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Assessment of sperm concentration and motility in turbot (Scophthalmus maximus)
Aquaculture, 101 (1992) 177-185 Elsevier Science Publishers B.V., Amsterdam
177
Assessmentof sperm concentration and motility in turbot (Scophthalmus maximus) M. Suquet, M.H. Omnes, Y. Normant and C. Fauvel Instittrt Francais pour I’ExpIoitationde la Mer, BP 70, 29280 Plouzank,France (Accepted I8 April 1991)
ABSTRACT Suquet, M., Omnes, M.H., Normant, Y. and Fauvel, C., 1992. Assessment of sperm concentration and motility in turbot (Scophthalmusmuximus). Aquaculture, 101: 177-l 85. Several techniques al!owing an assessment of turbot sperm quality in terms of concentration and motility were assessed. They included techniques adapted to experimental objectives as well as practical gross evaluation techniques which could be used easily in production operations. Sperm concentration could not be accurately estimated by spermatocrit. Counting in a Malassez’s cell was an accurate but time-consuming technique. Sperm concentration could be evaluated by spectrophotometry at a 420 nm wavelength. Samples with a viscous aspect had a higher concentration (54.6 x lo9 + 5.4 spermatozoa/ml) than liquid ones (20.0 x IO9 f 2.4). Increasing dilution from 1: 10 to 1: 1000 affected sperm motion in terms of activation and duration of movement. At dilution I : IO. sperm moliiit); presented a linear decrease with time. Hence, the duration of movement could characterize sperm mution. Using these methods, sperm samples collected during the first stripping of the uaiural spawning season wtre described (volume I .6 k 0.2 ml; concentration 38.3 x IO9 + 5.9 spermatozoa/ml; motility 6.06 f I .08 min.s.; and spermatocrit 40.4 f 5.8%).
lNTRODIJCTION
In the literature, sperm quality has often been estimated by the concentration of spermatozoa and their motility observed after activation of the initially motionless spermatozoa. Milt concentration has been assessed by three techniques: counting in a hemocytometer chamber (Biiyiikhatipoglu and Holtz, 198$; Leung-Trujillo and Lawrence, 1987)) spermatocrit (Bouck and Jacobson, 1976; De Montalembert et al., 1980; Munkittrick and Moccia, 1987 ) and spectrophotometric evaluation (Biliard et al., 197 I ). Sperm motility has been estimated on an arbitrary scale (Billard et al., 1977; Goodall et al., 1989) by the percentage of motile cells (Levandusky and Cloud, 1988), by total duration of movement (Dupli:Tsky, 1982) or by a combination of both parameters (Baynes et al., 198 1). The purpose of this work was to test techniques to assess sperm quality in 0044-8486/92/$05.00
0 1992 Elsevicr Science Publishers B.V. All rights reserved.
178
M. SUQUET ET AL.
turbot in terms of concentration and motility. This technical adjustment to turbot will permit future description of sperm output in this species. MATERIAL AND METHODS
Sperm was collected by stripping ripe males after drying the genital area. Urine was carefully discarded and semen was collected in l-ml syringes. Evaluation of spermatozoa concentration Counting ardspectropkotometric estimation. For both techniques, semen was diluted twice in a solution of Triton X-100 (0.25%). The first dilution ( 1: 1)
avoided sperm aggregation which was observed during dilution in distilled or sea water. The second ( 1:200) brought spermatozoa to counting concentration. For counting in a Malassez’s cell, 0. l-ml samples of this solution were transferred into cells and spermatozoa were counted after decantation. By spectrophotometry, the absorption spectrum of the samples was determined using a Beckman Acta C-3 spectrophotometer. The spectrum of diluted sperm was compared with those of seminal fluid, urine and Triton X100. The optimal wavelength allowing assessment of sperm concentration was defined for minimal interference between specific absorption of diluted spermatozoa and that of associated fluids. Sperm concentrations of 36 males, previously evaluated by counting in a Malassez’s cell, were then assessed by means of a Beckman DU-5 spectrophotometer. Spermatocrit. The sperm of 25 males was individually transferred into sedi-
mentation cones. Spermatocrit (packed cell volume x lOO/total semen volume) was measured by means of micropipettes after centrifugation at 10 OOOg for 10 min. Evaluation of viscosity.A practical criterion for evaluation of spermatozoa
concentration was also tested on sperm of 150 males. Concentration of samples was evaluated by spectrophotometry and their visual aspect (viscous, liquid or undetermined) was observed. Estimation of motility
To test the effect of different dilutions on spermatozoa motility, 10 ~1of 45 individual semen samples were observed on a coverslipped glass slide after activation by 0.1, 1 and 10 ml sea water (dilution rate 1: 10, 1: 100 and 1: 1000). Motility was assessed at intervals of 30 s using an arbitrary scale on which 0 represents 0%, 1 fi O-25%, 2 A 50%, 3 =.=50.75%, 4 -h 75-100% of motile spermatozoa.
ASSESSMENTOF SPERM CONCENTRATION AND MOTILITY IN TURBOT
179
Validation To confirm these methods, the first stripping products collected at the beginning of the natural spawning season were described. Compared to the following ones, these samples present the highest values for volume and concentration of semen (Suquet et al., unpublished data). Twenty-five males (mean weight 2.2 2 0.3 kg), which had not been stripped for 1 year before the experimental period, were sampled. Sperm concentration and motility were assessed by the techniques mentioned above. Means are presented with their 95% confidence intervals. They were tested using a one-way ANOVA and, when differences were significant, a Tukey test was used. RESULTS
Evaluation ofspermatozoa concentration Counting. A lo-min decantation time was necessary to observe sperm in the same focal plane. Counting a minimum of 300 spermatozoa per Malassez’s cell on four samples, from two equivalent dilutions of the same semen, allowed the evaluation of concentrations with an acceptable coefficient of variation (2 to 20%, mean 6%). Spectrophotometric evaluation. Minimal interference was observed between specific absorption of diluted. spermatozoa and those of seminal fluid, urine and Triton X- 100 at a 420 nm wavelength (Fig. 1). Using this constant wavelength, a significant correlation was observed between optical density (OD) of 35 diluted semen samples and spermatozoa concentration (C ) : C (spz/ ml) = (4 OD - 0.15)i011; Y = 0.97 (Fig. 2). A 24-h storage at 4°C of the
(
CI-”
I
-
Spermatozoa
--
Semmol fluId
____
LJrtne
1
. . . . . . . Trlton
k 0
o-
, 350
I
I
I
450 550 650 ,&!As;E_ LENGTI-I ( nm:
I 750
I
Fig. 1. Absorption spectra of spermatozoa, seminal fluid, urir.e and T&on X- 100.
M.SUQUETET AL.
180
20
40
60
Concentration
iOC
80
(x lO’spz/ml)
Fig. 2. Relation between optical density at a wavelength of 420 nm and spermatozoa concentration.
0 0
/I‘\ 60i-5 “” b 2
40-
-
cO
0
0
0
E a’, $-
0
20-
0
- l-Y-.-L--_-_ ----‘---T--
0
IO
20
Concentration
30
40
50
60
70
(xl Ogspz/ml)
Fig. 3. Relation between spermatocrit and spermatozoa concentration.
ASSESSMENT
OF SPERM CONCENTRATION
AND MOTILITY
IN TURBOT
181
60 SO4530-
2 s Aspect Fig. 4. Relation betuzen aspect of sperm and spermatozoa concentration: 3, undetermined (mean concentration with 95% confidence interval). 1
1,
viscous; 2, liquid;
TABLE 1 Effect of dilution on sperm motility Dilution rate 1:lO
I:100
Initial motility (class)
Movement duration (min.sI
Initial motility
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
I0.00
4
7.00 9.30 2.30 2.00 2.08 5.30 3.00 3.30 3.00 5.00 4.30 2.30 4.30 3.30
(class )
4 4 3 4 3 4 3 4 3 4 4 2 4 4
I: 1000
Movement duration (min.s)
Knitial motility (class)
Movement duration (min.s)
5.30 4.30 4.40 2.00 1.30 1.30 3.30 2.30 2.30 3.30 2.00 3.00 1.30 3.30 2.30
3 I I I 2 2 I 1 3 I I 3 I I 3
5.30 1.oo 1.oo 1.oo 0.30 1.30 1.30 1.30 2.00 1.oo 0.30 2.00 1.oo 1.30 2.30
M. SUQUET ET AL.
182
I’
0
0
I
I”
2 Duration
1
I”
4 of movement
0
I
r
n
n
1
8
6 (miilutes)
Fig. 5. Sperm motility in relation to time (three examples showing different decreasing schemes are presented). TABLE2 Mean values observed for the first sampling of sperm collected during the natural spawning season (data f 95%confidenqe interval) Volume (ml) Concentration ( x IO9spz/ml)
1.6kO.2 38.3 f 5.9
Motility (min.s) Spermatocrit (%)
6.06k 1.08 40.4 k5.8
first dilution ( 1: 1) did not modify the optical density. However, absorption measurement of samples frozen after the first dilution were unsuccessful since the solutions lost their homogeneity at thawing. Spermatocrit. No significant correlation was observed between sperm density and spermatocrit: r = 0.07 (Fig. 3). Estimation of viscosity.Samples with a viscous aspect had a significantly higher concentration (mean 54.6 x LO92 5.4 spermatozoa/ml) than liquid (mean 20.0 2 2.4) or undetermined ones (mean 2 1.1 II 4.4): PC 0.001 (Fig. 4). It
ASSESSMENT OF SPERMCONCENTRATIONAND MOTILITY IN TURBOT
183
was not possible to discriminate liquid and undetermined aspects in terms of concentration.
Estimation of motility Increasing dilution affected sperm motility (Table 1 ), but this difference was only significant between dilutions 1: 10 and 1: 1000 in terms of initial motility (P
Validation Mean values collected during the first sample of the natural spawning season are presented in Table 2. DISCUSSION
Evahation of spermatozoa concentration When assessing milt density by counting in a Malassez’s cell, four samples of at least 300 spermatozoa each are necessary to observe an acceptable coefficient of variation. However, it takes 2 h to evaluate the sperm concentration of only one fish. Thus, although accurate, this method cannot be used for practical estimation. Since spermatocrit is not correlated with spermatozoa concentration as in salmonids (Bouck and Jacobson, 1976) or pike (De Montalembert et al., 1%O), this parameter can not be used to evaluate bperm concentration in turbot. As in rainbow trout (Billard et al., 197 1 ), a spectrophotometric evaluation allows an accurate estimation of milt concentration. At 420 nm there is minimal interference between absorption of sperm and associated fluids. This method appears to be best adapted for experimentation since a IJV monitor is unusual in the field. According to results observed in carp (Clemens and Grant, 1965 ), samples with a viscous aspect present a significantly higher concentration of sperm than the liquid or undetermined ones. Though subjective, sperm aspects can easily be classified. This fast estimation should be useful in the field to deter s mine the volume of sperm necessary for fertilization.
Estimation of motiIi,ty In contrast to the results of Gray ( 1928), working on sea-urchin spermatozoa, a highL mIdilution enhanced neither the initiation of motility nor the duration of movement. After cryopreservation for 1 month, no motion was observed in spermatozoa of gilthead sea bream when the diluent/sperm ratio
184
M. SUQUET ET AL.
was high (Chambeyron and Zohar, 1990). Dilption could remove the protec-
tive role of seminal plasma, as suggested for mammals (Harrison et al., 1978 ) and fishes (Billard, 1982, 1983 ). For a 1: 10 dilution the motility decrease appears to be linear. The intercept is a constant value (motility class 4) and the slope is only a function of duration of movement. Hence, the time when al! movement ceased can be used as a practical motility criterion, so that intermediate measurements are not necessary. Validation Data collected for turbot during the first sample of the natural spawning season are higher than for salmonids (Billard et al., 197 1; Scott and Baynes, 1980) and equivalent to those observed in the sea bass (Billard et al., 1977; Zohar et al., 1984). As in these species, a high individual variability was observed. Sperm quality in turbot can be reliably characterised by the techniques described in this paper. Relation of these criteria to fertilization ability of spermatozoa will be studied in further research. ACKNOWLEDGEMENTS
We wish to thank Mr. Y. Harache for helpful comments on the manuscript.
REFERENCES aynes, S.M., Scott, A.P. and Dawson, A.P., 1981. Rainbow trout, Wmo gairdnerii Richardson, spermatozoa: effects of cations and pH on motility. J. Fish Biol., ‘r9: 259-267. Billard, R., 1982. Importance des protCines du liquide coelomique sur la fertilite des gam&es cke~ la truite arc en ciel et possibilitd de substitution. Bull. Fr. Pi&., 284: 168-l 77. Billard, R., 1983. Effects of coelomic and seminal fluids and various saline diluents on the fertilizing ability of spermatozoa in the rainbow trout, Safmo gairdneri. J. Reprod. Fertil., 68: 77-84. Billard, R., Breton, B. and Jalabert, B., 1971. La production spermato&nttique chez la truite. Ann. Biol. Anim. Biochim. Biophys., ll(2): 199-2 12. Billard, R., DuPont, J. and BarnabC,G., 1977. Diminution de la motilitb et de la duree de conservation du sperme de Dicentrarchus fa6ra.x L. (Poisson, TCltostten) pendant la pCriode de spermiation. Aquaculture, 11: 363-367. Bouck, G.R. and Jacobson, J., 1976. Estimation of salmonid sperm concentration by microhematocrit technique. Trans. Am. Fish. Sac.. 105(4): 534-535. Biiyiikhatipoglu, S. and Holtz, W., 1984. Sperm output in rainbow trout (Salmo gairdnerii) effect of age,timing and frequency of stripping and presence of females. Aquaculture, 37: 63-71.
ASSESSMENTOF SPERM CONCENTRATION AND MOTILITY IN TURBOT
185
Chambeyron, F. and Zohar, Y., 1990. A diluent for sperm cryopreservation of gilthead seabream, Sparus aurata. Aquaculture, 9: 345-352. Clemens, H.P. and Grant, B.G., 1965. The seminal thinning response of carp ( Cyprinus carpio) and rainbow trout (Saho gairdnerii) after injections of pituitary extracts. Copeia, 2: 174177. De Montalembert, G., Marcel, J. and Billard, R., 1980. La spermiation chez he brochet. 1. Evolution de la quantite de sperme recolte au @oursde la saison de reproduction. Bull. Fr. Piscic., 276: 90-103. Duplinsky, P.D., 1982. Sperm motility of northern pike and chain pickerel at various pH values. Trans. Am. Fish. Sot., 117: 768-771. Goodall, J.A., Blackshaw, A.W. and Capra, M.F., 1989. Factors affecting the activation and duration of motility of the spermatozoa of the summer whiting (Silfagu cifiata). Aquaculture, 77: 243-250. Gray, M.A., 1928. The effect of dilution on the activity of spermatozoa. J. Exp. Biol., 5: 337344. Harrison, R.A.P., Dott, H.M. and Foster, G-C., 1978. Effect of ionic strength; serum albumin and other macromolecules on the maintenance of motility and the surface of mammalian spermatozoa in a simple medium. J. Reprod. Fertil., 52: 65-73. Leung-Trujillo, J.R. and Lawrence, A.L., 1987. Observations on the decline in sperm quality of Penaeussetiferusunder laboratory conditiolts. Aquaculture, 65: 363-370. Levandusky, M.J. and Cloud, J.G., 1988. Rainbow trout (Safmo gairdneri) semen: effect of non-motile sperm on fertility. Aquaculture, 75: I 7 1-I 79. Munkittrick, K.R. and Moccia, P.D:j 1987. Seasonal changes in quality of rainbow trout (Salmo gairdneri) semen: effect of a delay in stripping on spermatocrit, motility, volume and seminal plasma constituents. Aquaculture, 64: 147- 156. Scott, A.P. and Baynes, S.M., 1980. A review of the biology, handling and storage of salmonid spermatozoa. J. Fish Biol., 17: 707-739. Zohar, Y., Billard, R. and Weill, C., 1984. La reproduction de la daurade (Sparus aurata) et du bar (Dicentrarchus fabrax): connaissance du cycle sexuel et controle de la gametogenese et de la ponte. In: G. Barnabe et R. Billard (Editors), L’Aquaculture du Bar et des Sparides. INRA Publ., Paris, pp. 3-24.
177
Assessmentof sperm concentration and motility in turbot (Scophthalmus maximus) M. Suquet, M.H. Omnes, Y. Normant and C. Fauvel Instittrt Francais pour I’ExpIoitationde la Mer, BP 70, 29280 Plouzank,France (Accepted I8 April 1991)
ABSTRACT Suquet, M., Omnes, M.H., Normant, Y. and Fauvel, C., 1992. Assessment of sperm concentration and motility in turbot (Scophthalmusmuximus). Aquaculture, 101: 177-l 85. Several techniques al!owing an assessment of turbot sperm quality in terms of concentration and motility were assessed. They included techniques adapted to experimental objectives as well as practical gross evaluation techniques which could be used easily in production operations. Sperm concentration could not be accurately estimated by spermatocrit. Counting in a Malassez’s cell was an accurate but time-consuming technique. Sperm concentration could be evaluated by spectrophotometry at a 420 nm wavelength. Samples with a viscous aspect had a higher concentration (54.6 x lo9 + 5.4 spermatozoa/ml) than liquid ones (20.0 x IO9 f 2.4). Increasing dilution from 1: 10 to 1: 1000 affected sperm motion in terms of activation and duration of movement. At dilution I : IO. sperm moliiit); presented a linear decrease with time. Hence, the duration of movement could characterize sperm mution. Using these methods, sperm samples collected during the first stripping of the uaiural spawning season wtre described (volume I .6 k 0.2 ml; concentration 38.3 x IO9 + 5.9 spermatozoa/ml; motility 6.06 f I .08 min.s.; and spermatocrit 40.4 f 5.8%).
lNTRODIJCTION
In the literature, sperm quality has often been estimated by the concentration of spermatozoa and their motility observed after activation of the initially motionless spermatozoa. Milt concentration has been assessed by three techniques: counting in a hemocytometer chamber (Biiyiikhatipoglu and Holtz, 198$; Leung-Trujillo and Lawrence, 1987)) spermatocrit (Bouck and Jacobson, 1976; De Montalembert et al., 1980; Munkittrick and Moccia, 1987 ) and spectrophotometric evaluation (Biliard et al., 197 I ). Sperm motility has been estimated on an arbitrary scale (Billard et al., 1977; Goodall et al., 1989) by the percentage of motile cells (Levandusky and Cloud, 1988), by total duration of movement (Dupli:Tsky, 1982) or by a combination of both parameters (Baynes et al., 198 1). The purpose of this work was to test techniques to assess sperm quality in 0044-8486/92/$05.00
0 1992 Elsevicr Science Publishers B.V. All rights reserved.
178
M. SUQUET ET AL.
turbot in terms of concentration and motility. This technical adjustment to turbot will permit future description of sperm output in this species. MATERIAL AND METHODS
Sperm was collected by stripping ripe males after drying the genital area. Urine was carefully discarded and semen was collected in l-ml syringes. Evaluation of spermatozoa concentration Counting ardspectropkotometric estimation. For both techniques, semen was diluted twice in a solution of Triton X-100 (0.25%). The first dilution ( 1: 1)
avoided sperm aggregation which was observed during dilution in distilled or sea water. The second ( 1:200) brought spermatozoa to counting concentration. For counting in a Malassez’s cell, 0. l-ml samples of this solution were transferred into cells and spermatozoa were counted after decantation. By spectrophotometry, the absorption spectrum of the samples was determined using a Beckman Acta C-3 spectrophotometer. The spectrum of diluted sperm was compared with those of seminal fluid, urine and Triton X100. The optimal wavelength allowing assessment of sperm concentration was defined for minimal interference between specific absorption of diluted spermatozoa and that of associated fluids. Sperm concentrations of 36 males, previously evaluated by counting in a Malassez’s cell, were then assessed by means of a Beckman DU-5 spectrophotometer. Spermatocrit. The sperm of 25 males was individually transferred into sedi-
mentation cones. Spermatocrit (packed cell volume x lOO/total semen volume) was measured by means of micropipettes after centrifugation at 10 OOOg for 10 min. Evaluation of viscosity.A practical criterion for evaluation of spermatozoa
concentration was also tested on sperm of 150 males. Concentration of samples was evaluated by spectrophotometry and their visual aspect (viscous, liquid or undetermined) was observed. Estimation of motility
To test the effect of different dilutions on spermatozoa motility, 10 ~1of 45 individual semen samples were observed on a coverslipped glass slide after activation by 0.1, 1 and 10 ml sea water (dilution rate 1: 10, 1: 100 and 1: 1000). Motility was assessed at intervals of 30 s using an arbitrary scale on which 0 represents 0%, 1 fi O-25%, 2 A 50%, 3 =.=50.75%, 4 -h 75-100% of motile spermatozoa.
ASSESSMENTOF SPERM CONCENTRATION AND MOTILITY IN TURBOT
179
Validation To confirm these methods, the first stripping products collected at the beginning of the natural spawning season were described. Compared to the following ones, these samples present the highest values for volume and concentration of semen (Suquet et al., unpublished data). Twenty-five males (mean weight 2.2 2 0.3 kg), which had not been stripped for 1 year before the experimental period, were sampled. Sperm concentration and motility were assessed by the techniques mentioned above. Means are presented with their 95% confidence intervals. They were tested using a one-way ANOVA and, when differences were significant, a Tukey test was used. RESULTS
Evaluation ofspermatozoa concentration Counting. A lo-min decantation time was necessary to observe sperm in the same focal plane. Counting a minimum of 300 spermatozoa per Malassez’s cell on four samples, from two equivalent dilutions of the same semen, allowed the evaluation of concentrations with an acceptable coefficient of variation (2 to 20%, mean 6%). Spectrophotometric evaluation. Minimal interference was observed between specific absorption of diluted. spermatozoa and those of seminal fluid, urine and Triton X- 100 at a 420 nm wavelength (Fig. 1). Using this constant wavelength, a significant correlation was observed between optical density (OD) of 35 diluted semen samples and spermatozoa concentration (C ) : C (spz/ ml) = (4 OD - 0.15)i011; Y = 0.97 (Fig. 2). A 24-h storage at 4°C of the
(
CI-”
I
-
Spermatozoa
--
Semmol fluId
____
LJrtne
1
. . . . . . . Trlton
k 0
o-
, 350
I
I
I
450 550 650 ,&!As;E_ LENGTI-I ( nm:
I 750
I
Fig. 1. Absorption spectra of spermatozoa, seminal fluid, urir.e and T&on X- 100.
M.SUQUETET AL.
180
20
40
60
Concentration
iOC
80
(x lO’spz/ml)
Fig. 2. Relation between optical density at a wavelength of 420 nm and spermatozoa concentration.
0 0
/I‘\ 60i-5 “” b 2
40-
-
cO
0
0
0
E a’, $-
0
20-
0
- l-Y-.-L--_-_ ----‘---T--
0
IO
20
Concentration
30
40
50
60
70
(xl Ogspz/ml)
Fig. 3. Relation between spermatocrit and spermatozoa concentration.
ASSESSMENT
OF SPERM CONCENTRATION
AND MOTILITY
IN TURBOT
181
60 SO4530-
2 s Aspect Fig. 4. Relation betuzen aspect of sperm and spermatozoa concentration: 3, undetermined (mean concentration with 95% confidence interval). 1
1,
viscous; 2, liquid;
TABLE 1 Effect of dilution on sperm motility Dilution rate 1:lO
I:100
Initial motility (class)
Movement duration (min.sI
Initial motility
4 4 4 4 4 4 4 4 4 4 4 4 4 4 4
I0.00
4
7.00 9.30 2.30 2.00 2.08 5.30 3.00 3.30 3.00 5.00 4.30 2.30 4.30 3.30
(class )
4 4 3 4 3 4 3 4 3 4 4 2 4 4
I: 1000
Movement duration (min.s)
Knitial motility (class)
Movement duration (min.s)
5.30 4.30 4.40 2.00 1.30 1.30 3.30 2.30 2.30 3.30 2.00 3.00 1.30 3.30 2.30
3 I I I 2 2 I 1 3 I I 3 I I 3
5.30 1.oo 1.oo 1.oo 0.30 1.30 1.30 1.30 2.00 1.oo 0.30 2.00 1.oo 1.30 2.30
M. SUQUET ET AL.
182
I’
0
0
I
I”
2 Duration
1
I”
4 of movement
0
I
r
n
n
1
8
6 (miilutes)
Fig. 5. Sperm motility in relation to time (three examples showing different decreasing schemes are presented). TABLE2 Mean values observed for the first sampling of sperm collected during the natural spawning season (data f 95%confidenqe interval) Volume (ml) Concentration ( x IO9spz/ml)
1.6kO.2 38.3 f 5.9
Motility (min.s) Spermatocrit (%)
6.06k 1.08 40.4 k5.8
first dilution ( 1: 1) did not modify the optical density. However, absorption measurement of samples frozen after the first dilution were unsuccessful since the solutions lost their homogeneity at thawing. Spermatocrit. No significant correlation was observed between sperm density and spermatocrit: r = 0.07 (Fig. 3). Estimation of viscosity.Samples with a viscous aspect had a significantly higher concentration (mean 54.6 x LO92 5.4 spermatozoa/ml) than liquid (mean 20.0 2 2.4) or undetermined ones (mean 2 1.1 II 4.4): PC 0.001 (Fig. 4). It
ASSESSMENT OF SPERMCONCENTRATIONAND MOTILITY IN TURBOT
183
was not possible to discriminate liquid and undetermined aspects in terms of concentration.
Estimation of motility Increasing dilution affected sperm motility (Table 1 ), but this difference was only significant between dilutions 1: 10 and 1: 1000 in terms of initial motility (P
Validation Mean values collected during the first sample of the natural spawning season are presented in Table 2. DISCUSSION
Evahation of spermatozoa concentration When assessing milt density by counting in a Malassez’s cell, four samples of at least 300 spermatozoa each are necessary to observe an acceptable coefficient of variation. However, it takes 2 h to evaluate the sperm concentration of only one fish. Thus, although accurate, this method cannot be used for practical estimation. Since spermatocrit is not correlated with spermatozoa concentration as in salmonids (Bouck and Jacobson, 1976) or pike (De Montalembert et al., 1%O), this parameter can not be used to evaluate bperm concentration in turbot. As in rainbow trout (Billard et al., 197 1 ), a spectrophotometric evaluation allows an accurate estimation of milt concentration. At 420 nm there is minimal interference between absorption of sperm and associated fluids. This method appears to be best adapted for experimentation since a IJV monitor is unusual in the field. According to results observed in carp (Clemens and Grant, 1965 ), samples with a viscous aspect present a significantly higher concentration of sperm than the liquid or undetermined ones. Though subjective, sperm aspects can easily be classified. This fast estimation should be useful in the field to deter s mine the volume of sperm necessary for fertilization.
Estimation of motiIi,ty In contrast to the results of Gray ( 1928), working on sea-urchin spermatozoa, a highL mIdilution enhanced neither the initiation of motility nor the duration of movement. After cryopreservation for 1 month, no motion was observed in spermatozoa of gilthead sea bream when the diluent/sperm ratio
184
M. SUQUET ET AL.
was high (Chambeyron and Zohar, 1990). Dilption could remove the protec-
tive role of seminal plasma, as suggested for mammals (Harrison et al., 1978 ) and fishes (Billard, 1982, 1983 ). For a 1: 10 dilution the motility decrease appears to be linear. The intercept is a constant value (motility class 4) and the slope is only a function of duration of movement. Hence, the time when al! movement ceased can be used as a practical motility criterion, so that intermediate measurements are not necessary. Validation Data collected for turbot during the first sample of the natural spawning season are higher than for salmonids (Billard et al., 197 1; Scott and Baynes, 1980) and equivalent to those observed in the sea bass (Billard et al., 1977; Zohar et al., 1984). As in these species, a high individual variability was observed. Sperm quality in turbot can be reliably characterised by the techniques described in this paper. Relation of these criteria to fertilization ability of spermatozoa will be studied in further research. ACKNOWLEDGEMENTS
We wish to thank Mr. Y. Harache for helpful comments on the manuscript.
REFERENCES aynes, S.M., Scott, A.P. and Dawson, A.P., 1981. Rainbow trout, Wmo gairdnerii Richardson, spermatozoa: effects of cations and pH on motility. J. Fish Biol., ‘r9: 259-267. Billard, R., 1982. Importance des protCines du liquide coelomique sur la fertilite des gam&es cke~ la truite arc en ciel et possibilitd de substitution. Bull. Fr. Pi&., 284: 168-l 77. Billard, R., 1983. Effects of coelomic and seminal fluids and various saline diluents on the fertilizing ability of spermatozoa in the rainbow trout, Safmo gairdneri. J. Reprod. Fertil., 68: 77-84. Billard, R., Breton, B. and Jalabert, B., 1971. La production spermato&nttique chez la truite. Ann. Biol. Anim. Biochim. Biophys., ll(2): 199-2 12. Billard, R., DuPont, J. and BarnabC,G., 1977. Diminution de la motilitb et de la duree de conservation du sperme de Dicentrarchus fa6ra.x L. (Poisson, TCltostten) pendant la pCriode de spermiation. Aquaculture, 11: 363-367. Bouck, G.R. and Jacobson, J., 1976. Estimation of salmonid sperm concentration by microhematocrit technique. Trans. Am. Fish. Sac.. 105(4): 534-535. Biiyiikhatipoglu, S. and Holtz, W., 1984. Sperm output in rainbow trout (Salmo gairdnerii) effect of age,timing and frequency of stripping and presence of females. Aquaculture, 37: 63-71.
ASSESSMENTOF SPERM CONCENTRATION AND MOTILITY IN TURBOT
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