ICES mar. Sei. Symp., 201: 170-172. 1995

Preliminary results of different methods of artificial fertilization with fresh sperm of turbot (Scophthalmus maximus L.)

O . Chereguini, B. Peleteiro, R. Cal, and A . Garci'a

Chereguini, O ., Peleteiro, B., Cal, R., and Garcia, A. 1995. Preliminary results of different methods of artificial fertilization with fresh sperm of turbot (Scophthalmus maximus L.). - ICES mar. Sei. Symp., 201: 170-172.

Turbot (Scophthalmus maximus L.) is a species that does not readily achieve spon­taneous spawning in captivity, and as a result the extraction of gonadal products by stripping and artificial fertilization of the eggs has to be carried out. With the aim of improving sperm activation techniques in artificial fertilization, a series of fertilization experiments was carried out in dry conditions and with different diluents (35 ppt sea water, 20 ppt sea water, and Ringer saline solution). A sperm pool of 5 cc from four males and 160cc of eggs from one female from the same broodstock was used. The ratio of sperm was 0.02 cc sperm/1 cc eggs. The sperm pool was previously evaluated for its density (4.2 x 109 sperm ml“ 1) and motility (4) according to the scale of Sânchez-Rodriguez (1975). Significantly higher fertilization rates were found using 35 ppt sea water, but the hatching percentages did not vary significantly.

O. Chereguini: Istituto Espanol de Oceanografia, Apto. 240, 39080 Santander, Spain [tel: (+34) 42 321513, fax: (+34) 42 322620], B. Peleteiro and R. Cal: Istituto Espanol de Oceanografia, Apto. 1552, 36280 Vigo, Spain. A . Garcia: Istituto Espanol de Oceanografia, Apto. 22, 3070040 Murcia, Spain.

Introduction

Artificial fertilization has long been used in species such as salmonids and cyprinids, and more recently this tech­

nique has been applied in o ther species: sea bream (Alessio and Bronzi, 1975), Epinephalus (C hen et al.,

1977), and Indo-Pacific mackerel (B oonprakov and D h e b ta ra n o n , 1974).

In salmonids this technique seems to have been used in the Middle Ages; widely known as the dry m ethod, it was a quantity of eggs mixed with sperm and water added later to activate motility of the spermatozoa. Nevertheless, the technique did not yield good results owing to the excess of spermatozoa (about 107 sperm a­tozoa per egg). Thus, Nursall and H asler (1952) sug­gested reducing the quantity of sperm, since one male

can fertilize the eggs o f between 20 and 100 females

(Morley and Withler, 1969).H owever, it was N om ura (1964) who proposed the

technique of the diluent, la ter perfected by Billard et al. (1974) and Billard (1975), and in which the optimum

proportions of sperm, eggs, and diluent can be found.

Billard (1978) later obtained good results in artificially

fertilizing marine fish (sea bream and sea bass) using 20 ppt sea w ater at pH 8 -9 as a diluent, though even

be tter results were obta ined with D CBS4 diluent spe­

cially prepared for cryopreservation (Billard, 1984).T urbot (Scophthalm us m axim us) is a species which

does not readily spawn spontaneously in captivity. It is

therefore necessary to remove the gonadal products by stripping, and to obtain fertilized eggs by artificial ferti­lization.

The main aim of this experiment was to determine which m ethod of fertilization was the most effective for turbot: dry fertilization, wet fertilization with different

diluents, such as sea w ater at 35 pp t, sea w ater a t 20 ppt o r modified R inger’s saline solution.

Material and m ethods

A series of experiments was carried out in dry conditions

(“ FS” ) and with different diluents: 35 pp t sea water

(“ F H ” ), 20 ppt sea w ater (“ F V ” ), and modified R inger’s saline solution (“ F R ” ), which is a diluent used in cryo­

preservation. The composition of this solution was

4 0 .23mM KC1, 111.22mM NaCl, 2 .70m M CaCl2,

2.38 mM N aH C O v Four replicate experiments were

carried out for each treatm ent.Female no. 67 was used (5.660 kg, 57 cm) from the

ICES mar. Sei. Symp.. 201 (1995)

% Fertilization

Artificial fertilization o f turbot 171

Types of artificial fertilization

% Hatching100

FS FH FV FR FH FV FR

Types of artificial fertilization

Figure 1. Mean percentages and standard error of fertilization and hatching with artificial fertilization : dry (FS) and wet 35 ppt sea water (FH), 2 0 ppt sea water (FV), and modified Ringers saline solution (FR).

Table 1. One-way analysis of variance and multiple range analysis results for fertilization rates by fertilization type (arc-sine transformed data). Confidence level: 95.

Source of variation Sum of squares d.f. Mean square F-ratio Sig. level

Analysis of varianceBetween groups 926.85602 3 308.95201 22.744 0.0000Within groups 163.00688 12 13.58391Total (corrected) 1089.8629 15

Level Count Confidence average Homogeneous groups

Multiple range analysis FS 4 46.945000 *

FV 4 47.817500 *

FR 4 48.665000 *

FH 4 65.330000 *

Table 2. One-way analysis of variance and multiple range analysis results for hatching rates by fertilization type (arc-sine transformed data). Confidence level: 95.

Source of variation Sum of squares d.f. Mean square F-ratio Sig. level

Analysis of varianceBetween groups 661.7713 3 220.59044 1.379 0.2966

Within groups 1920.0534 12 160.00445Total (corrected) 2581.8247 15

Level Count Confidence average Homogeneous groups

Multiple range analysis FS 4 32.245000 *

FV 4 41.847500 *

FR 4 45.175000 *

FH 4 49.775000 *

172 O. Chereguini et al. ICES mar. Sei. Symp., 201 (1995)

broodstock submitted to a controlled photoperiod and tem pera tu re o f 14 ± 1°C along with males no. 45 (1.780 kg, 44cm ), no. 38 (1.750 kg, 46 cm), no. 35 (2 .130kg, 47cm ), and no. 50 (1.970 kg, 45 cm) from the sam e stock.

A fte r checking egg quality (85% viable eggs), 160ml of oocytes was obta ined from the female by stripping; 10 cc of eggs was distributed am ong 16 2-1 beakers

( —9000 oocytes per beaker).A check of the sperm pool was made on a small sample

using the T hom a cham ber to obta in the density and

motility after activation with sea w ater, according to the m ethod of Sânchez-Rodriguez (1975).

The sperm for the experim ent was obta ined by mixing the sperm extracted from the four males, providing a sperm pool of 5 ml, and a ratio o f 0.02 ml o f sperm m l-1 o f eggs was used.

A t the same time as the sperm was distributed, 100 ml of the diluent was added , except in the case of dry fertilization, in which 100 ml of sea w ater was added after 30m in; 45m in after fertilization the beakers were

filled to 21 with sea w ater at 35 ppt and 14.3°C, and put into a floating ring in a 5001 incubator at 14 ± 1°C.

Fertilization rates were determ ined from the buoyant eggs after 3 h, and the eggs which were not viable were syphoned from the bo ttom of the beakers. The eggs

which w ere not buoyant were taken out daily; 75% of

the w ater was renew ed on the first 2 days of incubation, and 50% on the following 2 days. The tem pera tu re was kept constant.

T he larvae hatched after 5 days of incubation, and the hatching percentages were then determ ined for each experim ent. The hatching rate was determ ined from the num ber o f fertilized eggs, not the initial num ber o f eggs.

Results and discussion

T he sperm pool showed a density of 4.2 x 109 sperm ato ­zoa m f 1 and motility was estim ated at 4, i.e . , between 70 and 75% of the sperm atozoa m ade rapid and p ro ­gressive movem ents. These values of density and moti­

lity concur with those obta ined by Fauvel et al. (1992) and Suquet et al. (1992).

T he average percentages of fertilization and hatching

are shown in Figure 1. A variance analysis on arc-sine transform ed data showed significantly higher fertiliz­

ation rates (p < 0.05) when 35 ppt sea w ater was used as

a d iluent com pared to the o the r trea tm ents (Table 1).

T here were no significant differences, however, be ­tween hatching rates for the four trea tm ents (Table 2).

While B oonprakob and D heb taranon (1974) achieved the same success rate with wet and dry fertilization of

Indo-Pacific mackerel as did Alessio and Bronzi (1975) of sea bream , our results of artificial fertilization in tu rbo t are different.

T he results shown are the latest of a series of experi­m ents carried out over a period o f one year. A m ong the

techniques used in artificial fertilization to improve sperm activation, wet fertilization with sea w ater at

35 ppt gave the best fertilization rate , indicating tha t it is

the most suitable diluent for artificial fertilization of turbot.

R eferences

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