automated sperm morphology analysis in fishes: the effect of mercury on goldfish sperm

Automated sperm morphology analysis in fishes: the

effect of mercury on goldfish sperm

K. J. W. VAN LOOK* AND D. E. KIME†

Department of Animal and Plant Sciences, University of Sheffield,Sheffield S10 2TN, U.K.

(Received 12 December 2002, Accepted 30 July 2003)

This study is the first to examine themorphology of fish sperm using automated spermmorphology

analysis (ASMA).The techniquewas applied to investigate the effect of an environmental pollutant,

mercury, on the sperm morphology of goldfish Carassius auratus, and the effects on sperm

morphology were compared with those on sperm motility. Goldfish sperm flagellar length was

significantly shortened after instant exposure to 100mg l�1 (368mM) mercuric chloride, while

curvilinear velocity (VCL) and the percentage of motile sperm were significantly decreased at

mercuric chloride concentrations of 1 and 10mg l�1 (3�68 and 36�8mM), respectively. After 24h

exposure to 0�001mg l�1 (0�0037mM)mercuric chloride, flagellar lengthwas significantly reduced in

38% of the spermatozoa. Following exposure to 0�1mg l�1 (0�37mM) mercuric chloride for 24h,

however, the majority of spermatozoa (98%), had significantly shortened flagella and increased

sperm head length, width and area. Sperm motility was also significantly decreased at 0�1mg l�1

(0�37mM) mercuric chloride, probably due to the significantly reduced flagellar length at this

concentration. This study shows that the morphological examination of fish sperm by ASMA

provides, not only, an excellent tool for monitoring reproductive disruption caused by environ-

mental pollution, but also has applications to other areas of fish reproductive biology, such as

cryopreservation and aquaculture. # 2003 The Fisheries Society of the British Isles

Key words: ASMA; automated sperm morphology analysis; CASA; fish sperm; mercuric

chloride; sperm motility.

INTRODUCTION

Sexual reproduction in organisms is dependent upon the production of highquality sperm. Damage to sperm structure by, for example toxicants, cryopre-servation or genetic mutation, could cause shortening of flagella or convolutedsperm heads, which may affect the motility and, hence, fertilizing ability ofsperm. Indeed, abnormal sperm head morphometry in the bull, boar andstallion has been related to reduced fertility (Sailer et al., 1996; Casey et al.,1997; Hirai et al., 2001), while rat sperm head morphology was significantlydisrupted by 1,3-dinitrobenzene, a testicular toxicant (Davis et al., 1994).

*Author to whom correspondence should be addressed at present address: Institute of Zoology,

Zoological Society of London, Regent’s Park, London NW1 4RY, U.K. Tel.: þ44 (0) 20 74496640; fax:

þ44 (0) 20 75862870; email: [email protected]

†Present address: Druimnich, Glenborrodale, Acharacle PH36 4JP, Scotland, U.K.

Journal of Fish Biology (2003) 63, 1020–1033

doi:10.1046/j.1095-8649.2003.00226.x,availableonlineathttp://www.blackwell-synergy.com

1020# 2003TheFisheries Society of theBritish Isles

Until recently, morphology of both mammalian and fish sperm were exam-ined and analysed using a range of manual techniques: light, scanning andtransmission electron microscopy, laser light-scattering spectroscopy and strobo-scopic illumination. These techniques, however, are subjective, time-consumingand the results highly variable. Automated sperm morphology analysis(ASMA) is a relatively new technique, which has increasingly been used toexamine sperm of a range of mammalian species, such as rat (Davis et al.,1994), bull (Gravance et al., 1996), dog (Dahlbom et al., 1997), alpaca (Buendıaet al., 2002), monkey (Gago et al., 1998) and man (Davis & Gravance, 1993). Theuse of ASMA for mammals has reduced the subjective nature and inherenttechnical variation of morphological determination. In addition, ASMAgenerates measurements of the flagellum, sperm head, midpiece and acrosome,some of which were difficult to obtain with the earlier techniques. ASMA hasnot, however, previously been applied to fish sperm.Research on fish sperm using the earlier methodologies has primarily con-

centrated on the structure and comparative morphology of the sperm, and onchanges in morphology following freezing or thawing and dilution in varioussolutions (Billard, 1983; Tanaka et al., 1995; Perchec et al., 1996; Dreanno,1998; Gage et al., 1998; Hara & Okiyama, 1998; Ishijima et al., 1998; Billardet al., 2000; Balshine et al., 2001; Vladic et al., 2002). For instance, Hara &Okiyama (1998) described the structure and morphology of sperm from 65species of Japanese fishes using scanning and transmission electron microscopy,while sperm flagellum length was measured in relation to sperm competition inAfrican cichlid species (Balshine et al., 2001) and in Atlantic salmon Salmo salar L.(Vladic et al., 2002). The changes in flagellum morphology of intact andfrozen and thawed Siberian sturgeon Acipenser baerii Brandt sperm duringmotility using darkfield microscopy and stroboscopic illumination have also beenassessed (Billard et al., 2000).There is increasing evidence that environmental pollution can affect repro-

ductive capability of wild animals (Damstra et al., 2002) and ASMA mayprovide a valuable technique for determining whether this involves abnormal-ities in sperm structure. Several studies have used laser light-scattering spectro-scopy and transmission electron microscopy techniques to investigate the effectsof pollutants on the morphology of mammalian semen (Mohamed et al., 1986;Rao, 1989; Ackerman et al., 1999). Spermatozoa of monkeys Macaca fascicularisexposed in vitro to 1mg l�1 methylmercury hydroxide, developed coiled flagella andkinks at the midpiece junction, as analysed by laser light-scattering spectroscopy(Mohamed et al., 1986). Similarly, impala Aepyceros melampus exposed to highcopper levels (126�5mgkg�1 copper in their livers) in a South African national parkhad a higher percentage of sperm with vacuoles in the neck region than controlanimals (Ackerman et al., 1999).Fish sperm differs in many respects from that of mammals (Kime et al., 2001)

and the ASMA methodology used for mammals is not directly applicable tofishes. In this study the ASMA methodology was adapted to examine themorphology of teleost sperm and the effect of pollutants on sperm was inves-tigated. As an example the effect on the morphology of goldfish Carassiusauratus (L.) sperm after exposure to mercuric chloride and the resulting changesin sperm motility were assessed.

SPERM MORPHOLOGY OF FISHES 1021

# 2003TheFisheries Society of theBritish Isles, Journal of FishBiology 2003, 63, 1020–1033

MATERIALS AND METHODS

MILT COLLECTION

Goldfish, obtained from a local supplier (JMC Aquatics, Dronfield, U.K.), were heldin stock tanks in a recirculation system at ambient temperature and photoperiod at theUniversity of Sheffield. In May, groups of six goldfish (40–80 g; females :males¼ 1 : 5 or2 : 4) were transferred to separate 50 l tanks at a constant temperature of 19�7� C,range� 0�3� C, and ambient photoperiod. Twelve hours prior to milt collection, 17 ml333 ng l�1 17,20b-dihydroxy-4-pregnen-3-one (Sigma, Poole, U.K.) were pipetted intothese tanks to stimulate milt production and increase milt volume (Dulka et al., 1987).Milt was stripped c. 3 h after sunrise by gentle abdominal pressure and collected in 50 mlcapillary tubes, which were held in plastic tubes on ice.

INSTANT (NO PREINCUBATION) EXPOSURE – SPERMMORPHOLOGY

No preincubation (NP) exposure refers to instant exposure (5 s prior to analysis ofmorphology) of spermatozoa to mercuric chloride in extender.

The goldfish milt samples were diluted 1 : 500 in goldfish extender (5�80 g l�1 NaCl,0�23 g l�1 KCl, 0�22 g l�1 CaCl2, 0�04 g l�1 MgCl2, 2�10 g l�1 NaHCO3, 0�04 g l�1

NaH2PO4 and 3�75 g l�1 glycine in distilled water, pH8�64; modified from Ravinderet al., 1997) and kept on ice. HgCl2 was added to goldfish extender to give final mercuryconcentrations of 0�001, 0�01, 0�1, 1, 10 and 100mg l�1 (0�0037, 0�037, 0�37, 3�68, 36�8 and368mM) when in contact with the sperm. Sperm were then further diluted (1 : 1) inmercuric chloride extender and 1 ml was then immediately pipetted into one well of a12-well multitest slide (ICN, Basingstoke, U.K.) and covered with a coverslip.

TWENTY-FOUR HOUR EXPOSURE – SPERM MORPHOLOGY

Twenty-four hour exposure refers to sperm that have been exposed to mercuricchloride for 24 h in extender before analysis of their morphology.

Goldfish milt was diluted 1 : 1000 in the mercuric chloride extenders and stored at 4� Cfor 24 h. After 24 h, 1ml of mercuric chloride-exposed sperm was pipetted into the well ofa multitest slide and covered with a coverslip.

AUTOMATED SPERM MORPHOLOGY ANALYSIS (ASMA)

Slides were viewed using a �20 negative-phase contrast objective (Olympus, Japan)and a green filter on an Alphaphot-2 Nikon microscope (Japan). A Sony CCD black andwhite video camera (SPT-M108CE; Japan) transferred the image to a Hobson spermtracker (Hobson Vision Ltd, Baslow, Derbyshire, U.K.). Sperm morphology was ana-lysed using ASMA software (Hobson Vision Ltd.; version Morph y) on the tracker.Settings for ASMA were calibration: 1�6, digitizer reference: 40 and 165, peak: 9, xL: 20,radius offset: �4, head size: 2 and 25, and learn data: yes. Approximately 200 sperma-tozoa were analysed in each sample. There was no significant difference (paired t-test,P¼ 0�79; Microsoft Excel 97) between the ASMA software flagellar length measurementsand manual ones (mean¼ 37�1mm, range¼ 7�7–54�3 mm, n¼ 20 and mean¼ 36�9mm,range¼ 8�8–52�9mm, n¼ 20, respectively). Reproducibility of flagellum length onfour samples with means of 23�1, 50�7, 51�3 and 54�3 mm was 0, 5�5, 0�2 and 2�1%,respectively (range of values: 23�1–23�1mm, n¼ 5; 48�7–53�8mm, n¼ 5; 51�3–51�5mm,n¼ 5; 53�8–56�4mm, n¼ 5). The ASMA software has also been validated with boarsemen (Thurston et al., 1999).

1022 K. J . W . VAN LOOK AND D. E . KIME

# 2003TheFisheries Society of the British Isles, Journal of FishBiology 2003, 63, 1020–1033

SPERM MOTILITY ANALYSED BY COMPUTER-ASSISTEDSPERM ANALYSIS

The computer-assisted sperm analysis (CASA) technique for analysing fish spermmotility is described in detail in Kime et al. (2001).For instant (NP) exposure of sperm to mercury, milt was diluted 1 : 100 in goldfish

extender (first dilution step) and kept on ice. HgCl2 was added to distilled water to givemercury salt concentrations of 0�002, 0�02, 0�2, 2, 20 and 200mg l�1 (0�0074, 0�074, 0�74,7�37, 73�7 and 737mM). The solutions were adjusted with HCl or NaOH to pH6�0 andused to activate the spermatozoa (1 : 1; second dilution step). Immediately, 0�7mlactivated sperm were transferred into one well of a 12-well multitest slide and coveredwith a coverslip coated with 1% bovine serum albumin (BSA) in distilled water. The finalconcentrations of the mercury solutions were 0�001, 0�01, 0�1, 1, 10 and 100mg l�1

(0�0037, 0�037, 0�37, 3�68, 36�8 and 368mM) when in contact with the sperm.For 24 h exposure, HgCl2 was added to goldfish extender to give mercury salt con-

centrations of 0�001, 0�01, 0�1, 1, 10 and 100mg l�1 (0�0037, 0�037, 0�37, 3�68, 36�8 and368mM), and adjusted with HCl or NaOH to pH8�64. Milt was then diluted 1 : 1 ingoldfish extender with added mercury (first dilution step) and stored at 4� C for 24 h. Thefinal concentrations of the solutions were 0�001, 0�01, 0�1, 1, 10 and 100mg l�1 (0�0037,0�037, 0�37, 3�68, 36�8 and 368 mM) when in contact with the sperm. After 24 h, spermwere activated (1 : 1; second dilution step) in distilled water and immediately, 0�7mlactivated sperm were transferred into the well of a multitest slide and covered with acoverslip coated with 1% BSA in distilled water.Sperm motility was videotaped, from the moment of final dilution until all movement

ceased, using a Sony CCD black and white video camera connected to an Alphaphot-2Nikon microscope. A �20 negative-phase contrast objective, giving a final magnificationof �50, and a green filter were used. Videotapes were analysed using CASA software(Hobson Vision Ltd.; version 7V2B) on a Hobson sperm tracker for 15 s intervals fromthe beginning of recording until the end of the sequence using the optimized settings forgoldfish (Kime et al., 2001). The settings used were search radius: 8�44, trail: 59, predic-tion: off, pause window: 0�8, video: pal(50), aspect: 1�49, refresh time: 1, thresholds:þ30/�100, filter weightings: 1 : 2, 2 : 3, 3 : 1, 4 : 1, immotile process: shape, maximum size:15, minimum size: 4�7, ratio: 1 and minimum track time: 0�8. The variables used foranalysing the motility of goldfish sperm were curvilinear velocity (VCL) and percentageof motile sperm, and the data presented are the 5–20 s tracking interval after activation.

STATISTICAL ANALYSIS

Prior to analysis, sperm morphology data were tested for normality using aKolmogorov-Smirnov test (GraphPad, 1995). Data were then statistically analysed byone-way ANOVA followed by Tukey’s multiple comparison post-test or by a Kruskal–Wallis test followed by Dunn’s multiple comparison post-test (GraphPad, 1995). Thesperm motility data were analysed by one-way ANOVA followed by Tukey’s multiplecomparison post-test or by a paired t-test (GraphPad, 1995). All motility results aremeans� S.E.

RESULTS

EFFECTS OF MERCURY ON THE FLAGELLUM

The frequency distribution of spermatozoa exposed instantly to 100mg l�1

(368mM) mercuric chloride was more skewed to the left, indicating that ahigher proportion of these sperm had shorter flagella than controls and0�001–10mg l�1 (0�0037–36�8 mM) mercuric chloride-exposed sperm (Fig. 1).The flagella of 100mg l�1 (368 mM) mercuric chloride-exposed sperm were



significantly shorter than controls (Fig. 2, box plots; one-way ANOVA usingtransformed data, P< 0�01), while 0�001–10mg l�1 (0�0037–36�8 mM) mercuricchloride-exposed sperm all had similar flagellar lengths to the controls. Themedian flagellum length of 100mg l�1 (368 mM) mercuric chloride-exposedsperm was 35�9 mm compared with 46�2 mm in controls.After 24 h exposure of spermatozoa to mercuric chloride, there were very

different patterns in the frequency distributions of flagellar length (Fig. 3).Control and 0�001mg l�1 (0�0037mM) mercuric chloride-exposed sperm had a

4 12 20 28 36 44 52 60 68

Flagellum length (µm)

% S

perm

atoz

oa

0

5

10

15

20

25

30

35

FIG. 1. Frequency distributions of flagellum length of no preincubation mercuric chloride-exposed gold-

fish sperm (. .6. . 0, –�þ – 0�001, –n– 0�01, –*– 0�1 and –&– 100mg l�1 mercuric chloride, n¼ 212).

A number of the treatments have been omitted from the figure for ease of interpretation.

Hg2+ (mg l–1)

Fla

gellu

m le

ngt

h (µ

m)

0

10

20

30

40

50

60

0

20

40

60

80

100

120

140

VC

L (

µm s

–1),

%

Mot

ile s

perm

a

r r rrs

st

tu u

ab ab ab ab abb

x x x

xy

xy

yz

z

0 0.001 0.01 0.1 1 10 100

FIG. 2. The effect of no preincubation mercuric chloride exposure on flagellum length of goldfish sperm

(box plot, n¼ 212), curvilinear velocity (VCL, * line plot, n¼ 82) and the percentage of motile

goldfish sperm (& line plot, n¼ 82). Boxes represent the median, 25th and 75th percentiles, and the

error bars represent the 10th and 90th percentiles (untransformed data). The data were transformed

(by squaring) before statistical analysis. For line plots results are mean� S.E. and represent the

5–20 s tracking interval after activation. For each plot, different letters indicate a significant

difference by Tukey’s multiple comparison post-test after one-way ANOVA (P< 0�05).



majority of normal length flagella, as indicated by their right-skewed frequencydistributions. Sperm exposed to 0�01mg l�1 (0�037mM) mercuric chloride hadboth short and normal flagella with no definite peak in their frequency dis-tribution. On the other hand, sperm exposed to 0�1–100mg l�1 (0�37–368mM)mercuric chloride had significantly shorter flagella, as shown by the left-skewed frequency distributions. Indeed, flagellum lengths of 0�1–100mg l�1

(0�37–368mM) mercuric chloride-exposed sperm were c. 35mm shorter thancontrols, which had a median flagellum length of 46�2 mm (Fig. 4, box plots;Kruskal–Wallis test, P< 0�0001).

EFFECTS OF MERCURY ON THE SPERM HEAD

There was no effect of NP exposure to 0�001–100mg l�1 (0�0037–368mM)mercuric chloride on sperm head length or width and, consequently, no effecton its area. Length of the sperm heads was c. 4�2 mm and their width wasc. 4�3 mm, while the area was c. 13mm2 in all treatments.By contrast, after 24 h mercury exposure there were significant increases in

length and width of the sperm head and, hence, also in its area. The head lengthand width of sperm exposed to 0�1mg l�1 (0�37mM) mercuric chloride hadsignificantly increased by 0�6 and 0�7 mm, respectively, compared to controls,which were 4�2 and 4�3 mm, respectively [Fig. 5(a), (b); one-way ANOVA, bothP< 0�001]. As a result, the head area of sperm exposed to 0�1mg l�1 (0�37mM)mercuric chloride was significantly larger by 1�9 mm2 compared to the controls,which had a mean head area of 13�1 mm2 [Fig. 5(c); one-way ANOVA,P< 0�001]. After 24 h exposure to 100mg l�1 (368 mM) mercuric chloride, thelength, width and area of sperm heads were still significantly increased incomparison to those of controls, but the effect was less marked than at0�1mg l�1 (0�37mM) mercuric chloride (Fig. 5; one-way ANOVA, P< 0�05 forlength and P< 0�001 for width and area). Spermatozoa exposed to 100mg l�1

Flagellum length (µm)

% S

perm

atoz

oa

0

5

10

15

20

25

30

4 12 20 28 36 44 52 60 68

FIG. 3. Frequency distributions of flagellum length of 24 h mercuric chloride-exposed goldfish sperm

(. .6. . 0, –�þ – 0�001, –n– 0�01, –*– 0�1 and –&– 100mg l�1 mercuric chloride, n¼ 204). A number of

the treatments have been omitted from the figure for ease of interpretation.



(368mM) mercuric chloride for 24 h had heads that were 0�3 mm longer, 0�6mmwider and 1�1 mm2 larger in area than controls.

COMPARISON OF GOLDFISH SPERM MORPHOLOGY ANDMOTILITY

After NP exposure to 0–10mg l�1 (0–36�8mM) mercuric chloride, the flagellumlength of goldfish sperm was 43�6–46�2mm, but at 100mg l�1 (368mM) mercuricchloride themedian flagellum length had decreased to 35�9mm (Fig. 2). VCL, on theother hand, had already decreased significantly at 1mg l�1 (3�68mM) mercuricchloride and the percentage of motile sperm at 10mg l�1 (36�8mM) (Fig. 2; one-way ANOVA, P< 0�01 for VCL and P< 0�05 for % motile).After 24 h exposure to 0�001 and 0�01mg l�1 (0�0037 and 0�037mM) mercuric

chloride, there were significant decreases in median flagellum length by 5�2 and12�9 mm, respectively, in comparison to the control; but no significant effect onVCL or the percentage of motile sperm (Fig. 4). Exposure of spermatozoa to0�1mg l�1 (0�37mM) mercuric chloride resulted in a sharp drop in flagellumlength: from a median flagellum length of 46�2 mm in the controls to 15�4 mm in0�1mg l�1 (0�37mM) mercuric chloride-exposed sperm (Kruskal–Wallis test,P< 0�001). This very significant reduction in flagellum length after 24 h expos-ure to 0�1mg l�1 (0�37mM) mercuric chloride was similarly reflected in themotility of the sperm. VCL of sperm decreased significantly at 0�1mg l�1

Hg2+ (mg l–1)

Fla

gellu

m le

ngt

h (µ

m)

0

10

20

30

40

50

60

VC

L (

µm s

–1),

%

Mot

ile s

perm

0

20

40

60

80

100

120

140a

bc

d e

f g

r

***

rr

s

s s

x x x

x

yy ***0 0.001 0.01 10.1 10 100

FIG. 4. The effect of 24 h mercuric chloride exposure on flagellum length of goldfish sperm (box plot,

n¼ 204), curvilinear velocity (VCL, * line plot, n¼ 44) and the percentage of motile goldfish sperm

(& line plot, n¼ 44). Boxes represent the median, 25th and 75th percentiles, and the error bars

represent the 10th and 90th percentiles. Different letters indicate a significant difference by Dunn’s

multiple comparison post-test after a Kruskal–Wallis test (P< 0�05). For the line plots, results are

mean� S.E. and represent the 5–20 s tracking interval after activation. Different letters on the line

plots indicate a significant difference by Tukey’s multiple comparison post-test after one-way

ANOVA (P< 0�05). ***, significant difference (P< 0�001) by a paired t-test between a second

control (not shown) and the highest concentration; these data were collected in a later experiment

to extend the range of concentrations tested.



(0�37mM) mercuric chloride and all sperm were immotile at concentrations of�1mg l�1 (3�68mM) mercuric chloride (one-way ANOVA and t-test, P< 0�001).The percentage of motile sperm was, however, not significantly affected byexposure to 0�1mg l�1 (0�37mM) mercuric chloride, with c. 60% of sperm stillmotile. All sperm were, however, immotile at concentrations of �1mg l�1

(3�68mM) mercuric chloride (one-way ANOVA and t-test, P< 0�001), while

Len

gth

of s

perm

hea

d (µ

m)

0

1

2

3

4

5

aabab bbcbcc(a)

Wid

th o

f sp

erm

hea

d (µ

m)

0

1

2

3

4

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6

a aabbcbcc c

(b)

Are

a of

spe

rm h

ead

(µm

2 )

0

2

4

6

8

10

12

14

16

aabab bbcc c(c)

0 0.001 0.01 10.1 10 100

Hg2+ (mg l–1)

0 0.001 0.01 10.1 10 100

0 0.001 0.01 10.1 10 100

FIG. 5. The effect of 24 h mercuric chloride exposure on (a) head length, (b) head width and (c) head area

of goldfish sperm (n¼ 204). Means� S.E. Different letters indicate a significant difference by

Tukey’s multiple comparison post-test after one-way ANOVA (P< 0�05).



the length of flagella varied between 7�7 and 12�8mm after exposure to1–100mg l�1 (3�68–368mM) mercuric chloride.

DISCUSSION

While ASMA has previously been applied to mammalian sperm, the presentstudy is the first in which it has been used to examine the morphology of fishsperm. The data presented here on its application to the study of the effects ofmercury on goldfish sperm clearly show that it is a very powerful methodologyfor determining the effects of pollution on reproduction of fishes. It could alsohave very widespread application in other areas of fish reproduction, especiallyin the field of cryopreservation, in which cryoprotectants or freeze–thaw regimesare known to cause morphological damage to the sperm (Billard, 1983; Billardet al., 2000).In the present study exposure of sperm to mercuric chloride was used as an

example of the application of ASMA. Two exposure regimes were chosen:instant exposure, to mimic the effect of mercury on sperm released into apolluted aquatic environment, and 24 h exposure, to mimic exposure of spermto bioaccumulated mercury within the male reproductive system. The datademonstrate the value of ASMA in showing morphological damage to goldfishsperm after exposure to mercury for both NP and 24 h. Flagellum length wassignificantly shortened after instantaneous exposure to 100mg l�1 (368mM)mercuric chloride or 24 h exposure to 0�001mg l�1 (0�0037 mM). Sperm headlength, width and area increased significantly after 24 h exposure to 0�1mg l�1

(0�37mM) mercuric chloride. This is in accord with previous mammalian andinvertebrate studies, which have described disruption to the flagellum whensperm were exposed in vitro or in vivo to heavy metals or organometalliccompounds (Mohamed et al., 1986; Rao, 1989; Au et al., 2001). For instance,rat sperm exposed in vitro to 10mg l�1 methylmercury showed coiled flagella,and kinks in the midpiece and flagellum regions (Rao, 1989). Similarly, coiledflagella and kinks at the midpiece junction were observed after 1mg l�1 methyl-mercury hydroxide was added in vitro to spermatozoa of M. fascicularis(Mohamed et al., 1986). Sea urchins Anthocidaris crassispina exposed in vivoto 0�01mg l�1 cadmium for 4 weeks had spermatozoa with short, incomplete‘broken’ tails (Au et al., 2001). These studies did not, however, suggest apossible mechanism by which the sperm flagellum was damaged.After instant exposure to 100mg l�1 (368 mM) mercuric chloride, flagellum

length of goldfish spermatozoa was slightly shortened from 46�2 to 35�9, whileafter 24 h exposure to 0�1mg l�1 (0�37mM) mercuric chloride, flagellum lengthwas significantly reduced to only 15�4 mm. This shortening or breakage offlagella may have been the result of the mercury directly disrupting themicrotubules of the flagellum (Sager, 1988). In the green alga Chlamydomonasreinhardtii the breakage of flagella is caused by microtubular severing due toincreased Ca2þ levels (Sanders & Salisbury, 1989; Salisbury, 1995). Alterna-tively, the microtubules of the goldfish sperm flagella may have been detachedindirectly by a mechanical force, such as a torsional load, shear or axialcompression and tension (Sanders & Salisbury, 1989). Mercuric chloride(mM l�1 concentration) induces twisting of the flagella in turbot Psetta maxima



(L.) sperm (Cosson et al., 1999), which may result in axial compression andtension, for example in the microtubules, causing them to sever and the flagellato break. Mercury may, on the other hand, have caused breakage of the flagelladue to its affinity for sulphydryl groups (Moore & Ramamoorthy, 1984).Proteins containing such groups are known to be present in the membranes ofthe flagellum, the flagellar matrix and outer longitudinal fibres (Nelson, 1960),and the mercuric chloride may have bound to or denatured these groups,indirectly affecting flagellar length.The length, width and area of sperm heads were significantly increased when

sperm were exposed to concentrations of �0�1mg l�1 (0�37mM) mercuric chlor-ide for 24 h. Mercury at these concentrations may have disrupted the plasmamembrane of the sperm head, thus increasing ion flow into the head andresulting in swelling. Au et al. (2001) found that the plasma membrane ofsperm heads of sea urchins exposed in vivo to 0�01mg l�1 cadmium becamemore convoluted, which may have been due to binding of cadmium onto thesperm plasma membrane, interfering with the proper functioning of calciumchannels during acrosomal reactions.Spermmotility is the result of the propagation of waves along the flagellum, which

are generated by a sliding microtubule mechanism (Gagnon, 1995). Mohamed et al.(1986) suggested that the effects of methylmercury, and possibly also mercury, onspermmotility were due to the interference of methylmercury (and mercury) with thedynein-microtubule sliding assembly. In the present study, flagellar length wassignificantly shortened by 100mg l�1 (368mM) mercuric chloride after NP mercuryexposure, but VCL and the percentage of motile sperm were significantly decreasedalready at 1 and 10mg l�1 (3�68 and 36�8mM), respectively. Mercuric chloride at1mg l�1 (3�68mM) may therefore have decreased sperm motility by interfering withthe sliding microtubule mechanism and reducing mitochondrial energy levels. Thus,whereas this concentration of mercury did not significantly shorten flagellar length, itmay have severed a few microtubules of the flagellum and, consequently, weakenedflagellar beat. On the other hand, a larger proportion of spermatozoa had shortenedflagella at 100mg l�1 (368mM) mercuric chloride, probably due to increased disrup-tion of microtubules by the higher mercury dose. In comparison, 24h exposure to0�1mg l�1 (0�37mM) mercuric chloride resulted in shortened or broken flagella in98% of spermatozoa and significantly decreased sperm motility. This concentrationof mercury probably caused complete severing of the microtubules at a weak point,resulting in the majority of flagella being shortened or broken and, as a consequence,significantly impaired sperm movement. It is, however, possible that sperm motilitycould have been reduced by a completely different mechanism, such as decreasingATP levels of the spermatozoa (Rurangwa et al., 2002) or changes in water permea-bility of the sperm (Preston et al., 1993).Both sperm morphology and motility are important factors influencing ferti-

lity and, hence, fertilization success in free-spawning teleosts. Clearly, if spermare morphologically disrupted their fertilizing ability and success of fertilizationare likely to be very low or non-existent. A relationship between morphologically-disrupted sperm and fertility has been shown in several mammalian species(Dahlbom et al., 1997). Abnormalities of shape and size of spermatozoa areknown to be associated with clinical infertility in the bull (Sailer et al., 1996)and man (Ross et al., 1971). When determining the fertility and fertilization rate



of an individual, however, both sperm motility and morphology should beconsidered, since morphology can provide an indication of the possible mechan-isms (structural or energy) by which motility is affected. Certainly, if themorphology of sperm is adversely impacted, such as when flagella are shortenedor broken, sperm may have decreased or absent motility and their ability tofertilize ova will be reduced or non-existent. Gomendio & Roldan (1991) showedthat flagellum length in mice was positively correlated with sperm velocity and thepresent data (Fig. 4) support these suggestions that a shortening of the flagellumreduces sperm velocity. Impala exposed to high copper levels (126�5mgkg�1

copper in their livers) demonstrated a higher percentage of sperm with vacuolesin the neck region than sperm of control animals (Ackerman et al., 1999). Theauthors suggested that the vacuoles in the neck region of sperm could detach theflagellum, thus hampering motility. Since the ability of sperm to reach the ovasuccessfully is very much dependent on sperm motility (Ackerman et al., 1999),fertilization success would be reduced.Mercury concentrations in fresh water range from 0�04 to 74 ng l�1 in lakes

and from 1 to 7 ng l�1 in rivers and streams (USEPA, 1997). Mercury levels can,however, be much higher in waters contaminated by point-source dischargesfrom chloralkali plants and mining sites, with levels in the range of0�15–0�70mg l�1 mercury (Jian & McLeod, 1992). Freshwater mercury levelsand the highest levels of point-source discharges of mercury are still lowerthan the concentrations used in this study [0�001–100mg l�1(0�0037–368mM)mercuric chloride]. Bioaccumulation, however, of mercury may lead to levelswithin an organism that are 100–10 000 times higher than the mercury concen-trations in their surrounding environment. Crucian carp Carassius carassiuslangsdorfii Temminck & Schlegel from polluted Japanese rivers bioconcentratedinorganic mercury from the water 10 000� 2600 times (Matsunaga, 1975). Malegoldfish exposed to 1 mg l�1 mercuric chloride via their holding water had 6, 17and 5mgkg�1 mercury in their testes, liver and muscle, respectively, after 6weeks, i.e. bioconcentration factors of 5000–17 000 depending on the tissue(K.J.W. Van Look, unpubl. data). Even background levels of mercury canresult in significant mercury bioaccumulation in fishes. A recent study of 24‘clean’ Massachusetts’ waterbodies found wet mass muscle mercury concentra-tions of 0�15mgkg�1 in brown bullheads Ameiurus nebulosus (Lesueur),0�31mgkg�1 in yellow perch Perca flavescens (Mitchill) and 0�39mgkg�1 inlargemouth bass Micropterus salmoides (Lacepede) (Rose et al., 1999). Theconcentrations of mercury causing shortening of flagella and increased spermhead lengths, widths and areas in this study are therefore comparable tobioaccumulated mercury levels in wild and laboratory-exposed fishes.ASMA is a powerful tool for assessing the morphometric and potential

reproductive quality of sperm due to the objectivity, accuracy, repeatabilityand randomness of choice of the technique compared to previous manualmethods. In addition, the technique developed does not require any specimenpreparation, such as fixing or staining, which may damage sperm structure andcause morphological artefacts. Sperm can be exposed to pollutants in vitro forNP, 12 or 24 h for example, before examining its morphology. This is muchmore rapid and cost-effective than whole animal exposure and, if milt can beobtained without sacrifice (as in cyprinids and salmonids), ASMA obviates the



use of whole animals for testing. Alternatively, milt may be stripped in the fieldfrom wild populations of fishes exposed to environmental pollution and themorphology of their sperm assessed. ASMA clearly has considerable potentialfor becoming a widely used, simple and accurate measure of the effects oftoxicants on fish sperm morphology and by implication, reproduction. It alsohas widespread application in other areas of study of fish reproductive biologyand aquaculture.

The authors gratefully acknowledge a grant (FAIR Project CT97 3755) from theEuropean Commission and a Hossein Farmy studentship to KVL from the Universityof Sheffield. The authors also thank two anonymous referees for their helpful comments.

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