Perfil de ácidos grasos de organismos juveniles de abulón azul (Haliotis fulgens) sometidos a restricción de lípidos e inanición

Eduardo Durazo^1*, María Teresa Viana²

¹ Facultad de Ciencias Marinas, Universidad Autónoma de Baja California, AP 76, CP 22800, Ensenada, Baja California, México. * Corresponding author: E-mail: edurazo@uabc.edu.mx.

² Instituto de Investigaciones Oceanológicas, Universidad Autónoma de Baja California, AP 453, CP 22800, Ensenada, Baja California, México.

Received February 2013, ]]> received in revised form July 2013,
accepted October 2013.

ABSTRACT

The fatty acid composition of juvenile green abalone (Haliotis fulgens) exposed to lipid restriction and long-term starvation was studied. Juvenile organisms were acclimated during 26 days and then randomly separated into three treatment groups. One group was fed a restricted diet containing low lipid content (0.14%), another was fed a rich diet containing the optimal lipid content (5.1%), and the third was kept under starvation conditions. After 90 days, the abalone fed the restricted diet showed a significant increase in 18:1n-9 content and a decrease in n-3 and n-6 polyunsaturated fatty acids (PUFA), but the total lipid level remained similar to that observed before the treatment, suggesting lipogenesis. On the other hand, no changes in total lipid content and fatty acid profile were found during the 90-day treatment using the rich lipid diet compared with the initial samples. Starved abalone showed that lipids did not constitute the main energy source and that the concentrations of long-chain PUFA did not change throughout the 90-day starvation period.

Key words: green abalone, formulated feed, dietary lipid level, starvation, fatty acid composition.

RESUMEN

Se estudió la composición de ácidos grasos de juveniles de abulón azul (Haliotis fulgens) sometidos a restricción de lípidos y a un periodo largo de inanición. Los organismos juveniles se aclimataron durante 26 días y posteriormente se separaron al azar en tres grupos de tratamiento. Un grupo se alimentó con una dieta baja en lípidos (0.14%), otro se alimentó con una dieta rica en lípidos (5.1%) y el tercer grupo se mantuvo en condiciones de inanición. Después de 90 días los abulones alimentados con la dieta baja en lípidos mostraron un incremento del contenido de 18:1n-9 y una disminución de los ácidos grasos poliinsaturados (PUFA) n-3 y n-6, pero el contenido de lípidos totales permaneció similar al observado antes del tratamiento, sugiriendo lipogénesis. Por otra parte, después de 90 días no se encontraron cambios en cuanto al contenido de lípidos totales y el perfil de ácidos grasos en el tratamiento con la dieta rica en lípidos en comparación con las muestras iniciales. Los abulones en inanición mostraron que los lípidos no constituyen la fuente principal de energía y que las concentraciones de PUFA de cadena larga no cambian a lo largo de un periodo de inanición de 90 días.

Palabras clave: abulón azul, alimento formulado, nivel de lípidos en la dieta, inanición, composición de ácidos grasos.

INTRODUCTION

Abalone species are distributed worldwide and they are found in temperate as well as in tropical waters. Due to overexploitation the natural populations of some commercial species, with high market value, are declining dramatically (Guest et al. 2008). Considerable research has thus been conducted on their culture and nutrition over the past 30 years with success.

The green abalone (Haliotis fulgens) is a herbivorous gastropod with high cultivation potential. The distribution of this species includes southern California and the west coast of the Baja California Peninsula, Mexico. There is growing interest in farming and marketing this highly prized product, especially in the USA and Chile. In addition, the unusual energy and lipid metabolism of H. fulgens has led to several studies (Floreto et al. 1996, Durazo-Beltrán et al. 2004, Viana et al. 2007, Hernández et al. 2013). Dietary lipids, in addition to being an energy source, are the source of essential fatty acids that are not synthesized by the organism and that are necessary for cellular metabolism and maintenance of the membrane structure (Corraze 2001). In most aquatic organisms, including abalone, the main function of both n-3 and n-6 polyunsaturated fatty acids (PUFA) appears to be structural, and their use as an energy source is considered to be limited (Floreto et al. 1996, Sargent et al. 2002). Some reports on the green abalone have shown that neutral lipids are metabolized to produce energy only when the other sources, like proteins and carbohydrates, are depleted. Proteins are preferentially used as an energy source to maintain the lipid reserves (Segawa 1993, Durazo-Beltrán et al. 2004). On the other hand, several investigations with different abalone species (Haliotis discus hannai, H. fulgens, H. laevigata, and H. rubra) have shown that the growth rate and fatty acid composition of the different tissues are affected by both the quality and the quantity of dietary lipids (Uki et al. 1986, Durazo-Beltrán et al. 2003a, Grubert et al. 2004). It has been suggested that H. fulgens is able to desaturate and elongate the fatty acids 18:2n-6 and 18:3n-3 to synthesize the long-chain polyunsaturated fatty acids (LC-PUFA) 20:4n-6 (arachidonic acid) and 20:5n-3 (eicosapentanoic acid), respectively (Durazo-Beltrán 2003a, 2003b).

To our knowledge, no information is available on long-term starvation experiments on abalone, and it has been pointed out that more studies are needed on the lipid requirements of green abalone and their association with parameters such as growth rate, inanition, reproduction, and metabolism (Nelson et al. 2002, Viana et al. 2007). Thus, the main goal of the present work was to study the effect of the level of dietary lipids and of a long period of starvation on the fatty acid composition of muscle tissue of cultured juvenile H. fulgens.

MATERIALS AND METHODS

Juvenile H. fulgens (298 ± 30 mg; 28.0 ± 0.2 mm) were obtained from a commercial farm (BC Abalone, Ejido Eréndira, Baja California, Mexico). The organisms were acclimated for 26 days in a flow-through system consisting of 3.8-L plastic containers supplied with aerated and filtered seawater (300 mL min^-1), and fed a standard formulated diet (table 1). Eight abalone were placed in each container and maintained under constant temperature (20.0 ± 1.2 °C) and a photoperiod of 12 h light/12 h darkness. After the acclimation period, three treatment groups were randomly separated. One group was offered a rich lipid diet (RLD), another was offered a low lipid diet (LLD), and the third was kept under starvation conditions. All experiments were carried out in triplicate.

Total lipid content in diets and muscle was determined by extraction using chloroform:methanol (2:1, v/v) following the extraction method described by Folch et al. (1957). Analysis of fatty acid methyl esters (FAME) was performed according to Christie (1993) using a Hewlett Packard 6890II gas chromatograph equipped with a flame ionization detector and a capillary column (Omegawax 320, Supelco/Sigma-Aldrich; 30 m x 0.32 mm, film thickness 0.25 mm). Fatty acids were identified by comparing their retention times with those of well-characterized FAME standards (37-Component FAME Mix, PUFA1, PUFA3, Supelco/Sigma-Aldrich). Each fatty acid concentration was estimated from the corresponding chromatogram area using an internal standard (19:0) and the Agilent ChemStation (version E.02.00.493) software package.

All data were subjected to one-way analysis of variance. Differences were considered statistically significant at P < 0.05. Means were compared after analysis of variance by Tukey range tests. All statistical analyses were carried out using Minitab v16.2 (Minitab Inc., State College, PA, USA). The results are reported as mean ± standard error of the mean.

RESULTS

The two experimental diets (RLD and LLD) were formulated to have similar protein/energy ratios, but different lipid content (table 1). The levels of monounsaturated fatty acids (MFA), PUFA, and LC-PUFA in both RLD and LLD were lower than those found in a standard diet (table 2), whereas the level of saturated fatty acids (SFA) was higher in RLD. The level of 18:2n-6 in LLD was 24-fold lower than that in RLD, resulting in a higher n-3/n-6 ratio in LLD (0.30) than that observed in RLD (0.02), mainly due to the content of 18:2n-6 from corn oil in the lipid mixture.

The lipid profiles of the abalone muscles were determined before and after each treatment (table 3). There were significant differences in the fatty acid profiles after the experimental feeding and starvation periods. At the beginning of the experiment, the most abundant fatty acids in muscle tissue were 16:0, 16:2n-6, 18:0, 18:1n-9, 20:5n-3, and 22:5n-3. After 90 days, the organisms fed LLD showed a higher amount of 18:1n-9 and a decrease in n-3 and n-6 LC-PUFA and the n-3/n-6 ratio, while the total lipid level was similar (P > 0.05) to that observed before the experiment. Abalone fed RLD showed similar values of SFA, 18:1n-9, 18:1n-7, 18:2n-6, 20:1n-9, 20:2n-6, 20:3n-6, 22:4n-6, 22:6n-3, and total lipids (P > 0.05) to those observed in abalone fed the standard diet until starting the treatment. No changes (P > 0.05) in the fatty acid profiles and total lipid content of abalone were observed after 50 and 70 days of starvation. Nevertheless, abalone starved for 90 days showed significantly lower SFA, MFA, and total lipid contents (P < 0.05).

The fatty acid profiles reflected the lipid source used for the experimental diets. LLD was designed to contain the lowest possible amount of lipids. It is not possible for a diet to be lipid-free because it is difficult to eliminate all the fat present in the diet components (Mai et al. 1995). Nevertheless, the total lipid level was much lower (0.14% w/w) than that contained in RLD (5.1% w/w) or the recommended amounts.

Before treatment, the most abundant fatty acids of the muscle tissue were 16:0, 16:2n-6, 18:0, 18:1n-9, 20:5n-3, and 22:5n-3, with a balanced n-3/n-6 ratio (1.18), in agreement with a previous report (Durazo-Beltrán et al. 2003a). This profile is originated by the dietary lipids from fish and vegetal sources, since abalone tend to accumulate n-3 LC-PUFA as 20:5n-3 and 22:5n-3 (Uki et al. 1986). At the end of the LLD treatment, the muscle tissue of abalone showed an increase in the amount of 18:1n-9 and a decrease in the n-3 and n-6 LC-PUFA contents, while the total lipid level remained unchanged when compared with the content before the treatment. The absence or presence of small amounts of n-3 and n-6 LC-PUFA in the diet and the occurrence of high levels of 18:1n-9 in the tissue could be an indication of essential fatty acid deficiency, and suggest negligible Δ6-desaturase activity which is needed to synthetize 18:2n-9 and 20:2n-9 (Sargent et al. 1995, Ibeas et al. 1996). Consistently, Atlantic salmon fed a diet containing insufficient amounts of n-3 and n-6 PUFA for four months showed that nearly 50% of the fatty acids in liver triacylglycerols corresponded to 18:1n-9 (Ruyter et al. 2000).

The total lipid content and fatty acid profile of abalone fed LLD suggest possible lipogenesis from carbohydrate or protein (Durazo-Beltrán et al. 2003a), which tends to reduce the LC-PUFA content and has been associated with tissue growth under fatty acid deficiency. Abalone fed RLD showed similar values of SFA, 18:1n-9, 18:1n-7, 18:2n-6, 20:1n-9, 20:2n-6, 20:3n-6, 22:4n-6, 22:6n-3, and total lipids to those recorded after the acclimatization period during which a standard diet was used (i.e., before treatment), but the levels of n-3 and 20:4n-6 LC-PUFA were lower. Starvation or restricted feeding is not unusual to marine invertebrates when food is scarce or unavailable over a long period of time, and the main response is a reduction in the metabolic rate to conserve energy (Hochachka and Somero 1984). In addition to the general reduction in total body lipids, starvation induces highly variable changes in muscle lipid content (McCue 2010). Our results show that the total lipid content and fatty acid profiles of abalone starved for 50 and 70 days remain similar to those fed the standard diet before the treatment. This observation suggests that during the first 70 days of starvation, the muscle lipids are not used as an energy source. A similar pattern has been reported for starved eel (Boetius and Boetius 1985) and shrimp (Sánchez-Paz et al. 2006). Previous studies on green abalone showed that lipid accumulation combined with weight loss indicates that during starvation, carbohydrate and protein, rather than lipids, are used as the principal source of energy (Viana et al. 2007), a condition that may be associated with a low oxidative metabolism (Segawa 1993) of adipose tissue. After 70 days of starvation, however, the total lipid level began to decrease and declined approximately 26% within the next 20 days (90 days starvation). The initial levels of LC-PUFA remained essentially unchanged throughout the 90-day starvation period. In contrast, after 90 days of starvation, the total lipid, SFA, MFA, and PUFA contents all decreased. LC-PUFA, the main lipid constituents of cell membranes because they are a major component of phospholipids, appear to be preferentially conserved in order to maintain the structural integrity of membranes and physiological needs (Sargent et al. 1995, Zabelinskii et al. 1999). The tendency to conserve n-3 LC-PUFA has been reported in starved fish and abalone where SFA and MFA are preferentially mobilized, whereas the levels of LC-PUFA like 20:5n-3, 22:5n-3, and 22:6n-3 remain almost constant (Navarro and Gutiérrez 1995, Durazo-Beltrán et al. 2004). Although lipids in the green abalone seem to be primarily used for growth and gonad maturation (Ottaviani et al. 2011), during long-term starvation lipids can serve as an energy source.

In conclusion, our results show the high metabolic flexibility of abalone and their capacity to adapt to different situations, such as restricted feeding or starvation, where LC-PUFA supply is insufficient and lipogenesis may occur in order to meet the most important physiological requirements.

ACKNOWLEDGMENTS

This project was financed by Universidad Autónoma de Baja California (internal project 0365). We thank the commercial farm BC Abalone for their kind donation of the abalone used in our experiment.

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