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Author's personal copy Ecotoxicology and Environmental Safety 84 (2012) 293–298
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Cryopreserved semen in ecotoxicological bioassays: Sensitivity and reliability of cryopreserved Sparus aurata spermatozoa Adele Fabbrocini a,n, Raffaele D’Adamo a, Francesco Del Prete b,c, Antonio Luca Langellotti c, Francesca Rinna b, Fausto Silvestri b, Gerarda Sorrenti a, Valentina Vitiello b,c, Giovanni Sansone b,c a
Consiglio Nazionale delle Ricerche, Istituto di Scienze Marine, via Pola, 4, 71010 Lesina, Foggia, Italy Dipartimento delle Scienze Biologiche, Universita degli Studi di Napoli Federico II, via Mezzocannone 16, 80134 Napoli, Italy c Centro Interdipartimentale di Ricerche per la Gestione delle Risorse Idrobiologiche e per l’Acquacoltura, Universita degli Studi di Napoli Federico II, via Universita 100, 80055 Portici (NA), Italy b
a r t i c l e i n f o
abstract
Article history: Received 19 April 2012 Received in revised form 24 July 2012 Accepted 25 July 2012 Available online 11 August 2012
The aim of this study was to evaluate the feasibility of using cryopreserved S. aurata semen in spermiotoxicity tests. Cryopreservation is a biotechnology that can provide viable gametes and embryos on demand, rather than only in the spawning season, thus overcoming a limitation that has hindered the use of some species in ecotoxicological bioassays. Firstly, the sperm motility pattern of cryopreserved semen was evaluated after thawing by means of both visual and computer-assisted analyses. Motility parameters in the cryopreserved semen did not change significantly in the first hour after thawing, meaning that they were maintained for long enough to enable their use in spermiotoxicity tests. In the second phase of the research, bioassays were performed, using cadmium as the reference toxicant, in order to evaluate the sensitivity of cryopreserved S. aurata semen to ecotoxicological contamination. The sensitivity of the sperm motility parameters used as endpoints (motility percentages and velocities) proved to be comparable to what has been recorded for the fresh semen of other aquatic species (LOECs from 0.02 to 0.03 mg L 1). The test showed good reliability and was found to be rapid and easy to perform, requiring only a small volume of the sample. Moreover, cryopreserved semen is easy to store and transfer and makes it possible to perform bioassays in different sites or at different times with the same batch of semen. The proposed bioassay is therefore a promising starting point for the development of toxicity tests that are increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies. & 2012 Elsevier Inc. All rights reserved.
Keywords: Sea bream Spermatozoa Cryopreservation Sperm motility Ecotoxicological test
1. Introduction Current strategies in monitoring programs for marine–coastal areas usually require the integration of chemical analyses and biological testing in order to better evaluate the bioavailable fraction of toxicant actually interacting with living organisms (Macova et al., 2010; Coulaud et al., 2011). In terms of biological parameters, reproductive success being a crucial factor in determining the survival of a species, ecotoxicological bioassays using gametes and embryos of aquatic species are widely employed (Losso et al., 2007; MamindyPajany et al., 2010). Toxicants elicit a variety of effects depending on the species and the different synergistic and/or antagonistic effects of bioavailable substances present in combination. Therefore test
n
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[email protected] (A. Fabbrocini).
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batteries need to include organisms representing different phyla and different trophic levels (Macken et al., 2009). Organisms used in ecotoxicological bioassays should also be ecologically relevant and easily available all year round, and they should have well-known and above all homogeneous physiological responses. Moreover, they should come from the monitored area, or at least from similar environments, so that it is not necessary to alter the chemical and physical characteristics of the tested matrices and therefore the reactivity of the toxicants present. In addition, the relative endpoints should be selected so that they can be accurately, predictably and reliably measured (Chapman, 2002). Although ecotoxicological tests have been standardised for gametes and embryos from a range of aquatic species, in many cases their availability (limited to the spawning season), the difficulty in collecting and storing them until the tests are carried out and above all their potentially high biological variability still limit their use (Paredes and Bellas, 2009; Schipper et al., 2008).
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Cryopreserved gametes and embryos can play a useful role in strategies to overcome these limits. Indeed, successful cryopreservation procedures provide viable gametes and embryos that can be easily transferred and stored for prolonged periods (Suquet et al., 2000; Chao and Liao, 2001). The last ten years have seen an increasing number of studies involving the cryopreservation of sperm and embryos from a range of aquatic organisms (Sansone et al., 2002; Paredes and Bellas, 2009; Chen et al., 2010). These have generated successful cryopreservation protocols, enabling the all-year-round availability of biological samples from many invertebrate and vertebrate species, whose usefulness in ecotoxicological tests can now be tested. The sea bream Sparus aurata is a euryhaline species commonly found in both marine and brackish environments, intensively reared in the Mediterranean region (FAO, 2008). Due to its high commercial importance, its physiological responses to toxicant exposure have been evaluated (Cirillo et al., 2011). Its sperm morphology and motility have also been widely studied (Beira~ o et al., 2010; Lahnsteiner et al., 2010), and experimentally verified cryopreservation protocols have been available for some time (Fabbrocini et al., 2000; Cabrita et al., 2005). The aim of this study was to assess the post-thawing motility patterns of cryopreserved S. aurata spermatozoa, and to evaluate the feasibility of their use in ecotoxicological bioassays. The effects of cadmium on the main parameters describing sperm motility were evaluated by means of both visual and computer assisted analyses, to assess their relative sensitivity with a view to their use as endpoints in spermiotoxicity bioassays. 2. Materials and methods 2.1. Experimental design In the first phase of the research the sperm motility patterns of cryopreserved semen were evaluated immediately after thawing and in 1 h and 2 h post-thawing incubated samples. The percentage of spermatozoa with optimal motility was assessed with reference to indicators measured by visual and computer-assisted analyses, verifying whether the indicator values were maintained long enough to enable their evaluation in spermiotoxicity tests, i.e. 60–120 min of incubation, as recommended by USEPA (United States Environmental Protection Agency) (2000) and ASTM (American Society for Testing and Materials) (2004). In the second phase of the research, spermiotoxicity tests were performed on cryopreserved semen, using cadmium as a reference toxicant, in order to evaluate the relative sensitivity of the same motility indicators as endpoints. All the assays carried out with sea bream were performed in accordance with national and institutional guidelines for the protection of wildlife animal welfare. 2.2. Semen collection The semen was collected by abdominal stripping from adult male sea bream bred at the Azienda Ittica Ugento and Azienda Ittica Caldoli, two fish farms situated in the Puglia Region (Southern Italy). Six collections were made at monthly intervals in two successive reproductive seasons (December–February). For each collection, 10–50 sea bream were stripped. Artificial seawater (35%) at a ratio of 100:1 was added immediately after collection to a sample from each subject to activate the spermatozoa and evaluate the initial motility. Semen was selected by visual assessment of motility, as described in section 2.4.1. Samples found to have poor motility (with a percentage of progressive vigorously motile spermatozoa below 90%, delayed motility activation, i.e. requiring more than 30 s) or low sperm concentration, or to be contaminated by faeces or urine were discarded. Subsequently, after positive evaluation, two pools of semen, each from the five best males, were made upin each collection. Therefore, 12 pools, making a total of 60 different semen samples, were cryopreserved and used in the next phase of the research. 2.3. Cryopreservation procedure Semen was cryopreserved in accordance with the procedure described in Fabbrocini et al. (2000). Semen was diluted at a ratio of 1:6 in 1% NaCl (motility inhibitor medium) containing 5% dimethyl sulfoxide (DMSO), inserted by Pasteur pipette into 0.25 ml plastic straws, frozen at a gradient of 10 1C min 1 from 0 to 150 1C, and finally immersed in liquid nitrogen. The freezing procedure was performed in a CryoMeds 8023 programmable freezer (Thermo Scientific Forma, Inc.).
2.4. Motility evaluation parameters Straws were thawed at a rate of 15 1C sec 1 (7 s in a water bath at 35 1C); the thawing procedure was standardized by using straws in which a thermocouple had been inserted before freezing. Thawed samples were randomly pooled (5 straws/pool, irrespective of the pre-freezing origin) and incubated undiluted at þ 4 1C for 1 h and 2 h. On thawing and at the end of the two incubation times, sperm motility was activated by diluting aliquots of semen in 35% ASW (artificial sea water) at 18 1C (ASTM (American Society for Testing and Materials), 2004) at a final rate of 1:100. BSA (bovine serum albumin, fraction V) at a concentration of 0.05% was used as an anti-sticking agent; the presence of anti-sticking agents in the activating medium (ASW) is believed to prevent sperm from adhering to the slide, which would alter the sperm motility recording (Kime et al., 2001). 2.4.1. Visually-assessed sperm motility For each assessment of motility, the semen was activated as described in Section 2.4 directly on an uncoated glass slide and immediately observed by microscope (40 10 magnification). The percentage of progressive vigorously motile spermatozoa (PVM), calculated in relation to the total amount of cells (including immotile and poorly motile cells) observed in each field was assessed by two trained workers; a blind evaluation of the samples was also performed, in order to reduce observers’ subjectivity. For the visual evaluation of the motility patterns of cryopreserved semen six different pools were analysed (n¼ 6). 2.4.2. Computer-assessed sperm motility parameters For each assessment of motility, semen was activated in a small polyethylene vial (Eppendorf), as described in Section 2.4. 1.5 ml of semen were immediately pipetted on to a multi-chamber counting slide (10 mm thick; Leja, The Netherlands). Sperm movement was recorded using a 100 frame sec 1 camera (Basler, 782 582 resolution) attached to a microscope (Nikon Eclipse E600) with a phasecontrast objective (10 10 magnification) and connected to a computerised motion analysis system, the Sperm Class Analysers (SCA, Microptic, s.l., Spain). The SCA acquisition parameters were set as follows: maximum area¼400 mm2; minimum area¼ 50 mm2; frame rate¼ 100 s 1; total captured images ¼100. The following motion parameters were assessed: (a) Curvilinear Velocity (VCL, mm sec 1); (b) Straight-line Velocity (VSL, mm sec 1); (c) Average Path Velocity (VAP, mm sec 1); (d) Percentage of rapid sperm (RAP, curvilinear velocity above 100 mm sec 1). As the subpopulation of rapid sperm (in which VCL 4100 mm sec 1) has been shown to be positively correlated with hatching rate in fertilisation trials with S. aurata cryopreserved semen (Beira~ o et al., 2011), additional parameters regarding this subpopulation were evaluated: (e) ALH-r: Amplitude of lateral head displacement (mm); (f) BCF-r: Timeaveraged rate at which the curvilinear sperm trajectory crosses its average trajectory (beats sec 1); (g) VCL-r: (Curvilinear Velocity, mm sec 1); (h) VSL-r: (Straight-line Velocity, mm sec 1); (i) VAP-r: (Average Path Velocity, mm sec 1). For each semen sample, motility records were taken in 3 different microscopic fields. Each record consisted of the mean of three replicates from each field, each analysing from 250 to 500 sperm tracks. Records were carefully checked for sample drifting. For the computer assisted evaluation of the motility pattern of cryopreserved semen ten different pools were analysed (n¼ 10). 2.5. Spermiotoxicity tests Cryopreserved straws were thawed and sperm motility after activation in ASW was rapidly assessed by microscope, as described in Section. 2.2. Successfully cryopreserved straws were pooled (5 straws/pool, irrespective of the pre-freezing origin) and used to carry out the bioassays. Thawed semen was diluted 1:6 in 1% NaCl containing scaled concentrations (0.01–100 mg L 1) of cadmium as Cd(II) solution (prepared from standard solution for atomic absorption spectroscopy, Baker Italy, Milan) and incubated at þ4 1C for up to 60 min. 1% NaCl was used as a negative control. Each trial was run in triplicate. At the end of the incubation, sperm motility was triggered by dilution in ASW and the motility parameters were evaluated by visual assessment and by SCAs as described in Sections 2.4.1 and 2.4.2 respectively. Five independent trials were performed using five different cryopreserved semen pools. 2.6. Statistical analysis The sperm motility parameter values on thawing are expressed as mean values7 SD. Prior to analysis, percentage data were arcsine square-root transformed. Data were tested for normality using Cochran’s test and for homogeneity of variance
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300
100 50 0
t0
1h
2h
a
60
b
40 20 PVM
t0
1h
RAP
2h
µm sec-1
250 200 150 100 50 0
t0
1h
35
2h beats sec-1
µm
3 2 1 0
t0
1h
2h
30 25 20 15 10 5
ALH-r
0
BCF-r
Fig. 4. (A) Amplitude of lateral head displacement (ALH-r, mm) and (B) beat/cross frequency (BCF-r, beats sec 1) of rapid spermatozoa on thawing (t0) and after 1 h and 2 h of post-thawing incubation at þ 4 1C.
Table 1 Levels of LOEC (Low Observed Effect Concentration), EC50 with the relative confidence limits (95%) and COV% (coefficient of variation) for each tested end point during exposure to cadmium. End points*
LOEC mg L 1
EC50 mg L 1
COV%
PVM RAP VCL VSL VAP
0.02 0.03 0.03 0.03 0.03
1.67(1.11–2.44) 2.45(1.77–3.33) 3.87(2.84–5.28) 3.51(2.55–4.84) 3.27(2.39–4.47)
39 28 26 29 27
n PVM ¼progressive and vigorous motile spermatozoa; RAP ¼% of rapid spermatozoa (VCL4 100 mm sec 1); VCL ¼ curvilinear velocity; VSL¼straight line velocity; VAP ¼ average path velocity.
Fig. 1. Percentages of progressive and vigorously motile (PVM) and rapid (RAP, VCL4100 mm sec 1) activated spermatozoa on thawing (t0) and after 1 h and 2 h of post-thawing incubation at þ 4 1C. Values with different superscript letters are significantly different (P o0.01).
300
VAP-r
VSL-r
4
a
0
VCL-r
Fig. 3. Curvilinear (VCL-r), straight line (VSL-r) and average path (VAP-r) velocities (mm sec 1) of rapid spermatozoa on thawing (t0) and after 1 h and 2 h of postthawing incubation at þ 4 1C.
5
80
2h
150
3.1. Motility evaluation of thawed semen The sperm motility parameter values recorded by both visual and SCAs assessment on thawing and after 1 h and 2 h at þ4 1C are shown in Figs. 1–4. As shown in Fig. 1, the percentage of spermatozoa found to be progressive vigorously motile (PVM) by visual assessment did not show any significant relationship to incubation time after thawing. In contrast, the percentage of rapid spermatozoa (RAP, curvilinear velocity above 100 mm sec 1) as assessed by SCAs was significantly affected by incubation time (F2,27 ¼9.87). Although no differences were recorded by post hoc Dunnett’s test after 1 h at þ4 1C, in 2h-incubated samples RAP was significantly lower than the control. The curvilinear velocity (VCL) of semen on thawing ranged from 110 to 260 mm sec 1, straight-line velocity (VSL) from
1h
200
3. Results
% 100
t0
250 µm sec-1
using Shapiro-Wilk’s test, and then analysed by one-way ANOVA. When significant effects were shown to occur, Dunnett’s post-hoc test was performed. P o0.01 was considered to be significant. STATISTICA (Vers. 8.0, 2008, StatSoft, Inc.) software was used for statistical analyses. The responses of each tested endpoint in the spermiotoxicity tests (PVM, RAP, VCL, VSL, VAP, VCL-r, VSL-r, VAP-r, ALH-r, BCF-r) were corrected for effects observed in controls by applying Abbott’s formula (ASTM (American Society for Testing and Materials), 2004). Data were then expressed as EC50 values with 95% confidence limits, calculated using the Trimmed Spearman–Karber statistical method (ASTM (American Society for Testing and Materials), 2004). Furthermore, for each tested endpoint the coefficient of variation (COV%) of the EC50 value was calculated using the results from the five different trials in order to evaluate its replicability. The LOEC (low observed effect concentration) values for each endpoint were calculated by Dunnett’s method (USEPA (United States Environmental Protection Agency), 2000).
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70 to 200 mm sec 1, and average path velocity (VAP) from 100 to 200 mm sec 1 (Fig. 2). Although a decrease in velocity parameters after 1 and 2 h incubation time was observed, no significant differences were recorded by ANOVA. Regarding the velocity parameters of the subpopulation of rapid sperm (VCL-r, VSL-r, VAP-r, Fig. 3), again no differences in relation to post-thawing incubation time were recorded. ALH-r and BCF-r values (Fig. 4) were not significantly affected by incubation time either, remaining at around 3 mm and 27 beats sec 1 respectively. Since the motility parameters did not change significantly in the first hour after thawing, an exposure time of 60 min was selected in the spermiotoxicity bioassays for all the tested endpoints. 3.2. Spermiotoxicity tests
VCL
VSL
VAP
Fig. 2. Curvilinear (VCL), straight line (VSL) and average path (VAP) velocities (mm sec 1) of activated spermatozoa, on thawing (t0) and after 1 h and 2 h of post-thawing incubation at þ4 1C.
Table 1 shows the LOEC (Low Observed Effect Concentration) values and the EC50 levels with the relative confidence limits (95%) and COV% (Coefficient of Variation) for each tested endpoint during exposure to cadmium.
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The visually assessed endpoint (PVM) showed an LOEC of 0.02 mg L 1 of cadmium and an EC50 of 1.67 mg L 1. The LOEC values of the computer-assessed endpoints (RAP, VCL, VSL, VAP) were in all cases 0.03 mg L 1, while EC50 levels ranged from 2.47 mg L 1 for RAP to 3.87 mg L 1 for VCL. The coefficient of variation (COV%) was 39% for PVM, and from 26 to 29% for the computer-assessed endpoints. In conditions of up to 50 mg L 1 of cadmium exposure, the endpoints for the subpopulation of rapid sperm (VCL-r, VSL-r, VAP-r, ALH-r and BCF-r) showed no significant differences with respect to the control, and therefore they are not shown in Table 1.
4. Discussion In this study we evaluated whether the motility parameters of cryopreserved S. aurata semen meet the requirements for use in ecotoxicological tests. As spermiotoxicity test protocols involve an incubation phase before motility evaluation, not only motility on thawing but also longevity, i.e. the ability to reactivate motility by dilution in appropriate media at some time after thawing, need to be considered. In the present study neither the visual nor the computer-evaluated motility parameters of cryopreserved semen were found to change significantly when motility was activated up to one hour after thawing, a significant decrease in the percentage of rapid sperm (VCL 4100 mm sec 1) being recorded only when semen was activated two hours after thawing. Therefore its longevity can be considered sufficient for spermiotoxicity tests (USEPA (United States Environmental Protection Agency), 2000; ASTM (American Society for Testing and Materials), 2004). As cryopreserved semen is expected to be used mainly for artificial reproduction, much attention has been paid to the post-thawing motility of sperm, which must be high enough to guarantee sperm-egg interaction (Suquet et al., 2000; Chao and Liao, 2001). However the longevity of cryopreserved fish semen has also been evaluated, and no changes in the sperm motility of the sea bream P. major (Chen et al., 2010) or in the fertilisation rate of the catfish C. garepinus (Kova´cs et al., 2010) were found in the first few hours after thawing. Physiological alterations in sperm quality may result in a decreased fertilisation rate. Sperm quality, especially the sperm motility pattern, is thus commonly considered a reliable predictor of the fertilisation ability of a semen sample (Kime et al., 2001; Fauvel et al., 2010; Beira~ o et al., 2011) and computer-assisted evaluation of sperm motility patterns is increasingly being used to investigate the effects of toxicants and environmental matrices (Au et al., 2000; Dietrich et al., 2010 and 2011; Li et al., 2010a,b). Toxicants may affect the spermatozoa of aquatic species, leading to an impairment of sperm motility in various ways: induction of oxidative stress, inhibition of sperm enzyme activity, impairment
of endocrine mechanisms and thus of spermatogenesis, and alteration of mitochondria ultrastructure and therefore ATP supply (Runnalls et al., 2007; Sarosiek et al., 2009; Li et al., 2010b). Moreover, toxicants, mainly heavy metals, may affect the membrane system responsible for osmotic regulation and motility activation, i.e. water channels or aquaporins (Abascal et al., 2007). Table 2 shows literature data on the effects of cadmium on the motility of fresh or refrigerated semen of various aquatic species. Although the studies differ slightly from ours in terms of biological system (i.e. the type and cryopreservation of semen), exposure conditions and endpoints, some useful comparisons with our data can be made. Regarding the velocity parameters (VCL, VSL, VAP), the LOECs recorded in our study fall within the same range as those recorded by Au et al. (2000) for the sea urchin A. crassispina, and by Li et al. (2010a) for the sturgeon A. gueldenstaedtii. The EC50 levels recorded in our study for the velocity parameters and RAP are comparable to those reported for the sea urchin P. lividus by Fabbrocini et al. (2010). Despite being a blind analysis, visually assessed PVM should be considered a subjective evaluation parameter compared to those recorded by computer assisted analysis. Nevertheless, as it gives an overall picture of semen sample quality, it proved to be a sensitive endpoint, and may be considered comparable to or even more sensitive than the ‘‘total motile spermatozoa percentage’’ evaluation parameter recorded by computer assisted analyses in rainbow trout, sturgeon and common carp (Dietrich et al., 2010, Li et al., 2010a; Dietrich et al., 2011 respectively). In general, we observed no significant differences with respect to controls for cadmium concentrations below 0.02 mg L 1. These results are in agreement with those of Li et al. (2010b), who found no effect on sperm motility and no antioxidant response in sturgeon sperm exposed to single heavy metals at environmental concentrations. On the other hand, data reported in Fabbrocini et al. (2010) concerning the motility endpoints of P. lividus sperm show that their sensitivity and ability to discriminate with respect to environmental matrices was comparable to that of commonly used bioassays, in spite of the relatively high EC50 level for the reference toxicant. In the light of this consideration and the results presented here, tests on environmental samples based on motility parameters of cryopreserved sea bream semen will be planned and performed. Furthermore, we found that the responses to toxicity of cryopreserved semen exposed for 1 h at þ4 1C were comparable to those of freshly collected semen exposed for 1–2 h at room temperature or for 24 h at þ4 1C. The short duration of the test and its sensitivity even at low incubation temperatures are advantages of the proposed method, as the toxicity levels of environmental samples containing volatile compounds are sometimes difficult to estimate and require rapid bioassays (Dinnel et al., 1981; Chapman et al., 2002). Finally it may be observed that the COV% exceeded 30% only with the PVM parameter, and then only slightly. Environment Canada (1990)
Table 2 Toxicity levels of cadmium on motility parameters of fresh semen reported in literature. End points* PVM
RAP VCL
VSL
**
Species
Exposure
Conc. (mg L 1)
Decrease
Reference
O.mykiss A.gueldenstaedtii Cyprinus carpio P.lividus A.crassispina A.gueldenstaedtii P.lividus A.crassispina P.lividus
4 h, þ4 1C 2 h, r.t. 24 h, þ4 1C 1 h, 18 1C 1 h, 18 1C 2 h, r.t. 1 h, 18 1C 1 h, 18 1C 1 h, 18 1C
10 0.05 50 2.16 5 0.05 2.61 1 2.38
67% 30% 63% EC50 LOEC 38% EC50 LOEC EC50
Dietrich et al., 2010 Li et al., 2010a Dietrich et al., 2011 Fabbrocini et al., 2010 Au et al., 2000 Li et al., 2010a Fabbrocini et al., 2010 Au et al., 2000 Fabbrocini et al., 2010
n PVM ¼progressive and vigorous motile spermatozoa; RAP ¼% of rapid spermatozoa (VCL 4100 mm sec 1); VCL¼ curvilinear velocity; VSL¼straight line velocity; VAP ¼average path velocity. nn Data assessed by computerised analyses.
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suggests a COV% acceptability limit of less than 20% for standardised tests, but recognises that ‘‘higher values (e.g. 30%) may be more realistic for some tests’’, as test replicability may be affected by many other factors, such as the degree of standardisation. The data presented here can be regarded as a preliminary survey to evaluate the feasibility of using cryopreserved semen in spermiotoxicity tests. Although these COV values are based on a low number of replicates (but are representative of a high number of thawed straws), in our opinion they represent an encouraging result that justifies broadening the research to include the use of cryopreserved biological indicators in toxicity tests. To our knowledge, although it has been proposed (Chao and Liao, 2001; Paredes and Bellas, 2009), the only cryopreserved indicators that have been successfully used in ecotoxicological tests are oyster and clam larvae (Mc Fadzen, 1992). Considering the number of aquatic species for which gamete cryopreservation procedures have been developed, (Suquet et al., 2000; Chao and Liao, 2001), and the fact that this test requires neither eggs nor standardised protocols for artificial fertilisation, it could easily be extended to species from a range of different environments, meeting the need for ecologically relevant biological indicators (Chapman, 2002).
5. Conclusions In conclusion, the motility of cryopreserved spermatozoa of sea bream S. aurata did not significantly change when activated up to one hour after thawing, thus exhibiting a longevity that is sufficient for them to be used in spermiotoxicity tests. The motility parameters used as endpoints in the bioassay with cadmium as a reference toxicant were characterised by good reliability and a sensitivity (LOECs from 0.02 to 0.03 mg L 1) comparable to that recorded for the fresh semen of other aquatic species. The maximum exposure time (60 min) is shorter than what has been reported for refrigerated semen. Cryopreserved semen can be easily stored and transferred, making it possible to perform bioassays in different sites or at different times with the same batch of semen. Motility assessments are rapid, easy to perform, require small volumes of the sample for testing, and do not require a specially equipped facility, since they can even be performed as part of in-the-field monitoring programs. All these observations considered, the proposed bioassay is a promising point of departure for the development of toxicity tests that can be increasingly tailored to the needs of ecotoxicology and environmental quality evaluation strategies.
Acknowledgements The Authors are grateful to Mrs M. A. Maselli for her invaluable support. Azienda Ittica Caldoli and Azienda Ittica Ugento kindly supplied sea bream semen. This research was supported by Puglia Regional Administration, (Explorative Projects, grant no. PE060 to A. Fabbrocini). Mr George Metcalf revised the English text. References Abascal, F.J., Cosson, J., Fauvel, C., 2007. Characterization of sperm motility in sea bass: the effect of heavy metals and physicochemical variables on sperm motility. J. Fish Biol. 70, 509–522. ASTM (American Society for Testing and Materials), 2004. Standard guide for conducting static acute toxicity tests with echinoid embryos. In: Annual Book of ASTM Standards. Philadelphia, PA, E 1563-04, pp.18. Au, D.W.T., Chiang, M.W.L., Wu, R.S.S., 2000. Effects of cadmium and phenol on motility and ultrastructure of sea urchin and mussel spermatozoa. Arch. Environ. Contam. Toxicol. 38, 455–463. Beira~ o, J., Pe´rez-Cerezales, S., Martinez-Pa´naramo, S., Herra´ez, M.P., 2010. Detection of early damage of sperm cell membrane in gilthead seabream (Sparus aurata) with the nuclear stain YO-PRO 1. J. Appl. Ichthyol 26, 794–796.
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