsin Department of Natural Resources for their help in the collection of lake sturgeon spermatozoa. The assistance of Dr. Joni Torsella in statistical analyses was appreciated. This document has been reviewed in accordance with U.S. Environmental Protection Agency and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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References Billard, R., Cosson. M.P., 1992. Some problems related to the assessment of sperm motility in freshwater fish. J. Exp. Zool. 261, 122-131. Boyrrs, S.P., Davis, R.O.. Katz, D.F., 1989. Automated Semen Analysis. Curr. Probl. Obstet. Gynecol. Fertil. 12, 165-200. Christ, S.A.. Toth, G.P.. McCarthy, H.W., Torsella, J.A., Smith, M.K., 1996. Monthly variation in sperm motility in common carp. Cyprinus carpio, assessed using computer-assisted sperm analysis (CASA). Journal of Fish Biology, 48, 1210-1222. Ciereszko, A., Dabrowski, K., Lin, F., Doroshov, S.I., 1994. Identification of trypsin-like activity in sturgeon spermatozoa. J. Exp. Zool. 268, 486-49 I. Clemens, HF., Grant, F.B.. 1965. The seminal thinning response of carp (C~prinus ctrrpio) and rainbow trout (Snlnlo ~~crird,zerii) after injections of pituitary extracts. Copeia 2, I74- 177. Davis, R.O., Katz, D.F., 1993. Operational standards for CASA instruments. .I. Androl. 14, 385-394. Detlaff, T.A., Ginsburg, AS., Schmalhausen, 0.1.. 1993. Embryonic development: Gametes. In: Sturgeon fishes: Developmental biology and aquaculture. Springer-Verlag, pp. 61-64. Gallis, J.L., Fedrigo, E., Jatteau, P.. Bonpunt, E., Billard, R., 1991. Siberian sturgeon, Aciprrlsrr haeri, spermatozoa: effects of dilution, pH. osmotic pressure, sodium and potassium ions on motility, In: Williot, P. (Ed.). Acipenser. Cemagref Pub]., pp. 143-15 I Ginsburg, A.S.. 1972. Fertilization in fishes and the problem of polyspermy. Israel Program for Scientific Translations, p. 140. Moriaawa, S.. Moriaawa, M., 1988. Induction of potential for sperm motility by bicarbonate and pH in rainbow trout and chum salmon. .I. Exp. Biol. 136, 13-22. Oda, S., Morisawa. M., 1993. Rises of intracellular Ca+’ and pH mediate the initiation of sperm motility by hyperosmolality in marine teleosts. Cell Motil. Cytoskeleton 25, 17 I - 178. Perchec. G.. Cosson, _I.. Jeulin, C., Paxion. C., Andre, F., Billard, R., 1995. Alteration of carp (Cypriuts ctrr/?io) apermatoLoa motility by urine contamination during sampling. Proceedings of the Fifth International Symposium on the Reproductive Physiology of Fish, The University of Austin, TX, 2-8 July 1995. Fish Symposium 95. Austin, p. 135. Redondo-Miller. C.. Coason. M.-P., Cosson, J., Billard, R., 1991. In vitro maturation of the potential for movement of carp spermatozoa. Mol. Reprod. Dev. 29, 259-270. Suquet. M., Billard. R., Cosson, J., Dorange, G.. Chauvand, L., Mugnier, C., Fauvel, C., 1994. Sperm features in turbot (.Sco/~h/h~~/rtuu nu~irm~s): a comparison with other freshwater and marine fish species. Aquatic Living Resources 7, 283-294. Suquet. M., Dorange, G., Omnes, M.H., Normant. Y., Le Roux, A., Fauvel, C., 1993. Composition of the seminal fluid and ultrastructure of the spermatozoon of turbot (Sco@?tha/mus nmxinnrs). Journal of Fish Biology 42. 509-5 16. Toth. G.P., Christ, S.A.. Torsella, J.A., McCarthy, H.W., Smith, M.K., 1995. Computer-aaaisted sperm motion analysis of sperm from the common carp (Cyprinus carpio). Journal of Fish Biology 47, 9X6- 1003. Wirier. B.J.. 1971. Statistical principles in experimental design. McGraw-Hill, New York.
Aquaculture
154 (1997) 337-348
Objective analysis of sperm motility in the lake sturgeon, Acipenser fulvescens: activation and inhibition conditions Gregory P. Toth a,*, Andrzej Ciereszko b,c, Suzanne A. Christ a, Konrad Dabrowski b ’ MolecularEcology Research Branch, Ecological Exposure Research Division, National Exposure Research Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH 45268, USA b School of Natural Resources, The Ohio State Uniuersity, 210 Kottman Hall, 2021 Coffey Road, Columbus, OH 43210, USA ’ Centre for Agrotechnology and Veterinary Sciences, Polish Academy of Sciences, IO-718 Olsztyn-Kortowo, Poland Received
10 February 1997; accepted 10 February 1997
Abstract An objective analysis of the duration of motility of sperm from the lake sturgeon, Acipenser has been performed using computer-assisted sperm motion analysis at 200 frames/s. Motility was measured in both 1993 and 1994. The percentage of activated motile sperm and their velocities ([ 19931 curvilinear velocity: 313 pm/s; straight-line velocity: 129 pm/s) were unchanged after 5 min in Tris/glycine buffer, pH 9.0, with 10 mM added Na+. Qualitative, visual measurements revealed motility lasting for 30 min in this medium. The percentage of motile cells, but not the velocities of the motile cells, was reduced by the addition of potassium to the Tris/glycine buffer. The percentage of motile sperm was inhibited by 50% at 0.5 mM added K+. Elemental analysis of seminal plasma revealed the following concentrations: P (3.02 mM); K (5.78 mM); Ca (0.16 mM); Mg (0.21 mM); Na (25.6 mM); Zn (0.76 FM); and Cl (5.41 mM). Differences between two years of milt collection were not significant. 0 1997 Elsevier Science B.V.
fulcescens,
Keywords:
Sturgeon;
Sperm motility; CASA
* Corresponding author. U.S. EPA, MD-642,
National
Exposure
Research
45268. 0044-8486/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved PII SOO44-8486(97)00066-5
Laboratory,
Cincinnati,
OH
338
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1. Introduction Both the basic study of sturgeon reproductive biology and the production of domesticated fish would be aided by a quantitative evaluation of the motility of sturgeon sperm. In contrast to the spermatozoa of teleosts that spawn in fresh water (common carp, Cyprinus carpio; rainbow trout, Oncorhynchus mykiss; yellow perch, Perca jlaL;escens), which remain motile for less than I min post-activation, the spermatozoa of Chondrostei such as sturgeon retain their motility and their fertilizing capacity for several hours after activation (Ginsburg, 1972; Detlaff et al., 1993). Gallis et al. (199 1) reported on the effects of dilution, pH, osmotic pressure, and sodium and potassium ions on the activation of sperm from the Siberian sturgeon, Acipenser baeri. Under physiological conditions, K+ concentration, but not osmolality, seemed to be the principal cause of sperm immotility. Until now, the evaluation of sturgeon sperm motility has relied on subjective, qualitative assessments of activated sperm movement (Persov, 1941 [as reported in Detlaff et al., 19931; Gallis et al., 1991). Previously computer-assisted sperm motion analysis technology has been used to evaluate optimal activation conditions and seasonal variation for sperm from the common carp (Toth et al., 1995; Christ et al., 1996). Computer digitization of the sperm head after videotaping under phase contrast microscopy allowed accurate and reliable measurements of the physical parameters of sperm motion, curvilinear and straight-line velocity. The percentage of motile sperm was measured using software criteria (minimum straight-line velocity) to define movement apart from drift and shaking with no forward progression of the sperm head. The present study describes the application of this technology to the evaluation of semen from the lake sturgeon, Acipenser fuluescens, captured in the Wolf River, Wisconsin, in May, 1993 and April, 1994. Our objectives were to (1) visually assess the (given the report of Gallis et al. duration of motility across Na ’ ion concentrations (1991) on the lack of an effect of Na+ concentration on intensity but no report on duration), (2) objectively measure the percentage of motile sperm and their velocities after videomicroscopy at 200 frames/s between 5 and 12 s after activation in media of and (3) measure the concentration of ions in varying Na+ and Kf concentrations, seminal plasma in relation to their regulatory role in sperm activation. Given that the dependence of motility activation on ionic conditions and the seminal plasma ion concentrations were examined over two consecutive years, variation in motion endpoints across years could be explained in terms of seasonal temperature effects. Although the 1993 and 1994 studies differed with respect to sperm storage period, recognition of these annual changes was possible.
2. Materials and methods Lake sturgeons (A. fuluescens) were obtained from the Wolf River (Wisconsin) near Shawano dam (May 1993, April 1994). Semen was collected within 5 min after capture from nine males, 121-140 cm length, 15-22 yrs old (R. Bruch, Wisconsin Department of Natural Resources). Fish capture and semen collection were as described in Folz et al.
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339
r
Sturgeon #930000 1
/ VCL Histogram
- 1994
1
0 0
100
300 200 Curvilinear Velocity
400
Sturgeon #940081 \ Fig 1. Distributions year.
of curvilinear
velocities
for individual
sperm from each of two fish from each sampling
(1983). Rather small volumes (l-5 ml) of milt were obtained from individual fish in 1993. However, in 1994, more semen was obtained (4-32 ml). Extreme care was used in collection to avoid contamination with urine. Perchec et al. (199.5) have shown that the ratio of high velocity to low velocity sperm changes significantly immediately with the addition of urine to catheter-collected semen. Fig. 1 shows that the distributions of curvilinear velocities from one fish from each sampling year were unimodal and contained no sperm of low velocity.
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I54 (1997) 337-348
Semen was stored on melting ice (0°C) and transported within one day to the laboratory where it was stored for an additional day, under an atmosphere of oxygen (Ciereszko et al., 1994). Analyses of sperm motility were conducted 3 days post collection in 1993, while the 1994 studies were conducted on sperm stored for 9 days. 2.1. Preparation
of seminal plasma and analysis
The subsamples of milt (0.5 ml) from nine males were centrifuged (4°C) at 7000 g for 10 min. Three samples of pooled seminal plasma were formed, each consisting of three individual seminal plasma samples. Ionic composition of seminal plasma was measured by the inductively couple plasma (ICP) emission spectrophotometry method with the use of a ARL-3560 spectrometer (Applied Research Labs, Valencia, CA). 2.2. Manual assessment
of sperm motility
The milt from 1993 (n = 3) was diluted 100 times with 20 mM Tris, 30 mM glycine, pH 9.0, supplemented with NaCl (O-50 mM). The duration of motility (time from activation to less than 1% motility) was recorded at 12°C. 2.3. Dilution and actiuation of semen All solutions and microscopic equipment were kept in an environmental chamber maintained at 10°C. Semen was diluted 1:500 (1993) to 1:750 (1994) for activation in 50 mM Tris/glycine buffer, pH 9.0, with either 0, 10, or 25 mM Na+ added. For the inhibition studies 0, 0.1, 0.3, 0.6, or 1.0 mM K+ was added to the Tris/glycine buffer. 2.4. Videomicroscopy Less than 5 ~1 of the activated sperm suspension was loaded into a 20-pm deep P-Cell semen analysis chamber (Fertility Assoc., MA) and was videotaped on an Olympus BH2 microscope (phase contrast; 50 X total magnification) using a 200 Hz video camera (Motion Analysis, CA) and a high speed (200 Hz) video cassette recorder (V-32 VTR, Nat, Japan). An internal stopclock in the video tape recorder was started when the milt was mixed with an activating medium to record the time elapsed following initiation of motility. Videotaping commenced by 5 s post activation and was carried out to 30 s. Activated suspension of each sample was added to new analysis chambers, 2 and then 5 min post-activation to assess the time course of motility. Two repetitions of each milt sample were performed for each activation medium. 2.5. Computer-assisted
sperm motion analysis
The CellTrak/S computer-assisted sperm analysis system (CellTrak, Version 3.20) from Motion Analysis was used for measurement of sturgeon sperm motion endpoints. A detailed description of the derivation of the motion measurements can be found in Boyers et al. (1989). Table 1 lists the user-adjusted calibration settings. The VP110
G.P. Toth et al./Aquaculture Table 1 Cell trak research
341
154 (1997) 337-348
system settings
Frame rate Duration of data capture Minimum path length Minimum motile speed Maximum burst speed Distance scale factor Camera aspect ratio ALH path smoothing factor Cent. X search neighborhood Cent. Y search neighborhood Cent. cell size minimum Cent. cell size maximum Path max. interpolation Path prediction percentage Depth of sample Video processor model
200 frames/s 25 frames 25 frames 25 microns/s 1000 microns/s 1.2769 microns/pixel 1.0220 23 frames 2 pixels 2 pixels 4 pixels 16 pixels 2 frame 0% 20 microns VP1 10
video processing unit was operated in the quad edge mode since digitized sperm heads were elliptical (the horizontal edges contained more pixels than the vertical edges for sperm in some orientations). Output from the CellTrak analysis includes (1) the percentage of motile cells (those that have straight-line velocities over the minimum threshold velocity [25 pm/s]), (2) the curvilinear velocity (VCL) (the time-average velocity of the sperm head along its actual trajectory), and (3) the straight-line velocity (VSL) (the time-average velocity of the sperm head along a straight line from its first detected position to its last detected position), and (4) the amplitude of lateral head
0 Plot showing Actual sperm head motion (dotted) 0 Smoothed
head motion for ALH computations
(line)
238.73 1 Fig. 2. Diagram of an actual track from a sturgeon computerized motion endpoints.
sperm analysis
illustrating
the various sperm
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154 (1997) 337-348
displacement (ALH), the displacement of the actual track from a calculated path. Fig. 2 shows an actual sample path with the associated velocity vectors. 2.6. Statistical
average
analysis
Data were sufficiently normal 197 1). Effects of time, added computer-assisted sperm motion variance. Multiple comparisons
and the variance homogeneous (Levene’s test) (Wirier, Na+ concentration, and added K+ concentration on endpoints were each analyzed by a one way analysis of were done according to the Tukey procedure.
3. Results 3.1. Sperm motility - 1993 -
-----.-.-
3x0
25(x)-
3
2m-
I
2 J
% “E
1500-
: ‘i s
1m-
0 0
I 10
I Xl
I 30
I 40
-47 so
60 Na+[mMl
Fig. 3. Effect of sodium ion on duration of sperm motility - subjective observations.
G.P. Toth et al./Aquaculture Table 2 1993 Sturgeon
sperm motility endpoints
1.54 (1997) 3.37-348
343
(n = 2)
Time (min)
0 mM Na+
lOmMNa+
2.5 mM Na,!
% Motile sperm
0 2 5
,s7.0+ 1.9” ,56.5 f 11.3” ,36.0k3.0”b
,58.1 +6.6h ,65.7 f 3.3” ,55.5 * 10.8’
78.5 + 9.3”b \ 14.G 10.9b ,13.3+9.@
VCL (pm/s)
0 2 5
,312.9*42.3” ,318.3i5.9” ,306.5 * 24.3”
,315.7+6.4” ,313.3*6X’ ,304.3 + 11.5”
,308.3f 13.1” ,320.6 + 15.4” ,251.0*31.3“
VSL (km/s)
0
r 128.8 + 26.0“
5
,127.9+ 13.0“ ,73.6 f 5.0ab
r120.0i2.8” ,98.0+3.0.‘h ,93.7 f 13.8”
rl 15.6k 12.8” ,50.7 f 27.0h p3.9 + 13Xb
Linearity
0 2 5
,41.2*2.7” ,40.5 i 3.4” ,23.9 f 3.3”
,38.2?0.8” ,32.9f3.3”h ,31.6*3.8”
(37.7 f 5.8 d ,15.8f7.2b r17.4+3.5”
ALH
0 2 5
,4.05 * 0.37” ,4.29 i 0.06” ,4.42 i 0.40”
,4.38 f0.23” ,4.70+0.2Fh ,4.50*0.17”
,4.25 + 0.03“ ,5.18*0.29b ,4.81 +0.55”
“Superscripts with the same letters within a time (row) are not significantly different ( p I 0.05). ,Subscripts with the same letters within a treatment (column) are not significantly different ( p 5 0.05) Values are means f population standard deviations.
observed sperm in 10 mM Na+ described above (i.e., the longest subjectively-observed duration was observed with 10 mM Na+). The curvilinear velocity remained essentially unchanged over time and concentration of added Naf, while the straight-line velocity decreased steadily over time with no added Na+ or 25 mM Na’ (changes were insignificant based on variation between the two samples at 2 min). Two minutes after activation, addition of 25 mM Na+ brought about significant changes in linearity and ALH relative to those samples with no added Na+. 3.2. Sperm motility - 1994 - Qect
qf added sodium
Although velocities lower than those of 1993 were evident in the 1994 data set (Table 3), the percentages of motile sperm ranged within the 1993 data set. The addition of 25 mM Na+ to the Tris/glycine buffer resulted in no loss of the percentage of motile sperm during the first 5 min. A significant reduction in motile sperm in relation with time was seen with no added Na+, while 10 mM added Na+ reduced motility means by more than half. The lack of statistical significance of the latter observation should be viewed in the context of the high variability. Velocities and sperm trajectory pattern indices (linearity and ALH) were unchanged across media Na+ concentrations or across time. 3.3. Sperm motility - 1993 - effect
qf added potassium
The effect of added K+ on sturgeon sperm motility is shown in Fig. 4. A clear dose response is seen with a 50% reduction in the percentage of motile sperm at approxi-
344 Table 3 1994 Sturgeon
G.P. Toth et al./Aquaculture
sperm motility endpoints
154 (1997) 337-348
(n = 5)
Time (min)
OmMNa+
10 mM Na+
25 mM Na:
0 2 5
,564 + 22.1” ,,26.4+23.5 ,12.2+8.61”
,51.8+25.5” ,45.8 f 25.5 ,21.0+27.7”
49.8 + 24.6” ,34.9* 18.5 ,42.4+ 18.0a
VCL (pm/s)
0 2 5
217.2*68.5# 207.6 f 63.5 201.5+62.0
210.7i61.0 214.3k53.6 199.3rt41.3
218.lk44.2 228.1 f 56.3 213.8552.7
VSL (I*.m/s)
0 2 5
61.9+ 14.5 55.8 + 24.9 46.7 f 18.5
67.4+ 11.1 47.8 + 14.6 53.4+ 19.7
57.4* 12.0 48.9 + 12.6 41.3k7.4
Linearity
0 2 5
32.6 + 14.9 30.6+ 18.1 25.8 + 14.1
35.4+ 13.9 25.0* 12.2 28.7 + 13.6
28.3 + 8.6 23.9+ 10.7 21.7+8.8
ALH km
0 2 5
3.68 kO.35 3.84kO.30 4.19f 1.14
3.51 kO.20 3.5orto.30 3.44 f 0.34
3.82+0.26 3.84 + 0.3 1 3.54+0.44
% Motile sperm
‘Superscripts with the same letters within a time (row) are not significantly different ( p I 0.05). &Subscripts with the same letters within a treatment (column) are not significantly different ( p < 0.05). Comparisons within and across time not shown were not significant. Values are means + SD.
STURGEON
SPERM
MOTILITY
Effect of Added
K’
60 -
- 240
40 -i
- 160
________-------.o-_,,
0
-M-
0.3 K’ Concentration Percent Motile
.. *...
“C.
0.6 (mM) --0--
“SL
Fig. 4. Effect of potassium ion on CASA-measured sperm motility. Motility means with the same letters are not significantly different ( p I 0.05) (n = 2). Error bars denote standard deviations.
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345
Table 3 Seminal plasma ion concentrations
Na
K P Cl Ca Mg Zn
I993 Mean (mM) f SD
I994 Mean (mM) f SD
25.6 * 2.8 5.78 i 0.49 3.02 + 0.37 5.41 * 2.79 0.16+0.05 0.2 I * 0.02 0.00076 f 0.000 I5
x1.8+7.0 6.97 + I .42 3.665 1.37 2.3 I k I .28 0.13kO.02 0.22 f 0.07 0.00076 + 0.00030
mately 0.5 mM added Kf (Tris/glycine)(n = 2). Curvilinear velocity and straight-line velocity were unchanged for the remaining motile cells across all concentrations of potassium. 3.4. Seminal plasma inn corlcentrutions Table 4 shows the ion concentrations determined for seminal plasma from the lake sturgeon in two spawning seasons. There were no significant differences between ion concentrations, although chloride levels were twice as high in 1993 as in 1994.
4. Discussion The present study of sperm motility in the lake sturgeon, A. ,jidce.scer~~, provides for the first time an objective demonstration of the extended duration of motility, previously reported by Gallis et al. (1991). Gallis et al. (1991) described the sperm from the Siberian sturgeon, A. baeri, showing subjective sperm motility evaluations in domesticated fish. The activation medium consisting of the Tris/glycine buffer with 10 mM added Na+ provided no loss in the percentage of motile sperm after 5 min and no decrease in the velocities of the sperm. Visual microscopic observation revealed maintenance of motility over 30 min. Duration of motility in our studies exceeded that described by Gallis et al. (1991), where ‘intensity’ was decreased within the first minutes after activation. Gallis et al. (1991) examined motility at room temperature while our subjective study was conducted at 12°C. Billard and Cosson (1992) reported that increased temperature (range: 5” to 26°C) increased the beat frequency of trout sperm and decreased the duration of forward movement. A temperature of 12°C is more relevant to natural spawning activities. Several years of observations with Wolf River lake sturgeon indicate spawning being associated with a relatively narrow range of water temperature (9- 11“C, R. Bruch, personal communication). Nevertheless, the duration of sperm motility at either temperature exceeds that of sperm from teleosts that spawn in fresh water (trout < 30 s; carp < 120 s). As described by Detlaff et al. (1993). the increased duration of sturgeon sperm motility and fertility increases the effectiveness of natural spawning which takes place in strong river currents. Our results show swimming
346
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velocities (straight-line velocities of 120-130 pm/s and curvilinear velocities of 310-320 pm/s> similar to those described for other fish sperm, both freshwater and marine. Redondo-Miiller et al. (1991) reported trout sperm curvilinear velocities of approximately 250 km/s. There were two objectives in studying the motility characteristics of sturgeon sperm. One was the characterization of the factors influencing activation of the sperm and the second was the setting of reference values for fresh sperm for a given location and date of spawning. Given the inability to conduct on site experimentation with electronic equipment and microscopes that were not field-ready, attainment of the first objective depended upon transport of sperm samples to the distant laboratory and attainment of the second goal was left for another time. The assumption was made that given moderate motility (percentage of motile sperm > 50%), long-term storage (9 days) would not influence the nature of the activation and inhibition of sturgeon sperm. Sample size was limiting and no argument is made that this is a weakness of the study. However, the present studies have adequate sample size to illuminate factors influencing activation and inhibition of sturgeon sperm as previously described subjectively by Gallis et al. (1991). In the present work, a half-maxima1 inhibition of motility activation has been reported following the addition of 0.5 mM K+ to the Tris/glycine buffer. This observation confirms and extends those of Gallis et al. (1991) who reported complete inhibition in the presence of 0.1 mM potassium ion. Confirmation that the osmotic pressure of seminal plasma (38 mosM/kg, see below) is not limiting to sperm activation, the present study shows that activation of sturgeon sperm occurs over a theoretical range of 50 to 100 mosM/kg. Gallis et al. (1991) reported a mean osmotic pressure for seminal plasma of the Siberian sturgeon of 38 mosM/kg. This value is far below those of the freshwater, amphihaline, and marine species of teleosts (range: 232-400 mosM/kg) as listed by Suquet et al. (1994). The major elemental seminal plasma constituents are described for the lake sturgeon. These observations extend those of Gallis et al. (1991) who reported mean sodium and potassium concentrations of 28 mM and 2.5 mM, respectively, in Siberian sturgeon seminal plasma. The sodium and chloride concentrations in the lake sturgeon (25-30 mM and 2-5 mM, respectively) mark the major contributions to the differences between the chondrosts and teleosts - Na: 140 mM and Cl: 150 mM in rainbow trout (Morisawa and Morisawa, 1988) - Na: 133 mM and Cl: 129 mM in turbot (Suquet et al., 1993). The potassium concentration (6 mM) of lake sturgeon seminal plasma resembles those of marine teleosts, such as turbot (3.8 mM) (Suquet et al., 1994). It is of interest that the added Na+ concentrations eliciting no loss of motility 5 min post-activation differed between the study years (1993, 10 mM Na+; 1994, 25 mM Na+). Given that the seminal plasma ions contributing most to osmotic pressure (Na+ and Kf) appeared to be higher in 1994, it is suspected that the sperm from the 1994 sampling could be activated with a higher osmolality medium - i.e., a 25 mM Na+ addition to Tris/glycine - since the osmotic change would remain constant. Clemens and Grant (1965) reported on a seminal thinning response (or hydration) in carp and rainbow trout after injections of pituitary extracts. Changes in the water and salt content
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154 IIYY7) 337-348
347
and sperm cell density of semen occurred in direct proportion to doses of these extracts. It could be hypothesized that since the sturgeon spawned early in 1994 that the hydration of their testes would not have been as substantial as at a later time. While the ion concentrations in the present study were not significantly different across years, it is felt that the reported trends are not inconsistent with the hypothesis. Several advantages are gained by the application of computer-assisted sperm motion analysis (CASA) to the measurement of sturgeon sperm motility endpoints. For adequate measurement of oscillating biologic systems, the sampling rate should be two to four times the frequency of the event (Boyers et al., 1989; Davis and Katz, 1993). The high speed videomicrography (200 frames/s) used in the present study allowed adequate sampling of trajectories of fish sperm with an initial flagellar beat frequency between 40 and 60 Hz. Previous references to fish sperm motility have used only straight-line velocities and beat frequencies (Billard and Cosson, 1992; Oda and Morisawa, 1993). The latter employed a computer-assisted analysis system (CellSoft@), but was not working at video speed above 30 Hz (which are not required for straight-line velocity). Measurement of physical components of the trajectory of the sperm such as curvilinear velocity and linearity provides indices of pattern which may be more robust indicators of the fertilizing capacity. In the present study, automated tracking of the phase-contrast-illuminated sperm head required digitization of the entire sperm head (4 edge analysis in contrast to the 1 edge analysis used for carp sperm [Toth et al., 199.51) since the sturgeon sperm head is elliptical. Tracking of one permanent XY quadrant (in the 1 edge mode) could allow biased tracking of the center of the sperm head, allowing oscillation between ellipse axis maxima and minima.
5. Conclusions Computer-assisted sperm motion analysis of activated lake sturgeon sperm revealed peak durations of sperm motility with unchanging velocity parameters for up to 5 min after addition of various concentrations of Na ion to Tris/glycine buffer. Added potassium ion inhibited activation with an apparent EC,, of 0.5 mM K+. This ability to objectively analyze sturgeon sperm is already being used in other studies of cryopreservation in the lake sturgeon.
Acknowledgements We thank Ron Bruch, Steven Fajfer, and the personnel of the Wisconsin Department of Natural Resources for their help in the collection of lake sturgeon spermatozoa. The assistance of Dr. Joni Torsella in statistical analyses was appreciated. This document has been reviewed in accordance with U.S. Environmental Protection Agency and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
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References Billard, R., Cosson. M.P., 1992. Some problems related to the assessment of sperm motility in freshwater fish. J. Exp. Zool. 261, 122-131. Boyrrs, S.P., Davis, R.O.. Katz, D.F., 1989. Automated Semen Analysis. Curr. Probl. Obstet. Gynecol. Fertil. 12, 165-200. Christ, S.A.. Toth, G.P.. McCarthy, H.W., Torsella, J.A., Smith, M.K., 1996. Monthly variation in sperm motility in common carp. Cyprinus carpio, assessed using computer-assisted sperm analysis (CASA). Journal of Fish Biology, 48, 1210-1222. Ciereszko, A., Dabrowski, K., Lin, F., Doroshov, S.I., 1994. Identification of trypsin-like activity in sturgeon spermatozoa. J. Exp. Zool. 268, 486-49 I. Clemens, HF., Grant, F.B.. 1965. The seminal thinning response of carp (C~prinus ctrrpio) and rainbow trout (Snlnlo ~~crird,zerii) after injections of pituitary extracts. Copeia 2, I74- 177. Davis, R.O., Katz, D.F., 1993. Operational standards for CASA instruments. .I. Androl. 14, 385-394. Detlaff, T.A., Ginsburg, AS., Schmalhausen, 0.1.. 1993. Embryonic development: Gametes. In: Sturgeon fishes: Developmental biology and aquaculture. Springer-Verlag, pp. 61-64. Gallis, J.L., Fedrigo, E., Jatteau, P.. Bonpunt, E., Billard, R., 1991. Siberian sturgeon, Aciprrlsrr haeri, spermatozoa: effects of dilution, pH. osmotic pressure, sodium and potassium ions on motility, In: Williot, P. (Ed.). Acipenser. Cemagref Pub]., pp. 143-15 I Ginsburg, A.S.. 1972. Fertilization in fishes and the problem of polyspermy. Israel Program for Scientific Translations, p. 140. Moriaawa, S.. Moriaawa, M., 1988. Induction of potential for sperm motility by bicarbonate and pH in rainbow trout and chum salmon. .I. Exp. Biol. 136, 13-22. Oda, S., Morisawa. M., 1993. Rises of intracellular Ca+’ and pH mediate the initiation of sperm motility by hyperosmolality in marine teleosts. Cell Motil. Cytoskeleton 25, 17 I - 178. Perchec. G.. Cosson, _I.. Jeulin, C., Paxion. C., Andre, F., Billard, R., 1995. Alteration of carp (Cypriuts ctrr/?io) apermatoLoa motility by urine contamination during sampling. Proceedings of the Fifth International Symposium on the Reproductive Physiology of Fish, The University of Austin, TX, 2-8 July 1995. Fish Symposium 95. Austin, p. 135. Redondo-Miller. C.. Coason. M.-P., Cosson, J., Billard, R., 1991. In vitro maturation of the potential for movement of carp spermatozoa. Mol. Reprod. Dev. 29, 259-270. Suquet. M., Billard. R., Cosson, J., Dorange, G.. Chauvand, L., Mugnier, C., Fauvel, C., 1994. Sperm features in turbot (.Sco/~h/h~~/rtuu nu~irm~s): a comparison with other freshwater and marine fish species. Aquatic Living Resources 7, 283-294. Suquet. M., Dorange, G., Omnes, M.H., Normant. Y., Le Roux, A., Fauvel, C., 1993. Composition of the seminal fluid and ultrastructure of the spermatozoon of turbot (Sco@?tha/mus nmxinnrs). Journal of Fish Biology 42. 509-5 16. Toth. G.P., Christ, S.A.. Torsella, J.A., McCarthy, H.W., Smith, M.K., 1995. Computer-aaaisted sperm motion analysis of sperm from the common carp (Cyprinus carpio). Journal of Fish Biology 47, 9X6- 1003. Wirier. B.J.. 1971. Statistical principles in experimental design. McGraw-Hill, New York.