Evaluation of marking efficiency of different alizarin red S concentrations on body fish structures in Oreochromis niloticus (Perciformes: Cichlidae) juveniles
Ana L. Ibáñez1*, Antonio Rodríguez-Canto1, Jasmín Cortés-Martínez1 & José L. García-Calderón1
The use of alizarin red S (ARS) marked tilapias could provide valuable fisheries management information to evaluate fish stocking events and may facilitate aquaculture management practices. As a new technique in fishes, the aim of this study was to compare and evaluate the chemical marks produced in tilapia juveniles by ARS through two treatments: 1) 12 hours of immersion and 2) immersion after osmotic induction. This was analyzed at three concentrations: 50, 75 and 100mg/l, and in three structures: otoliths, fish scales and caudal fin rays of Oreochromis niloticus juveniles. After three culture months 80% of specimens were analyzed and significant differences (p<0.05) in mark intensity were detected between treatments for otoliths and fin rays, but not for fish scales. Significant differences between concentrations were found for the 12h immersion treatment, while no significant differences were detected with osmotic induction. Our results showed that marks appeared at all concentrations, and none of the concentrations produced weak marks. Osmotic induction had a greater mortality than the 12h immersion procedure. After eight culture months the rest of the specimens were analyzed and the mark permanence was observed in all cases. According to the present results we recommend the marking process of 12h immersion treatment at 100mg/L concentration.
Key words: dye, tilapia, fish stocking, chemical mark, stock enhancement, alizarin red.
Resumen
El uso de alizarina roja S (ARS) para marcar tilapias podría proporcionar información valiosa para el manejo de su pesquería. Para evaluar pesquerías acuaculturales manejadas con siembras o repoblamientos de peces se comparó y evaluó la marca producida por la alizarina roja S, empleando dos tratamientos: 1) Inmersión en ARS durante 12h; e 2) Inmersión en ARS después de un choque osmótico. El análisis se realizó a tres concentraciones: 50, 75 y 100mg/l y en tres estructuras: otolitos, escamas y radios de la aleta caudal de Oreochromis niloticus. Ochenta por ciento de los ejemplares fueron cultivados durante tres meses y analizados posteriormente. Los resultados mostraron diferencias entre las concentraciones de la marca para el tratamiento de 12h de inmersión mientras que no hubo diferencias entre las concentraciones para el tratamiento con inducción osmótica. Se encontraron diferencias en la intensidad de la marca entre los tratamientos para otolitos y radios de las aletas pero para las escamas no hubo diferencias significativas. Todas las concentraciones produjeron marcas (desde débiles a intensas), sin embargo la concentración de 100mg/l no produjo marcas débiles. El tratamiento por inducción osmótica presentó mayores niveles de mortalidad. Después de ocho meses de cultivo el resto de los ejemplares fueron analizados y se observó la permanencia de las marcas en todos los casos. En vista de lo anterior, para los propósitos de marcaje se recomienda el uso del tratamiento de inmersión por 12h y una concentración de 100mg/l.
Palabras clave: pigmento, tilapia, siembra de peces, marcaje químico, mejoramiento pesquero. According to FAO (2009), worldwide percapita aquaculture fish consumption has rapidly increased between ]]>
Different chemicals have been successfully used for fish and specifically, several for cichlids. Klesius et al. (2006), used calceine to discriminate between a vaccinated population of tilapia (Oreochromis ]]>
) and other not treated populations. They found a detectable fluorescent band following a four- hour-bath in 500mg/l. Egger (2004) used tetracycline to validate the pattern of increment in the otoliths of Tropheus moorii Boulenger (1898), a native cichlid of Lake Tanganyika, where marking involved individually injecting each fish with 10mm (4.4mg/l) of tetracycline solution. Likewise, Barker & McKaye (2004), marked juveniles of the cichlid Amphilophus, with OTC ]]>
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Immersion in a dye, combined with osmotic induction, is a technique consisting of a brief salt bath prior to immersion in a fluorescent dye. This procedure dramatically reduces color absorption time, in comparison to conventional methods of marking. Simplicity and speed of osmotic induction make this method especially suitable for marking fish on a large scale where cost and logistics largely determine the utility of marking methods (Mohler 2003). Crook et al. (2007 & 2009) also evaluated osmotic methods ]]>
Macquaria ambigua using ARS. They found that the osmotic induction-marked fish possessed more intense marks and that these marks remained visible nine months after treatment. Mark production using ARS it is consider as nontoxic for several species (Lucas et al. 2008, Simon et al. 2009) and human consumption; also is acceptable to the public for commercial use (Rothlisberg & Preston 1992). ]]>
In Mexico, the federal government supports 40 fish farms that produce, on average, 111 million fry per year, 48% of which are tilapia. Although culture-based fisheries in Mexico are almost one century old, there is very little information available as to their influence on fish production (Ibáñez 2004, Ibáñez et al. 2011). The release of ARS-marked tilapias could provide ]]>
The aim of this study was to compare and evaluate the chemical mark produced by ARS through two treatments: 1) 12h of immersion and 2) immersion after osmotic induction. This was analyzed using three different concentrations: 50, ]]>
Material and Methods
Fish transportation ]]>
Juveniles of the Stirling strain of Oreochromis niloticus L. were obtained from a fish farm at Zacatepec, in the State of Morelos, Central South Mexico. They were transported to the Fisheries laboratory at the Metropolitan University in Mexico City, in plastic bags filled with fresh water and oxygen. Fish were acclimated for seven weeks in an aerated 200-l tank with a Fluval filter model 305 (volume of 710 l/h) and fed to satiation twice a day each day with ]]>
Experimental design: ]]>
et al. (2007) protocol; for this, fish were immersed in a 5% sea-salt solution for five minutes, followed by a five seconds rinse in aquarium water, and then, a 10 minute immersion in the ARS solution of 10-l with the respective 50, 75 and 100mg/l concentration. ]]>
After the marking ]]>
Structure preparation and examination: Scales were cleaned and placed between two glass slides ]]>
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To assign a mark intensity value based on an ordinal scoring system, the following five classifications were established: no mark, weak mark, moderate mark, intense mark and very bright mark (modified from Crook et al. 2007). In order to determine significant differences between the ARS concentrations used, Kruskal-Wallis tests were used; furthermore, a Mann-Whitney U-test was used to determine ]]>
Fish were treated in compliance with the regulations and protocols of our University (Lineamientos para la conducción ética de la investigación, la ]]>
Results
The results shown ]]>
Mortality: Mortalities reached 12.24% in the 12h ARS immersion treatment (Treatment 1), and 22.4% in the osmotic induction- ARS immersion treatment (Treatment 2). ]]>
General mark results for both treatments: Moderate and intense marks were the most frequent results in both treatments (38.8 and 36.1%, respectively) for the whole period. ]]>
Figs. 1, 2). Mark intensity was not significantly different between duplicates for the two treatments (Treatment 1: p=0.509; Treatment 2: p=0.964). There were significant differences between treatments for caudal fin (p=0.001) and otoliths (p=0.019), but not for scales (p=0.302). Significant ]]>
Treatment 1: Mark intensity in the 12h immersion treatment of otoliths showed wide variability, while most scales had moderate intensity marks (74.1%). Likewise, most caudal fin marks were intense (63.0%). It is important to mention that the 20% frequency in the no mark results showed in
figure 1a, applies to control specimens. In the 50mg/L batch, 62.5% of the marks were moderate while very bright marks were not observed. Most of the 75mg/l concentration batch showed either intense (57.7%) or moderate marks (30.8%); while intense (42.1%) and very bright marks (31.6%) increased in the 100mg/l group. At this higher concentration, no weak marks were formed (Fig. 1b). This treatment showed differences between 50 and 75mg/l and also between 50 and 100mg/l for otoliths (p=0.003 and p=0.001, respectively). Also, differences in ARS ]]>
Treatment 2: The osmotic induction treatment did not produce very bright marks in any case (Figs. 2, 3); ]]>
Fig. 2a). At 50 and 75mg/l most marks were moderate with 83.3% and 73.3%, respectively. On the other hand, at 100mg/l, 83.3% of the marks were intense. ]]>
Environmental parameters: During the eight-month culture period, water temperature ranged from 15.9 to 27.0°C (21.0±2.3), pH from 8.9-9.3 (9.3±0.3), oxygen ranged from 4.7 to 6.4mg/l (5.2±1.0), salinity from 5.0 to 8.2 PSU (6.5±1.0) and total dissolved solids from 3.4 to 8.6g/l (7.4±1.3). Temperature and oxygen, in particular, fluctuated widely during the day. Most of ]]>
Mark permanence: As ]]>
In summary, mark intensity did not differ between replicates. There were differences between treatments in the results obtained for caudal fins and otoliths, but not for scales. ARS concentrations only produced different intensities in treatment 1. Osmotic induction treatment did not result in very bright marks. Different ARS concentrations produced marks going from weak to intense with the exception of the 100mg/l, which did not produce weak marks.
Discussion
In this study, mortality resulted greater than in other similar studies (Leips et al. 2001, Bashey 2004, ]]>
et al. 2007, Crook et al. 2009). Size of fish could be an explanation of these results since, in those studies, fish were smaller and we observed that small fish were less vulnerable to the osmotic immersion shock. As a consequence of exposing the tilapia to high salinity water, the fish cells loose a certain amount of water. When the tilapias return to the ARS solution, the consequential osmotic differences resulted in the rapid uptake of dye, as water was replaced ]]>
et al. 2007). Nevertheless, osmotic induction marking with ARS produced more mortality than the 12-hour immersion treatment; we do not discard that lower salt immersion times might produce less mortality. According to Crook et al. (2007) increasing the immersion time beyond 3.5min may not necessarily improve ]]>
This experiment has demonstrated ]]>
et al. (2009) the growth and mortality of European glass eel Anguilla anguilla 192 days after marking with oxytetracycline and alizarin red S were not significantly different between the two treatments and not different to the unmarked A. anguilla. The study provided evidence that both marking methods ]]>
A. anguilla stage. Therefore, it must be studied the species specific effects of the marking techniques on growth and mortality rates.
The affinity of the ARS to calcium ]]>
Bashey ]]>
et al. (2009) also reported the presence of weak marks in non-exposed controls using osmotic immersion treatments. Even so, auto-fluorescence does not present a problem because it is easily distinguished from chemical marks. Natural fluorescence is due in large part to substances like flavins, porphyrins, lipofuscin and (in plants) chlorophyll. Lipofuscin is the breakdown product of old red blood cells with fluorescent red under green excitation. Elastin and collagen also could produce autofluorescence. Elastin contains several fluorophores, one of which ]]>
et al. 1980). This is a similar fluorophore to the one found in collagen. The elastine in presence of a saline solution, even for 10min followed by a 5min wash, shifts the usual green autofluorescence emission of elastin into a red emission (Neumann & Detlef 2002). The mentioned substances could have produced the autofluorescence in the control specimens of shock osmotic treatment; nevertheless, to be certain it will be necessary to design a particular study to explore this aim.
Mark permanence is decisive in dye election. In this study ARS showed eight months permanence. According to Simon et al. (2009) fish marks with ARS and oxytetracycline were clearly visible after a period of two years in specimens of Anguilla anguilla. The visibility of the marks eight months after marking ]]>
Macquaria ambigua and Thymallus thymallus has been retained from months to years (Beckman & Schulz 1996, Crook et al. 2007).
The concentrations ]]>
The present results ]]>
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In conclusion, both treatments and all concentrations produced useful marks, but the 100mg/treatment did not produce weak marks. Control in the 12-hour immersion did not mark while auto-fluorescence appeared in the osmotic treatment control. The osmotic treatment resulted in greater mortality rates than the 12h immersion; thus, a concentration of 100mg/l combined with a 12h immersion in ARS can be recommended for hatchery produced O. niloticus in this size range. ]]>
Acknowledgments
We thank the valuable help in the English adaptation to Anselmo Galindo Molina and to three anonymous referees who really improved the first version of the manuscript. Financial support was obtained from the Universidad Autónoma ]]>
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*Correspondencia: Ana L. Ibáñez: Universidad Autónoma Metropolitana-Iztapalapa, Departamento de Hidrobiología, Av. San Rafael Atlixco 186, Col. Vicentina. México, D.F. 09340, México; ana@xanum.uam.mx Antonio Rodríguez-Canto: Universidad Autónoma Metropolitana-Iztapalapa, Departamento de Hidrobiología, Av. San Rafael Atlixco 186, Col. Vicentina. México, D.F. 09340, México; roca@xanum.uam.mx ]]>
Jasmín Cortés-Martínez: Universidad Autónoma Metropolitana-Iztapalapa, Departamento de Hidrobiología, Av. San Rafael Atlixco 186, Col. Vicentina. México, D.F. 09340, México; hada_30@hotmail.com José L. García-Calderón: Universidad Autónoma Metropolitana-Iztapalapa, Departamento de Hidrobiología, Av. San Rafael Atlixco 186, Col. Vicentina. México, D.F. 09340, México; jlgc@xanum.uam.mx 1. Universidad Autónoma Metropolitana-Iztapalapa, Departamento de Hidrobiología, Av. San Rafael Atlixco 186, Col. Vicentina. México, D.F. 09340, México; ana@xanum.uam.mx, roca@xanum.uam.mx, hada_30@hotmail.com, jlgc@xanum.uam.mx
Received 13-II-2012. Corrected 09-VIII-2012. Accepted 13-IX-2012.