Artículos

 

Effect of the seaweed Macrocystis pyrifera and a formulated diet on growth and fatty acid composition in the green abalone, Haliotis fulgens, under commercial culture conditions

 

Efecto de la macroalga Macrocystis pyrifera y una dieta formulada sobre el crecimiento y la composición de ácidos grasos en el abulón azul, Haliotis fulgens, en condiciones de cultivo comercial

 

Eduardo Durazo-Beltrán¹, Jorge F. Toro-Vázquez², Carlos Vásquez-Peláez³ and María Teresa Viana^4*

 

¹ Facultad de Ciencias Marinas Universidad Autónoma de Baja California Apartado postal 453 Ensenada, CP 22800, Baja California, México.

² Facultad de Ciencias Químicas Universidad Autónoma de San Luis Potosí, Ave. Dr. Manuel Nava No. 6 San Luis Potosí, CP 78210, SLP, México.

³ Facultad de Medicina Veterinaria y Zootecnia Universidad Nacional Autónoma de México Ciudad Universitaria México, CP 04510, DF, México.

⁴ Instituto de Investigaciones Oceanológicas Universidad Autónoma de Baja California Apartado postal 453 Ensenada, CP 22800, Baja California, México. *E-mail: viana@uabc.mx

 

Recibido en enero de 2003;
aceptado en julio de 2003.

 

Abstract

The effect of a formulated diet (FD), a seaweed diet (SW), and a mixture of both (FD + SW) on growth, survival rate, and fatty acid content in the tissue of juvenile Haliotis fulgens abalone grown under commercial culture conditions, was analyzed over a 329-day period. Survival and growth rate in terms of length and weight were different with each of the diets evaluated, being significantly higher (P < 0.05) with the mixed diet (FD + SW), followed by SW and FD. Since feed intake was not evaluated in this preliminary study, differences in growth cannot be attributed to the dietary treatments; however, the importance of this work was to show the significant impact of the diet treatments on the tissue fatty acid profiles, suggesting that the treatments contribute to the composition of some fatty acids found in muscle. The polyunsaturated fatty acid (PUFA) 22:4n-6 was not detected in any of the diets, while 20:4n-3 was only present in SW and 22:5n-3 only in FD. However, after the feeding experiment all these PUFAs were present in abalone tissue. The possible synthesis of PUFAs from dietary n-3 and n-6 fatty acids of shorter hydrocarbon chain is discussed.

Key words: Haliotis fulgens, commercial culture, formulated diet, Macrocystis pyrifera, fatty acids.

 

Resumen

Se analizaron los efectos de una dieta formulada (FD), de una dieta de macroalgas (SW) y de la mezcla de ambas (SW + FD) sobre el crecimiento, la tasa de supervivencia y la composición de ácidos grasos en el tejido de juveniles de abulón azul, Haliotis fulgens, en un cultivo comercial durante un periodo de 329 días. La tasa de supervivencia y el crecimiento en términos de longitud y peso, mostraron diferencias significativas entre los tratamientos estudiados, siendo significativamente mayores (P < 0.05) para la dieta FD + SW, seguida por las dietas SW y FD. Dado que la tasa de consumo no pudo ser evaluada en este experimento preliminar, las diferencias encontradas no pueden ser atribuidas a los diferentes tratamientos; sin embargo, la importancia de este trabajo consistió en mostrar el impacto de los tratamientos sobre los perfiles de ácidos grasos contenidos en el tejido de los abulones, lo cual sugiere que los tratamientos contribuyeron en la composición de algunos de los ácidos grasos encontrados en el músculo. El ácido graso poliinsaturado (PUFA) 22:4n-6 no fue detectado en ninguna de las dietas, mientras que el 20:4n-3 estuvo presente sólo en la SW y el 22:5n-3 sólo en la FD. Sin embargo, al finalizar el experimento todos estos PUFAs estuvieron presentes en el tejido de abulón. Se discute la posible síntesis de los PUFAs a través de los ácidos grasos n-3 y n-6 de cadena más corta presentes en la dieta.

Palabras clave: Haliotis fulgens, cultivo comercial, dietas balanceadas, Macrocystis pyrifera, ácidos grasos.

 

Introduction

Abalone nutrition research is conducted under strict experimental conditions; however, experimental diets can behave differently under commercial conditions. This happens when factors such as temperature, light, abalone manipulation, and the presence of natural food are not taken into consideration, leading to contradicting results (Viana et al., 1996). In fact, several studies have reported that seaweed performed significantly poorer compared with formulated diets (Viana et al., 1993a; Alarcón, 2000). Nevertheless, seaweed is still being used under commercial conditions (Fleming et al., 1996) based on its apparent performance, because no data are available to support the idea that seaweed is more nutritious than formulated diets. Several aspects of the use of seaweed in the culture of abalone have been questioned, such as the role that diatoms play in the nutrition of abalone. Diatoms grow attached to the seaweeds and ponds, and are available for abalone to grasp onto them to complement their nutritional requirements. Thus, any contribution addressing the role that seaweed plays on a commercial scale will help to determine abalone nutritional requirements and the possible substitution of seaweed by formulated diets under commercial conditions.

The goal of any abalone producer, as of most other aquatic organisms and livestock, is to grow the species of interest using formulated diets to optimize their growth efficiency, unless natural food is cheaply available. Any formulated diet should contain all the necessary nutrients to meet the nutrition requirements of the particular organisms, usually met with a variety of ingredients available in the market (Jobling, 2001a). Therefore, nutrition research is essential to determine their nutritional requirements in order to sustain adequate production.

Recently, several reports on abalone nutrition have resulted in the modification of diets in order to improve feed utilization. For example, protein levels have been reduced from 40% to 27% of the diet. Cellulose, previously reported to have a negative effect on growth (Uki and Watanabe, 1992), has now been shown to be efficiently used by abalone (Erasmus et al., 1997; Monje and Viana, 1998). Although progress has been made, lipids may be the nutrient less studied, probably because their recommended level of inclusion is no more than 5% of the diet. This requirement is usually met by incorporating fish and soybean meals as protein sources. Thus, the quantity and quality of fatty acids in the diet will depend on the type and level of protein source used. Nevertheless, it has been shown that abalone exhibit greater growth when n-3 and n-6 polyunsaturated fatty acids (PUFAs) such as 18:3n-3, 18:2n-6, 20:4n-6, and 20:5n-3 are present in the diet (Uki et al., 1986; Floreto et al., 1996; Mai et al., 1996).

In addition to using growth as a parameter to evaluate the effect of diets, it is important to study the muscle tissue composition, since the overall performance of the diets, including the feed ingredients, feeding frequency and handling, will be reflected in the composition of the muscle (Carter et al., 2001; Jobling, 2001b). Even under commercial conditions, tissue composition will show the presence of natural foods besides the formulated diets.

Therefore, the aim of the present work was to evaluate the effect of a formulated diet, a natural diet composed of seaweed and the combination of both, on the growth, survival and fatty-acid composition of the tissue of juvenile abalone Haliotis fulgens.

 

Material and methods

Experimental diets

The percentual and proximate compositions of the diets are presented in table 1. The formulated diet (FD) was balanced according to the recommendations of Mai et al. (1995) and Viana et al. (1996) (table 1). The vitamin and mineral mixtures used were those recommended by Hahn (1989). Fish silage was made as described by Viana et al. (1993b). The lipid content in the FD was the result of the corresponding lipid concentration present in the ingredients used. Butyl-hydroxy toluene was added to the formulation to prevent lipid oxidation. All ingredients were mixed with a blender (Robot Coupe® R10) until a homogeneous paste was obtained. The diet was then flattened into 2-mm-thick sheets using a pasta maker (Rossito Bisanti®). Pieces of 2 x 2 cm were then cut and dried (45°C for 24 h). The diet was stored in plastic containers in a cold, dry place for four weeks prior to feeding.

Fresh seaweed, Macrocystis pyrifera, from coastal areas close to Ensenada (Ejido Eréndira, Baja California, Mexico), collected and supplied weekly, was used as seaweed diet (SW). Prior to feeding, the seaweed was transported to the abalone farm and left in a large pond where it was washed thoroughly with seawater. Fresh seaweed samples were stored in sealed containers at -25°C for proximate and fatty acid analyses.

The mixed diet (FD + SW) was prepared with fresh seaweed and formulated diet at a ratio of 150% and 5% of the abalone biomass, respectively.

Experimental procedure

The study was carried out in a commercial abalone farm (BC Abalone, SA de CV, Ejido Eréndira, Baja California, Mexico). Three-month-old H. fulgens, with an average length of 5.9 ± 0.06 mm and average weight of 24.2 ± 1.5 mg, were held in a flow-through system (20 L min^-1) using seawater. Aeration was also constantly provided. Water temperature was 13.1°C and 21.1°C during winter and summer, respectively. The animals were confined in six 850-L rectangular fiberglass tanks with open seawater flow, filled to contain 300 L in each tank (two replicates per treatment) and 2000 abalone per tank. Abalone were fed daily as indicated before. The FD was offered for 12 h overnight every night, and the remaining FD was siphoned by the system or removed daily. Feed intake was not determined due to the difficulty of measuring under commercial conditions.

Abalone growth was evaluated as the total growth for the experimental period in terms of length and body weight. Whole-body wet weight was measured with an electronic balance (± 0.001 g) and shell length with an electronic digital caliper (± 0.05 mm), at 0, 141, 234, and 329 days. Survival rate (%S) was calculated for each treatment during the experimental period.

To prevent injury from handling abalone, MgSO₄ (4%) or 2-phenoxyethanol (1mL L^-1) were used as anesthetic (White et al. , 1996). To evaluate the effect of the diets on the chemical composition of the tissue, 40 abalone per experimental unit were collected at the end of the experiment.

Proximate analysis

Samples of the experimental diets used for each treatment and abalone muscle tissue were used to perform the analysis. Dry weight was calculated after drying the sample to constant weight at 100°C. Total nitrogen (N) was determined by the Kjeldhal method (AOAC, 1995), and crude protein was calculated by N x 6.25. Crude lipids were determined by extraction using chloroform-methanol-water (1:1:0.9 v/v), following Bligh and Dyer's (1959) method. Ash was determined gravi-metrically after burning the organic matter at 550°C for 18 h.

Fatty acid analysis

Aliquots of the lipid extract were first refluxed during 3 min in a 0.5 M KOH solution in methanol prior to methylation, achieved by refluxing again (3 min) in 14% boron trifluoride in methanol (BF₃-MeOH) (Metcalfe et al., 1966). Fatty acid methyl esters (FAMEs) were analyzed in a Hewlett Packard 5890II gas chromatograph equipped with a flame ionization detector (260°C). FAMEs were separated with a capillary column (Omegawax 320 by Supelco Inc.; 30 m x 0.32 mm, film thickness 0.25 mm), using hydrogen as carrier gas. The initial oven temperature was 140°C. Five minutes after sample injection (1 mL), the temperature was increased at a rate of 4°C min^-1 until 240°C was reached, temperature that was held for an additional 10 min. Fatty acids were identified using commercial standards, 37 Component FAME Mix (Supelco Inc.; GLC 87, Nu-Chek Prep), and marine oils (PUFA1 and PUFA3, Supelco Inc.), under similar conditions. An internal standard (23:0) was used to calculate the concentration for each fatty acid using the software package HP ChemStation rev. A.06 for Windows.

Statistical analysis

Survival, growth rates and fatty acid profiles among treatments were analyzed using a one-way ANOVA, followed by a SNK multiple comparison (Zar, 1999). For percentage values, data were transformed to arc sen. The analysis was performed using the statistical package SAS 6.08 (Cary, NC, USA).

 

Results

The overall performance of the abalone in terms of growth and survival was significantly higher when fed the mixed diet (FD + SW), compared with the SW diet and FD (table 2). Growth rates were significantly higher, only in terms of length, with the SW treatment than with the FD treatment; however, survival was significantly higher with the FD treatment than with the SW treatment.

Despite the differences in growth rate observed in abalone fed the experimental diets, the proximate composition of abalone muscle was quite similar (table 2). No significant differences in crude protein and ash contents were observed among abalone fed the dietary treatments. Significant differences in total lipid content of tissue were found among H. fulgens fed the dietary treatments. Abalone fed FD + SW contained higher total lipid level (5.36%) than those fed SW (4.88%) or FD (4.69%).

Among the fatty acid profiles, differences were observed in both the diet composition (table 3) and the tissue after the experimental feeding period (table 4). In this work, FD showed the highest content of fatty acids 15:0, 16:0, 16:1n-7, 16:3n-6, 18:0, 18:1n-9, 18:1n-7, 18:2n-6, 18:3n-3, 18:3, 20:1n-9, 22:1n-9, and 22:6n-3, whereas SW presented the highest content of 16:2n-6, 17:1n-7, 18:4n-3, 20:4n-6, and 20:5n-3. The abalone tissue from the FD treatment had a higher content of 16:3n-6 and 18:2n-6 than that observed in abalone fed with the other diets. Additionally, the PUFAs 20:3n-6, 20:4n-3, and 22:4n-6 were present in the abalone muscle from the FD treatment, though these fatty acids were not detected in the corresponding diet. Moreover, the 22:4n-6 is present in the abalone tissues, though this particular fatty acid is absent in the diets, showing a significant correlation between levels among this fatty acid (22:4n-6) and 20:4n-6 (r = 0.99; P = 9.3 x 10^-5). The SW treatment showed the highest content of 20:5n-3 and the lowest content of 18:2n-6. Likewise, 22:4n-6 and 22:5n-3 were present in abalone muscle from the SW treatment, though these fatty acids were not detected in the seaweed. Abalone fed with mixed diet presented the highest content of 13 of the 32 fatty acids reported.

 

Discussion

Survival rates obtained in this study (table 2) seem to be low compared to earlier studies, but not too different from those figures found under commercial conditions for a period of 329 days (Neori et al., 1998; Preece and Mladenov, 1999). The FD treatment resulted in lower growth in length and weight, while the mixed diet performed better. Different results were obtained in another study conducted under similar laboratory conditions and scale, with similar treatments and diets (Alarcón, 2000), in which the FD and the mixed diets performed equally well, and the SW diet resulted in the lowest growth with almost no survival after 45 days. Even though our FD treatment did not achieve a good growth, the results show a positive effect when used together with the seaweed. Moreover, since feed intake was not evaluated, the differences in growth cannot be attributed to the diet quality itself, but rather to the availability of feed plus environmental conditions (Jobling, 2001b). Unfortunately, the estimation of feed intake on a commercial scale is difficult since water is siphoned from the tanks under an open flow system; in addition, the 12-h exposure to food in the tanks, which has been shown to be the time for a proper feeding of abalone during the dark period, can lead to a high dry matter loss of pellets making the feed estimation imprecise. Moreover, abalone has a negative photo-taxis (Leighton, 2000) and, as a result, when too much light is present in the tanks, abalone gather in groups and concentrate in the darker areas of the tank. Here it was possible to observe that abalone in the FD treatment tanks were confined to the corners, whereas in the presence of seaweed, abalone were all over the tanks. Therefore, more studies are recommended to evaluate commercial systems using formulated diets that promote an equal distribution of abalone so that the food is available for all organisms in the tanks.

Differences were observed among the fatty acid profiles in both the diets and the abalone tissues after the experimental feeding period (tables 3, 4). Even though the amount of feed ingested is unknown, due to differences in the fatty acid profiles obtained in the tissue it is possible to suggest that abalone were feeding their corresponding treatments rather than grazing on food that grows naturally on seaweeds and ponds (i.e., diatoms, bacteria), because otherwise abalone would be ingesting lipids from sources different to the dietary treatments; however, in a long-term study like this, where more than 70% of the fatty acids reported in tissue was different among treatments, an effect of dietary fatty acid profile on the fatty acid profile in muscle would be expected.

The abalone tissue from the FD treatment had a higher content of fatty acids 16:3n-6 and 18:2n-6 than that observed in abalone fed with the other diets. Additionally, the PUFAs 20:3n-6, 20:4n-3, and 22:4n-6 were present in the abalone muscle from the FD treatment, even though these fatty acids were not detected in the corresponding diet. The presence of these n-3 and n-6 PUFAs in the muscle tissue suggests that H. fulgens may be able to synthesize PUFAs from the series of lower n-3 and n-6 fatty acids found in the diet. Likewise, 22:4n-6 and 22:5n-3 were present in abalone muscle from the SW treatment, even though these fatty acids were not detected in the seaweed. Studies on H. discus hannai established that this species is able to convert 18:2n-6 and 18:3n-3 into 22:4n-6 and 22:5n-3 (Uki et al., 1986). Moreover, it has been determined that H. laevigata and H. rubra are capable of producing C20 PUFAs from C18 PUFAs (Dunstan et al., 1996). Abalone fed with mixed diet had the highest content of 13 of the 32 fatty acids reported. The effect of dietary treatment on fatty acid content in abalone tissue is difficult to explain, because the PUFAs n-3 and n-6, required for normal growth and development of abalone (Uki et al., 1986; Floreto et al., 1996; Mai et al. , 1996), are metabolized by the same enzyme systems of sequential desaturation and elongation, resulting in a long chain of the n-3 and n-6 series (Lands, 1992; Cook, 1996; Bell, 1998). Therefore, in dietary studies it is important to consider the influence that one type of fatty acid can have on the metabolism of another. Based on the fatty acid profiles, we can suggest that H. fulgens is able to synthesize long-chain PUFAs from short-chained fatty acids, an ability that has already been reported in green abalone (Durazo-Beltrán et al., 2003).

The effect of specific lipids and fatty acids on growth and tissue composition should be studied under strict experimental conditions, with no presence of natural food and adequately measuring feed intake. More work is necessary to evaluate the use of seaweeds under commercial conditions in order to understand their role in abalone production to improve the production system for formulated diets.

 

Acknowledgements

We thank BC Abalone for allowing the experiment to be conducted at their farm. Roche provided the vitamin and mineral mixtures used in this experiment. This project was financed by CONACYT (projects 1925PB and G28119B) and UABC. Special thanks to Lou D'Abramo for his valuable comments and interest in the present work.

 

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