lindrical fibreglass tanks at a density of 80 larvae.l-1. In the first trial ... On the first experiment, feeding protocol for balanced and unbalanced diet treatments was ... tilage was stained with Alcian blue (40 minutes) and ossified bone was stained.
LARVI ’09 – FISH & SHELLFISH LARVICULTURE SYMPOSIUM C.I. Hendry, G. Van Stappen, M. Wille and P. Sorgeloos (Eds) European Aquaculture Society, Special Publication No. 38, Oostende, Belgium, 2009
AMINO ACIDS HAVE IMPORTANT ROLES IN LARVAL DEVELOPMENT OTHER THAN GROWTH M. Saavedra1, P. Pousão-Ferreira1, Y. Barr2, S. Helland2, M. Yúfera3, M.T. Dinis4, and L.E.C. Conceição4 1 2 3 4
Instituto Nacional de Investigação Agrária e das Pescas (INIAP/IPIMAR-CRIPSul), Av. 5 de Outubro, 8700-305 Olhão, Portugal Nofima Marin, Sjolseng, N-6600 Sunndalsora, Norway Instituto de Ciencias Marinas Andalucia (CSIC), Apartado Oficial 1510, Puero Real, Spain CCMAR, Universidade do Algarve, Campus Gambelas, 8005-139, Faro, Portugal
Introduction Amino acids are the most important energetic substrate during fish larval stages and therefore there is a high amino acid (AA) requirement (Rønnestad et al., 1999). AA composition in larval diets affects growth (Fauconneau et al., 1992) as well larval quality and performance (Aragão et al., 2007). Tyrosine is a semi-indispensable AA, precursor of dopamines and the adrenocortical hormones norepinephrine and adrenaline. Dopamines regulate central and peripheric nervous system activity and can therefore be related to the control of stress in the fish (Lehnert and Wurtman, 1993). Material and methods Two different trials using Diplodus sargus larvae were run to test the effect of an AA balanced diet and the effect of an AA balanced diet supplemented with tyrosine. Each experiment lasted 25 days and larvae were held in 200-l conical cylindrical fibreglass tanks at a density of 80 larvae.l-1. In the first trial three dietary treatments were tested: a control diet consisting on live feed, a diet with an AA profile similar to the AA profile of the larval carcass (balanced diet) and an AA unbalanced diet. On the second trial the balanced diet was used as a control and a balanced diet supplemented with tyrosine was tested. On the first experiment the AA balanced diet was given in the form of a casein microencapsulated diet whereas on the second experiment, besides the microencapsulated diet, rotifers were given liposomes incorporating free AA (FAA) in order to balance their AA profiles.
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On the first experiment, feeding protocol for balanced and unbalanced diet treatments was the same as the control until the 11DAH (5 rotifers.ml-1). From 12 to 14DAH the live food was decreased to half of the control and afterwards Brachionus plicatilis and Artemia were only given at 10% of the control. The microencapsulated diet was introduced at 8DAH (from 0.5g to 1.5g at the end of the experiment). On the second experiment rotifers were boosted with liposomes filled with FAA one hour before given to the larvae (5 rotifers.ml-1 until 14DAH and 2.5 rotifers.ml-1 from 15DAH). The microencapsulated diet was introduced at day 15 (1.5g to 2g). Liposomes were filled with free amino acids according to Barr and Helland (2007), using the free amino acid quantities detailed in Table I. Table I. Amino acid quantities added to the liposomes used to enriched rotifers from the control and tyrosine treatment (Tyr) for one meal. Data are expressed as g of AA per million of enriched rotifers. His Lys Arg Thr Met Phe Tyr Control 0.016 0.061 0.033 0.017 0.008 0.017 0.001 Tyr 0.047 0.183 0.100 0.050 0.023 0.052 0.003
On the first experiment ammonia excretion trials were done in fed and fasted larvae using 10 fish larvae per tank enclosed in 45 ml spherical glass vials for two hours. Five replicates from each treatment were used. Ammonia concentration was determined according to Berthelot (Grasshoff, 1983). Deformities at the vertebral column were also analysed in 90 larvae per treatment, at 25DAH. Cartilage was stained with Alcian blue (40 minutes) and ossified bone was stained with Alizarin Red (2 hours), according to Gavaia et al. (2000). Vertebral column was divided into three regions: trunk (1 to 11 vertebrae), caudal (12 to 20), and preurostyle (remaining three and urostyle). On the second experiment, a temperature stress test consisting on a sudden drop of temperature (21ºC to 10ºC for five minutes). Stress resistance was measured in terms of survival ten minutes after the temperature was risen. Ten larvae were used from each tank and trials were repeated three times for each tank (nine replicates per treatment). Results and discussion On the first experiment, significant differences in ammonia excretion were found between control and balanced diet treatments in fed larvae. Balanced diet seemed to present almost no difference between fed and fasted larvae and registered the lowest ammonia excretion of all treatments. On the same experiment, the frequency of deformed larvae at 25DAH was approximately 40% in the control group, 30% in the unbalanced diet group and 20% on the balanced diet group (Fig. 1). A significant high number of vertebrae fusions were found in the control treatment. Lordosis was found in control and unbalanced diet groups but not in the balanced diet group.
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Fig. 1. Deformities observed at the dorsal column in Diplodus sargus fed on control, a balanced and unbalanced diets (n=60 larvae per treatment). AV- Abnormal shape vertebra, HSS- Supranumeric haemal process, VC- Vertebral compression, VFVertebral fusion. Different letters represent significant differences for p < 0.05.
On the second experiment the stress test showed significant differences between treatments (Fig. 2).
Fig. 2. Diplodus sargus larval survival rate after being subjected to a temperature stress test. Values are mean and standard deviations (n=9). Different letters represent significant differences.
When submitted to a sudden drop of temperature at 25DAH, larvae fed a diet supplemented with tyrosine had significantly higher survival rate when compared with the other treatments. There is lack of information on this matter related to fish but laboratory studies strongly suggest that tyrosine supplementation may serve to reduce cognitive and behavioural effects of exposure to stress (Deijen et al., 1999).
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Conclusions An AA-balanced diet seems to reduce nitrogen excretion in fed larvae and decrease the incidence of skeletal deformities at the vertebral column. Using the same AA-balanced diet with a supplement of tyrosine there seems to be a higher survival rate when larvae are submitted to a stress test such as a sudden drop of temperature. The present study confirms the idea that there are AA requirements for metabolic processes other than growth. References Aragão C., L.E.C. Conceição, M. Lacuisse, M. Yúfera, and M.T. Dinis. 2007. Do dietary amino acid profiles affect performance of larval gilthead seabream? Aquatic Living Resources 20: 155-161. Barr Y. and S. Helland. 2007. A simple method for mass production of liposome, in particular large liposomes, suitable for delivery of free amino acids to filter feeding zooplankton. Journal of liposome research 17: 79-88. Deijen J.B., C.J.E. Wientjes, H.F.M. Vullinghs, P.A. Cloin, and J.J. Langefeld. 1999. Tyrosine improves cognitive performance and reduces blood pressure in cadets after one week of combat training course. Brain Res. Bull. 48: 203209. Fauconneau B., A. Basseres, and S.J. Kaushik. 1992. Oxidation of phenylalanine and threonine in response to dietary arginine supply in rainbow trout (Salmo gairdneri R.) Comp. Biochem. Physiol. Vol. 101A, Nº 2: 395-401. Gavaia P.J., C. Sarasquete, and M.L. Cancela. 2000. Detection of mineralized structures in very early stages of development of marine Teleostei using a modified Alacian blue-Alizarin red double staining technique for bone and cartilage. Biotech Histochem 75: 79-84. Grasshoff K. 1983. Methods of seawater analysis-Verlag chemie-NY, pp 317. Lehnert, H. and Wurtman, R.J. 1993. Amino acid control of neurotransmitter synthesis and release: Physiological and clinical implications. Psychother. Psychosom. 60: 18-32. Rønnestad I., A. Thorsen, and R.N. Finn. 1999. Fish larval nutrition: a review of recent advances in the roles of amino acids. Aquaculture 177: 201-216.
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