Otolith staining by oral administration of alizarin complexone with enteric-coated microcapsules for juvenile Japanese flounder Paralichthys olivaceus

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1 2007 * ** Otolith staining by oral administration of alizarin complexone with enteric-coated microcapsules for juvenile Japanese flounder Paralichthys olivaceus Tatsuya ISSHIKI and Satoshi KATAYAMA Otolith staining by oral administration of microcapsulated alizarin complexone MALC was examined as a fluorescent dye for juvenile Japanese flounder Paralichthys olivaceus to develop a new mass-marking method Three diet regimes MALC mixed with compound feed MALC diet ALC solution directly mixed with the compound feed DALC diet and ALC free control diet were employed for rearing experiments Ten juveniles TL -mm were used for each regime and reared for ten days Fluorescent marks of ALC were observed at the rate of % % of individuals reared with the MALC diet obviously lower than %- % of reared with DALC diet Beside that fish feeding MALC diet showed less growth rate than those feeding DALC diet suggesting that more improvements are needed in the MALC production for stable otolith staining % CHNO ALC ALC ALC ALC ALC ALC ALC ALC ALC * ** - --

2 ALC ALC ALC ALC ph mmmm ph HORIBA -C ph mg/ml mg/ml mg/mlalc ALCmg/mlml ml mlml mg/ml mg/ml Fig ALC ALC Acetone 20ml Cellulose Acetate Phthalate 2.0g Diethyl Phthalate 0.6g Mixing Mixing Mineral oil 100ml Ethanol 1ml Sucrose Esters of Fatty Acids 0.1g Quickly mixing without containing air for 2 min. ALC solution 10ml 95% Ethanol 2.0ml Mixing Dropping and mixing Mixing for 5 min. Chloroform 90ml Dropping and mixing Decantation Chloroform 90ml Dropping and mixing Decantation Drying of the precipitate Enteric-Coated Microcapsule contained ALC solution(malc) Preparation process of microcapsulesated ALC enteric capsule

3 ALC MALC MALC ALC DALC ALC L mmmm g kg ALC mg MALC DALC ALC % g g MALC Fig mg/ml ALCmlMALC g ALC ALCmg / MALC MALCg ALC mg gmalc g MALC g MALCml DALC mg/ml ALC ml g MALC ml ml ALC EP mm ALC G ALC ALC L ALCppm EP mm GALC ph Table ph ph value in stomach with contents and vacuous stomach of Japanese Flounder Paralichthys olivaceus juveniles Stomach with contents Vacuous stomach Sample size 8 25 Mean TL (mm) Mean BW(g) Mean ph ph range

4 ALC ph value in intestine with contents and vacuous intestine of Japanese Flounder Paralichthys olivaceus Intestine with contents Vacuous intestine Sample size 8 19 Mean TL (mm) Mean BW(g) Mean ph ph range Fp ph F p ph F p mg/ml mg/ml mg/ml ALC Fig MALC Table DALC MALC One-way ANOVA p One-way ANOVA p Tukey HSD DALC MALC MALC MALC DALC Fig Table DALC MALC ALC ALC ALC ALC DALC MALC DALC Growth for days of Japanese Flounder Paralichthys olivaceus juveniles reared in diets regimes ALC diets Initial TL range(mm) Final TL range(mm) Mean growth rate ANOVA Ⅰ (54.0) (55.7) 0.03 MALC diet Ⅱ (51.9) (53.2) 0.02 Ⅲ (52.2) (53.5) 0.02 Mean 0.03 Ⅰ (52.5) (55.5) 0.06 DALC diet Ⅱ (55.4) (58.6) 0.05 F= P<0.05 Ⅲ (53.1) (57.2) 0.07 Mean 0.06 Ⅰ (52.4) (55.5) 0.06 Control Ⅱ (53.9) (56.1) 0.04 Ⅲ (55.5) (58.2) 0.05 Mean 0.05 MALC diet: Microcapsulated ALC mixed with compound feed DALC diet: ALC solution directly mixed with the compound feed Control: ALC free compound feed

5 ALC Microcapsulesated ALC enteric capsule mg/l A MALC DALC DALC mmg %kg mg/kgdalc kg g ALC B Whole otolith of Japanese flounder fluorescent Paralichthys olivaceus observed under UV light Otolith from fish reared with MALC diet A and DALC diet B Arrows indicate ALC marks Scores of ALC mark radiation under UV light in otolith of Japanese flounder Paralichthys olivaceus reared each regime ALC mark radiation Marking rate ("+++"and "++") Ⅰ MALC diets Ⅱ Ⅲ Ⅰ DALC diets Ⅱ Ⅲ : strong radiation as marking; ++: good radiation as marking; +: feeble radiation; : no marking section

6 ALC ALC DALC ALC ALC ALC MALC MALC DALC ALC MALC DALC ALC MALC ALC / ALC ALCMALC phmalc phmalc ph ph% ph % ph ph ALC % ALC ph ph ph ALC ALC ALC ALC MALC DALC ph ph - mg/ml mg/ml mg/ml ALC MALC DALC MALCDALC ALC

7 ALC DKF- Tsukamoto KMass-marking of ayu eggs and larvae by tetracycline tagging of otoliths Bull Japan Sci Fish Takifugu raubripes ph