First development of various vegetable-based diets and their suitability for abalone Haliotis tuberculata coccinea Reeve

    First development of various vegetable-based diets and their suitability for abalone Haliotis tuberculata coccinea Reeve M.P. Viera, G. Courtois de Vic¸ose, L. Robaina, M.S. Izquierdo PII: DOI: Reference:

S0044-8486(15)30022-3 doi: 10.1016/j.aquaculture.2015.05.031 AQUA 631686

To appear in:

Aquaculture

Received date: Revised date: Accepted date:

17 September 2014 19 May 2015 21 May 2015

Please cite this article as: Viera, M.P., Courtois de Vi¸cose, G., Robaina, L., Izquierdo, M.S., First development of various vegetable-based diets and their suitability for abalone Haliotis tuberculata coccinea Reeve, Aquaculture (2015), doi: 10.1016/j.aquaculture.2015.05.031

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ACCEPTED MANUSCRIPT FIRST DEVELOPMENT OF VARIOUS VEGETABLE-BASED DIETS AND THEIR SUITABILITY FOR ABALONE Haliotis tuberculata coccinea

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Reeve

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M.P. Viera*, G. Courtois de Viçose, L. Robaina, M.S. Izquierdo

Grupo de Investigación en Acuicultura (GIA), Universidad de Las Palmas de Gran

Phone: (34) 928 132034 E-mail: [email protected]

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Corresponding author: M. P. Viera

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Canaria (ULPGC). Las Palmas, Canary Islands, Spain

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Address: Muelle de Taliarte s/n, 35214, Telde, Gran Canaria, Canary Islands, Spain

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Abstract

To date, European abalone aquaculture relies mostly on locally harvested fresh seaweeds which nutritional quality and abundance varies greatly, hence affecting abalone growth. Abalone artificial diets generally include fishmeal, limiting their utilization in ecologically sustainable aquaculture and affecting

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abalone quality and acceptance by the consumers. A six month feeding trial was conducted to assess the nutritional value of four different dried seaweed meals: Ulva lactuca (Chlorophyta), Gracilaria cornea (Rhodophyta), Laminaria digitata (Phaeophyta) and Palmaria palmata (Rhodophyta), as ingredients to all-vegetable-based formulated feeds for abalone Haliotis tuberculata coccinea (33.1 ± 0.8 mm and 4.7 ± 0.6 g). A mixed fresh algae diet of G. cornea and U. rigida, reared in an Integrated Multi-Trophic Aquaculture (IMTA) system, served as control. Survival rates were very high (95-98%), regardless of the diet fed. Enriched fresh algae produced a significantly higher growth for H. tuberculata coccinea (169% weight gain) than all the artificial diets (49-84% WG). Comparison among abalone fed the different formulated diets showed that the inclusion of P. palmata improved growth, condition index and dietary protein utilization. On the contrary, the use of L. digitata markedly reduced the efficiency of dietary protein since the protein-related nutritional index (PER), the percentage of protein deposited in the foot muscle as well as the meat to shell ratio recorded for animals fed this diet were the lowest, despite a higher feed intake. Large differences were found in the FA profile of fresh algae as compared with the three formulated diets. The n-3/n-6 ratio was much higher in the fresh algae and, consequently, in the foot tissues of abalone fed this diet in comparison to the one of those fed the formulated ones. The elevated contents of 20:4n-6 in the abalone fed the experimental diets and 20:5n-3 in abalone fed the fresh algae,

ACCEPTED MANUSCRIPT as well as their respective metabolites, suggest that abalone have the ability to desaturate and elongate LA to ARA and ALA to EPA. Further studies are required to improve the growth obtained with these vegetable based diets, especially concerning the use of different seaweed combinations and inclusion

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levels, as well as the diet processing methods to improve diets water stability.

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Keywords: abalone; growth; artificial diets; seaweeds; IMTA

1. Introduction

The European native abalone species (Haliotis tuberculata spp.) is a highly appreciated

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European shellfish product in both traditional (Mgaya and Mercer, 1994) and novel premium quality abalone markets (Dallimore, 2010). However, its availability is severely restricted due to

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lack of supplies from both wild resources and aquaculture production. Due to abundance of macro-algae in Western Europe, most European farms grow abalone by feeding them locally harvested fresh seaweeds (Fitzgerald, 2008). However, macroalgae nutritional quality and

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abundance varies greatly according to geographic location and time of sampling (Dawczynski et al., 2007), and greatly influences abalone growth rates, affecting the economic success of the

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on-growing activity (Bautista-Teruel and Millamena, 1999).

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Among the different nutrients, abalone requires adequate levels of high quality protein for soft tissue growth (Uki et al., 1985a; Mai et al., 1995a, b; Britz and Hecht, 1997; BautistaTeruel and Millamena., 1999; Gómez-Montes et al., 2003; Reyes and Fermín, 2003; Viana et al., 2007). The most common protein sources employed in abalone feeds include fishmeal,

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defatted soybean meal (Guzmán and Viana, 1998; Sales and Britz, 2002; Gómez-Montes et al., 2003; Thongrod et al., 2003; Naidoo et al., 2006; García-Esquivel et al., 2007), casein (Uki et al., 1985b; Viana et al., 1993; Mai et al., 1995b; Sales et al., 2003; Vandepeer et al., 2003) and Spirulina spp. (Uki et al., 1985b; Britz et al., 1996a; Bautista-Teruel et al., 2003;Thongrod et al., 2003; Naidoo et al., 2006; Troell et al., 2006). Few novel protein sources have also been tested at low inclusion levels (Vandepeer et al., 1999, 2003; Shipton and Britz, 2001; Sales and Britz, 2002; Reyes and Fermín, 2003). To gain advantage of the high nutritional value of algae, algal meals have been occasionally included in abalone feeds (García-Esquivel et al., 2007; Viana et al., 2007). Fishery by-products such as fish or abalone viscera silage have been also proposed as economic protein sources (López and Viana, 1995; Viana et al., 1996; Rivero and Viana, 1996; Guzmán and Viana, 1998). Additionally, different protein sources may be balanced by addition of synthetic amino acids such as methionine, threonine and arginine in order to fulfil the essential amino acid requirements of these species (Mai et al., 1995b; Guzmán and Viana, 1998; Serviere-Zaragoza et al., 2001; García-Esquivel et al., 2007). Among all the

ACCEPTED MANUSCRIPT sources tested as a single protein source, fishmeal is the only one that has been reported to support good growth performance (Fleming et al., 1996). Studies with cultured abalone have demonstrated that diet can have a significant effect on quality-related factors such as chemical composition, taste, texture and colour (Dunstan et al.,

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1996; Chiou and Lai, 2002; Allen et al., 2006). In particular, the lipid composition of abalone

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muscle is markedly affected by the diet (Uki et al., 1986; Dunstan et al., 1996). Moreover, lipids

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(especially long-chain polyunsaturated fatty acids, PUFA) are essential to determine the flavour and odour of seafoods (Lindsay, 1988). Thus, the use of artificial feeds containing fishmeal could give cultured abalone a much “fishier” flavour than diets containing vegetable sources (Dunstan et al., 1996). Indeed, a way of improving market acceptability and product quality

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includes feeding abalone on macroalgae immediately prior to sale (Dunstan et al., 1996; Kinkerdale et al., 2010).

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Moreover, abalone output in Europe is substantially focused on high quality and low volume niche markets, such as organic or eco-certified products, implying that, among other requirements, no fishmeal, pharmaceuticals or fertilizers are used. Hence, developing a vegetable based artificial feed for European abalone will have marketing benefits not only for

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consumers, who are increasingly environmentally sensitive, but also for producers, providing

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them a readily available and more stable nutritional feed, whereas validating the environmental and social sustainability of their farming operations (SUDEVAB, 2007; WWF, 2010).

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In the wild, abalone consume a variety of seaweeds, obtaining its required nutrients from a combination of algal species (Sales and Britz, 2001; Dlaza, 2008). These seaweeds are selected mainly according to their abundance and availability in the surrounding area (Nelson et al., 2002). In the case of the European abalone, red macroalgae such as Palmaria palmata and the

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green ones Ulva spp. are preferred. Nevertheless, other coarser and abundant seaweeds like kelps Laminaria spp., (Koike et al., 1979; Mercer et al., 1993) are commonly used as a bulk feed in abalone farms (Fitzgerald, 2008; Walsh and Watson, 2011; Hannon et al., 2013). Earlier studies have shown that fresh P. palmata, Ulva spp. or the rhodophyte Gracilaria spp. have a high dietary value for H. tuberculata spp., (Culley and Peck, 1981; Mercer et al., 1993; Viera et al., 2005, 2011), showing also that mixed diets produced far better growth rates than singlespecies diets, which is generally accepted for most abalone species (Simpson and Cook, 1998; Dlaza, 2008; Naidoo et al., 2006; Kinkerdale et al., 2010; Robertson-Andersson et al., 2011). Therefore, the objective of the present study was to test the nutritional value of dried Laminaria digitata (Phaeophyta), Palmaria palmata (Rhodophyta), Ulva lactuca (Chlorophyta) and Gracilaria cornea (Rhodophyta) meals, as dietary ingredients to obtain more sustainable formulated feeds for abalone (Haliotis tuberculata coccinea) production.

ACCEPTED MANUSCRIPT 2. Material and methods 2.1. Processing of seaweed meals, diets formulation and preparation Fresh G. cornea (G), U. lactuca (U), L. digitata (L) and P. palmata (P) (kindly supplied

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respectively by the Centro de Biotecnología Marina (CBM-ULPGC), Gran Canaria, Spain;

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South West Abalone Growers Association (SWAGA), Cornwall, UK; Martin Ryan Institute (MRI), Galway, Ireland and France Haliotis (FRHAL), Brittany, France), were cleaned, washed

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with freshwater to remove salts and epizoos and oven dried at 35ºC. The dried samples were finely ground using an electric fine mill (sieve size <0.1 mm) and frozen at -80 ºC until analysis. Algal meals were analyzed for nutrient composition and amino acid profile (Table 1).

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Based on these meals, three diets were formulated to contain 35% protein, 50% of which was contributed by the different selected seaweeds meals: U and G; U, G and L and U, G and P (Tables 2 and 3). The remaining protein and energy contents were supplied by soybean meal,

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corn gluten meal, spirulina and starch, such that each experimental diet had a similar lipid content (4%) and total energy (16 KJ mg-1). These nutrient levels have been reported by Britz and Hetch (1997), Viana et al. (2007) and Viera et al. (2011) as being optimal for abalone

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growth. Vitamin and mineral mixtures were used as recommended by Uki et al. (1985a). All the

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experimental diets were supplemented with synthetic L- methionine and lysine in order to match the amino acid profile of abalone muscle which was used as a guide to formulate the amino acid

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composition of the practical diets. Sodium alginate, which has been suggested to increase protein efficiency when feeding with seaweed (Kemp and Britz, 2012) was used as binder (Table 2).

Experimental diets were prepared by mixing pre-weighed finely ground ingredients

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including vitamins and minerals to produce a homogeneous mixture. The diets were then processed through a pasta machine (Parmigiana, RV3, Italia) into 2 mm thick strips from which 0.5 x 0.5 pieces were cut, dried at 38ºC for 24 h and stored at 4ºC until use. A mixed fresh algae diet of G. cornea and U. rigida served as control. Seaweeds were reared within the Grupo de Investigación en Acuicultura (GIA, Canary Islands, Spain) aquaculture research facility, in a flow-through integrated system collecting wastewater from fish and abalone ponds in a macroalgal biofilter (Viera et al., 2014). Experimental and control diets were sampled for proximate composition. The algal samples were processed as indicated above and artificial diets were held under the same conditions as fresh algal samples until analysis. All the diets were tested for their stability in seawater under the same conditions as the feeding experiment in tanks without abalone, by measuring the loss of dry matter of pellets after 17 h of immersion (16:00-9:00h) and of natural diet for a 3-days period.

ACCEPTED MANUSCRIPT 2.2. Abalone and experimental procedure Three hundred and sixty (30/replicate) 1-year-old H. tuberculata coccinea individuals with an average shell length and weight of 33.1 ± 0.8 mm and 4.7 ± 0.6 g respectively, were selected

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from the experimental hatchery production unit of GIA. Animals were blot dried, weighed to

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the nearest 0.1 mg (total fresh body weight: TFBW), measured with manual calipers with 0.1 mm accuracy (total shell length: SL) and distributed among triplicate tanks in a flow-through

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system. The experimental unit consisting of a plastic bucket (15x16 cm) hung in a 100-L rectangular tank (100x40x25cm) filled with seawater provided with constant aeration. Water flowed at 2.8 l/min and two PVC shelters were provided in each container. Water temperature

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ranged between 22.6-24.5 ºC and abalones were subjected to a natural photoperiod of approximately 12 h L / 12 h D. Animals were conditioned on the test diets for two weeks before data collection begun. The feeding trial was run for 176 days and dead abalones were daily

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recorded.

Animals in all treatments had access to their corresponding diet from Monday to Saturday. Artificial feeds were offered once daily (ad libitum) in the evening. Any remaining diet was

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collected every day at 9:00 h. except Sunday, by manually siphoning uneaten feed from tanks.

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Consumption was estimated on a dry weight basis by relating the dry weight of the uneaten food to the known dry weight of the feed provided. Consumption data were corrected for dry matter weight loss attributable to leaching, by allowing the diets to leach over a 17-h period (16:00-

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9:00 h) using a “control” rearing unit without abalone, and drying the remaining diet until constant weight. To guarantee ad libitum feeding in the control diet, fresh algae were supplied well in excess twice a week. To determine control feed intake, freshly collected algae to be fed

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to the abalone were blotted dry and accurately weighed as well as the remaining algae every third day. The weight of unconsumed food was deducted from the total weekly ration. Besides, weight of uneaten algae was corrected by calculating the natural change in weight of the algae in the control units during the same feeding period. The average daily intakes by individual abalone during the entire feeding trial were calculated by dividing the algal biomass eaten each week by the feeding days and the number of abalones in each experimental unit. SL and TFBW of each animal were recorded monthly. Abalone growth rates (growth rate day-1), as well as the following indices, were calculated for all treatments at the end of the trial: Shell growth rate (μm d-1) = (SL2-SL1) / days of culture x 1000 Specific growth rate, SGR (%d-1) = (LnW2-LnW1) / days of culture x 100 Weight gain, WG (%) = ((W2-W1) / W1) x 100 Feed conversion ratio, FCR = total feed intake (g dry) / total weight gain (g wet) Protein efficiency ratio, PER = (increase in body wet weight (g)) / (protein intake in dry weight (g))

ACCEPTED MANUSCRIPT where L1 is the initial mean length of animals; L2 is the final mean length of animals; W2 is the weight at time t (days of culture), and W1 is the initial weight. Besides, six abalones were collected from each experimental unit, and the soft tissue was

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shucked from the shell. Shell and meat were then weighed separately in order to calculate the

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meat to shell weight ratio (wet weight of soft flesh/wet weight of shell. SB/S in W/W) as an indicator of the abalone nutritional status. Abalone tissues were also sampled and frozen at -80

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ºC until analysis.

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2.3. Proximate, amino and fatty acid analysis

Homogenized samples of the seaweed meals, formulated diets, fresh algae and abalone (visceral mass and foot muscle) were analyzed in triplicate for nutrient composition. Amino acid

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was done on each experimental seaweed meal using an HPLC amino acid analyzer. Protein content was determined by Kjedahl method according to AOAC (2005) standard analyses. Total lipids were extracted with chloroform–methanol (2:1) as described by Folch et al. (1957). Fatty

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acids in the lipid extracts were transesterified to methyl esters (FAMEs) with 1% sulphuric acid:

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methanol complex (Christie, 1982). FAMEs samples were extracted into hexane and stored at 80 ºC. Fatty acids were analyzed in a Thermo Finnigan- GC Focus gas chromatograph equipped

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with a flame ionization detector (260ºC). FAMEs were separated with capillary column (Supercowax 28m x 0.32mm x 0.25 i.d.) using helium as the carrier gas under the conditions described by Izquierdo et al. (1989). Fatty acids of the experimental and control diets and abalone were also analyzed. The dry matter was determined by drying at 110 ºC until constant

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weight (AOAC, 2005). Ash content was determined by incinerating samples at 600 ºC for 24 h (AOAC, 2005).

2.4. Statistical analysis All data were tested for normality and homogeneity of variance. Means and standard deviations (SD) were calculated for each parameter measured. At the end of the trial, proximate composition of abalones, survival, growth performance and nutritional indices were calculated and statistically treated by one-way ANOVA and Tukey’s test was applied for multiple comparison of means at a 5% significance level (P< 0.05). All statistical analyses were applied using the Statgraphics Plus 5.1 (MANUGISTIES, Rockville, Maryland, USA) software.

ACCEPTED MANUSCRIPT 3. Results 3.1. Nutritional composition of diets and water stability Proximate composition and energy content of the formulated diets correspond well to the

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intended compositions and levels based upon the dietary formulations (Table 2). All artificial

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diets were isoproteic, containing approximately 35% crude protein, and isocaloric with similar gross energy values and protein: energy (PE) ratios. However, nutritional composition and

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energy content varied between artificial feeds and fresh; protein values and PE ratios being higher in formulated feeds as compared to those of the control treatment, whereas the fresh algae had a higher carbohydrate content than the formulated experimental diets. No differences

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were observed in gross energy, lipid and ash contents among the fresh algae and formulated diets (Table 3).

After 17 h of immersion in seawater, pellets stability ranged between 58-73%, with diet

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3.2. Fatty acid composition of diets

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UGL being 7-15% more water stable than other two artificial diets (Table 3).

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Large differences were found in the fatty acids (FA) profile of fresh algae as compared with the three formulated diets (Table 4). Linoleic acid (18:2n-6) was15 times more abundant in

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formulated diets than in fresh algae. On the contrary, formulated diets were lower in 16:0 and 16:1n-7 than in the fresh algae. Subsequently, then-3/n-6 ratio was much lower in formulated diets. Differences in FA profiles between formulated diets were less pronounced than those between fresh algae and practical diets. Both control and formulated diets presented very low

4).

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levels of eicosapentanoic acid (EPA) 20:5n-3 and docosahexaenoic acid (DHA) 22:6n-3 (Table

3.3. Abalone survival and growth Survival rates were very high (95-98%), regardless of the diet. In general, fresh algae produced a significantly better growth for H. tuberculata coccinea (169% WG) than all the other experimental diets (49-84% WG) (Table 5). Comparison among abalone fed the different formulated diets showed that growth was improved by the inclusion of P. palmata, whereas the lowest growth was obtained by the inclusion of L. digitata. Hence, at the end of the trial, animals fed diet UGP displayed the highest shell growth rate, specific growth rate and weight gain, whereas those fed diet UGL showed the lowest values (Table 5).

ACCEPTED MANUSCRIPT 3.4. Feed intake and feed utilization Regarding consumption, there was a significantly (P<0.05) higher feed intake of the fresh algae, followed by diets UGL, UG and UGP (Table 6). In relation to nutritional utilization,

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except with UG and UGP diets, there were significant differences among all diets for food

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conversion ratio (FCR), the highest and lowest values recorded for abalone fed diet UGL and the fresh one, respectively. Similarly, the trends in protein efficiency ratio (PER) were similar to

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those of FCR, being better in animals fed fresh algae and worse in those fed diet including L. digitata meal (Table 6). Regarding meat to shell ratio, the lowest value (2.6) resulted from feeding diet UGL, which produced the poorest growth performance, whereas there were no

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significant differences among the rest of the treatments (3.0-3.1) (Table 6).

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3.5. Effects of diets on the general nutrients and fatty acid composition of animals Nutritional analysis revealed that, except for moisture content, foot muscle composition of H. tuberculata coccinea was significantly (P<0.05) affected by the dietary treatments, whereas

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viscera composition did not differ significantly among the feeding regimes (Table 7). The

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percentage of protein deposited was significantly (P<0.05) highest in abalone fed diet containing P. palmata meal (UGP), followed by those fed fresh algae, and the lowest in those

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fed diet UGL. Overall, foot muscle contained much lower lipid levels (5-7%) than viscera (1920%), whereas the latter showed much lower protein content. Abalone fed the control diet showed significantly (P<0.05) lowest lipid levels stored in the foot muscle, the highest being found in those fed diet UGL. The carbohydrate content of the foot muscle was significantly

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lowest in animals fed diet UGP than that of abalone fed the rest of the feeding regimes. Ash content was significantly lower in animals fed fresh algae than that of abalone fed the formulated diets (Table 7). Fatty acid profiles of the H. tuberculata coccinea foot tissues are summarised in Table 8. The proportion of total saturated (41-49%), total monounsaturated (25-29%) and total polyunsaturated (22-29%) fatty acids were remarkably similar among all abalone tissues samples (both viscera and muscle). Palmitic acid (16:0) was the major fatty acid in all tissues (29-34%). Other abundant FA included 18:0.18:1n-9 and18:1n-7 (Table 8). Abalone fed the experimental diets showed accumulations of linoleic acid (LA 8-9%) in the foot muscle and elevated levels of its chain-elongation product 20:2n-6 (3%) compared with the tissues of abalone fed fresh macroalgae (3% LA and 0.2% 20:2n-6 respectively) (Table 8). In agreement with the dietary levels, foot tissues of abalone fed fresh algae presented remarkably higher levels of n-3 fatty acids, including 20:3n-3, 20:4n-3, 22:5n-3, and specially, eicosapentanoic acid (EPA) 20:5n-3 (3.2-5%) compared with those showed by abalone fed artificial diets (0.8-

ACCEPTED MANUSCRIPT 1.7%). Abalone fed all diets showed elevated levels of ARA (20:4n-6) relative to their feeds. For all treatments, the proportion of ARA was higher in the foot muscle than in the viscera, resulting in relatively higher ARA: EPA ratio in the foot, being also higher in abalones fed artificial diets relative to those fed the control onediet. Docosahexaenoic acid (DHA) (22:6n-3)

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was a minor FA (< 1%) in all tissue samples.

4. Discussion

Compound feeds have been reported to outperform fresh algae diets in abalone culture

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(Viana et al., 1993; Britz, 1996b; Corazani and Illanes, 1998; Bautista-Teruel and Millamena. 1999; Coote et al., 2000). In those studies, the low protein content and less balanced amino acid profile of the seaweed could not be sufficient to support abalone rapid growth, suggesting a high

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requirement of good quality protein in this species. Moreover, it has been suggested that this could be the reason for the long period of time to get abalone to marketable size (2-5 years) using macroalgae (Hahn, 1989; Robertson-Andersson, 2003; Johnston et al., 2005). However, in

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the present study, feeding the fresh algae resulted in maximum growth of abalone, indicating the

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high dietary value of the macroalgae reared in the IMTA. Indeed, the type of macroalgae consumed can significantly affect abalone growth by offering different proportions of their

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nutrients requirement. The good growth performance attained for these large abalones fed enriched seaweeds in the present study (169±15% WG) seems to be explained by the high protein content of the macroalgae produced under the high nitrogen culture conditions of the biofilter system (Boarder-Shpigel, 2001; Viera et al., 2005, 2011; Naidoo et al., 2006;

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Roberstson-Andersson et al., 2006, 2011). Furthermore, not only protein but also very high carbohydrates and low lipid levels, as well as energy content of the enriched seaweeds, matched abalone nutritional requirements (Fleming et al., 1996; Jackson et al., 2001; McBride et al., 2001; Sales and Janssens, 2004). Besides, the combination of both red and green algae species, each one with the typical biochemical composition of its phylum (Li et al., 2002; Dawczynski et al., 2007; Kinkerdale et al., 2010), may have a complementary effect to fulfil the micronutrient requirements of abalone, contributing to the good growth rates obtained. In fact, abalone fed mixed algal regimes, have been reported to perform significantly better than those fed with a single algal diet (Mercer et al., 1993; Dlaza, 2008; Naidoo et al., 2006; Roberstson-Andersson et al., 2011; Viera et al., 2011). Analysis of the proximate composition of the compound diets showed no differences in protein, lipid, gross energy values, protein:energy (PE) ratios and only a small difference in carbohydrates content, all of them being also within the levels recommended for several abalone species (Fleming et al., 1996; Jackson et al., 2001; McBride et al., 2001; Bansemer et al., 2014).

ACCEPTED MANUSCRIPT In comparing the artificial treatments, abalone growth was influenced by the type of seaweed meal incorporated into its diet. Animals fed the diet containing P. palmata as specific seaweed meal, performed better than the others and, particularly, those fed diets containing L. digitata. These results agree well with those obtained by Mercer et al. (1993) and Mai et al.

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(1995a, 1996) who reported the best performance on close European ormer, H. tuberculata fed

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P. palmata and the lowest on those fed L. digitata and L. sacharina. SGR values in this study

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(0.2-0.3%) were higher than in abalone H. roei fed various compound diets as reported by Boarder and Shpigel (2001), who found SGR values of 0.1 %, under similar experimental conditions. However, growth rates (21-32 μm day-1) were generally lower (21-56 μm day-1) than those obtain by Britz and Hetch (1997) in similar size H. midae fed with fish meal-based diets

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containing different proportions of protein and energy. Previous studies have shown that abalone fed formulated diets based on animal protein sources or a combination of plant and

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animal protein ones, yield better growth rates than those fed diets with protein sources of plant origin only (Britz, 1996b; Viana et al., 1996; Boarder and Shpigel, 2001; Bautista-Teruel, 2003). In agreement, Dlaza et al. (2008) reported the poorest performance (27 μm day-1) of postweaning abalone H. midae when fed an all-seaweed-based formulated feed compared to that

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recorded for those fed several fishmeal-based protein diets (46-61 μm day-1), claiming that

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seaweed proteins were less readily absorbed than animal based protein. Laminaria digitata including diet (UGL) was found to be water stable retaining at 73% of

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dry matter after the 17 h test for stability. However, diets UG and UGP leached a considerable proportion of their dry matter (34-42%), indicating low diet water stability, when compared with other compound diets (17-25-%, Jackson et al., 2001; 35-37%; Bautista- Teruel et al., 2003). This could be related to the higher moisture content of the former (19-20%) related to

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those previously tested (9-12%, Bautista-Teruel and Millamena, 1999; Jackson et al., 2001). Despite diet UGL showed the highest water stability, it also provided the lowest growth performance, whereas the one including P. palmata with the lowest water stability, produced the highest growth. Moreover, feed intake was also highest in abalone fed UGL, whereas protein deposition and the protein-related nutritional index (PER) were lowest. Thus, dietary inclusion of Laminaria digitata, despite did not negatively affected feed water stability, feed intake or dietary protein or essential aminoacid contents, significantly reduced dietary protein efficiency, suggesting a reduction in protein digestibility that could be partly responsible for the low growth obtained. Indeed, brown algae are reported to be digested more slowly than red algae for several abalone species (H. midae, Day and Cook, 1995; H. rubra, Foale and Day, 1992). Nevertheless, this growth and PER reduction could be also related to an specific leaching of dietary proteins even though dry matter leaching was low, as suggested by other authors (Viana et al., 1996; Edward and Cook, 1999; Jackson et al., 2001).

ACCEPTED MANUSCRIPT Animals fed fresh macroalgae ate significantly more than animals fed practical diets. This result could be due to the significantly lower PE ratio of the fresh algae related the artificial feeds, since feed intake is the main compensatory mechanism of herbivorous animals to satisfy energy requirement and being protein and carbohydrate, rather than lipid, the principal energy sources

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in abalone (Durazo-Beltrán et al., 2004; Viana et al., 2007). Moreover, the high moisture

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content of macroalgae results in reduced nutrient density, which may make it difficult for

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abalone to consume a sufficient amount to achieve a comparative nutrient intake (Bansemer et al., 2014). Additionally, previous investigations have demonstrated that dietary protein source may affect feed consumption in Haliotids (Uki and Watanabe, 1986; Viana et al., 1994), hence different attractiveness and palatability between fresh algae and artificial diets could have also

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affected consumption. The food conversion ratio (FCR) attained for the control treatment, agrees well with those obtained previously for this (H. tuberculata coccinea, Viera et al., 2005,

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2011) and other abalone species fed macroalgae (H. asinina, Kunavongdate et al., 1995; H. discus hannai and H. tuberculata, Shpigel et al., 1999). However, FCR (3-6) obtained for abalone fed formulated diets were higher than reported values (0.7- 1.8; Britz, 1996b; Shipton and Britz, 2001; Bautista-Teruel et al., 2003; García-Esquivel et al., 2007) probably due to the

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loss of some rasped pellets that could have broken up and drained out of the tank. Similar

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observations and even higher FCR results Similar and even higher FCR results (3-13) have been reported for H. asinina or H. midae fed formulated diets (Shipton, 2000; Reyes and Fermin,

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2003; Thongrod et al., 2003).

In the present study, PER was significantly better in abalone fed fresh algae, hence, suggesting a higher protein utilization efficiency. However, the meat to shell ratio (SB/S) of animals reared on fresh macroalgae was similar to those of animals fed diets UG and UGP,

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indicating that all of them produced healthy animals of high quality. Moreover, protein deposition being higher in animals fed UGP and similar in those fed UG, related to that obtained with fresh algae, further indicate the good protein quality of those diets, as tissue deposition cannot occur unless the requirements for essential amino acids are met (Durazo-Beltrán et al., 2003; Reyes and Fermin, 2003). Meat to shell ratio values (2.6-3.1) were similar and even higher than those recorded for Viera et al. (2011) for this specie (2.4-3.3) or by Mai et al. (1995a) for H. tuberculata (1.8-2.1) and H. discus hannai (2-2.4) fed with P. palmata and various levels of dietary lipids or by Sales et al. (2003) for H midae (2.9-3.2) fed different dietary crude protein level. Total lipid in the foot muscle were relatively low in agreement to their natural diets and to previous reports on both wild and cultured abalone (Nelson et al., 2002; Durazo-Beltrán, et al., 2004; Grubert et al., 2004). Abalone seem very efficient in assimilating lipids from their diets and, thus, diets with only 3-5% lipid have been recommended to promote high growth rate of abalone (Fleming et al., 1996; Bautista-Teruel et al., 1999; Shipton and Britz, 2001; Johnston et

ACCEPTED MANUSCRIPT al., 2005; Green et al., 2011).Viscera contained much higher lipid levels than muscle in agreement with previous studies (Webber, 1970; Mercer et al.,1993; Nelson et al., 2002 and Viera et al., 2011), and denoting the lipid storage function of the hepatopancreas/gonad assemblage (Uki et al., 1986b; Dunstan et al., 1996). In Haliotids, lipids are essential nutrients

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for growth and gonad maturation, but not a primary source of energy (Nelson et al., 2002).

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Moreover, muscle in the abalone foot is a major energy consumer due to daily movements and

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strong shell adhesion properties, free amino acids playing an important role in rapid energy production for shell adhesion, while the energy for slow locomotion mainly comes from carbohydrates (Mercer et al., 1993).

Overall, FA results for H. tuberculata coccinea in the present study were similar to those

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for the foot muscle of H. laevigata and H. rubra (Dunstan et al., 1996; Grubert et al., 2004), H. asinina (Jackson et al., 2001; Thongrod et al., 2003) and H. fulgens (Nelson et al., 2002;

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Durazo-Beltrán et al., 2004) fed macroalgae and /or artificial diets. In those studies, the main FA were 16:0, 18:0, 18:1n-7, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6, 20:5n-3 and 22:5n-3, and may reflect biochemical and dietary similarities of wild-caught representatives from Haliotidae. However, abalone fed fresh algae showed higher levels of n-3 fatty acids, specially, EPA 20:5n-

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3 in comparison to abalone fed formulated diets. The main PUFA in the formulated diets were

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n-6, while those in fresh algae were the more highly unsaturated n-3 PUFAs. Similarly, the ratio of n-3/n-6 was higher in the foot tissues of abalone fed fresh algae relative to those fed

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formulated diets, hence reflecting not only the composition of the diets (Uki et al., 1986b; Dunstan et al.,1996; Nelson et al., 2002) but also their highest nutritional value, since growth enhancement of H. tuberculata L. appeared to depend largely on n-3 PUFA, mainly 20:5n-3 and 18:3n-3 playing an important role in accelerating the growth of this species (Mai et al., 1996).

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In relation to the dietary levels, the elevated contents of 20:4n-6 in the abalone fed the experimental diets and 20:5n-3 in abalone fed the fresh algae, as well as their respective metabolites 20:2n-6, 20:3n-6, 20:4n-3, suggest that abalone have the ability to desaturate and elongate LA to ARA and ALA to EPA. This conversion of C18 PUFA to the C20 and C22 PUFAs they require, is present in many marine invertebrates including other Haliotis species, these herbivorous marine animals converting much more efficiently than carnivorous ones (Uki et al., 1986). Both control and formulated diets as well as all abalone tissues presented very low levels of DHA 22:6-n3 (Mai et al., 1996; Dawczynski et al., 2007; Viera et al., 2011; Courtois de Viçose et al., 2012), indicating that the composition of abalone is quite different to that of other marine animals, which have this fatty acid as one of the main tissue essential PUFA. Furthermore, it has been suggested that abalone are unusual compared with other marine animals in the importance of DPA rather than DHA (Viana et al., 1993; Dunstan et al., 1996). In summary, this study has shown that feeding H. tuberculata coccinea with seaweeds based diets resulted in high survival and good dietary protein utilization. The inclusión of P.

ACCEPTED MANUSCRIPT palmata as dietary ingredient is recommended to improve growth, soft body to shell ratio and dietary protein utilization. On the contrary, L. digitata should be avoided, at least in the level tested in the present study, since markedly reduced growth performance and increased feed conversion ratio, reducing the efficiency of dietary protein. Further studies are required to

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improve the growth rate obtained with these all vegetable diets, especially concerning the use of

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different seaweed combinations and inclusion levels, as well as the diet processing methods to

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improve stability.

Acknowledgements

The authors especially thank Dr. J.L. Gómez-Pinchetti from Centro de Biotecnología

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Marina, A. Fitzgerald, from South West Abalone Growers Association, Dr. A. Soler from Martin Ryan Institute and Dr. S. Huchette from France Haliotis, who kindly supplied the

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macroalgae experimental meals. This study has been financed by the FP 7-SME-2007-1/BSGSME. Project Sustainable Development of European SMEs engaged in Abalone Aquaculture –

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(SUDEVAB).

ACCEPTED MANUSCRIPT 5. References Allen, V.J., Marsden, I.D., Ragg, N.L.C., Gieseg, S., 2006. The effect of tactile stimulants on feeding, growth, behaviour, and meat quality of cultured blackfoot abalone, Haliotis

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iris. Aquaculture 257. 294-308.

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AOAC, 2005. Official Methods of Analysis of the Association of Analytical Chemistry. Washington, DC 1018 pp.

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Bansemer, M, S., Qin, J.G., Harris, J.O., Howarth, G.S., Stone, D.A.J., 2014. Nutritional requirements and use of macroalgae as ingredients in abalone feed. Reviews in Aquaculture 5, 1-15.

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Bautista – Teruel, M.N., Millamena, O.M., 1999. Diet development and evaluation for juvenile abalone, Haliotis asinina: protein/energy levels. Aquaculture 178, 117-126. Bautista-Teruel, M.N., Fermin, A.C., Koshio, S.S., 2003.Diet development and evaluation for

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juvenile abalone, Haliotis asinina: animal and plant protein sources. Aquaculture 219, 645-653.

Boarder, S.J., Shpigel, M., 2001. Comparative performances of juvenile Haliotis roei fed on

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enriched Ulva rigida and various artificial diets. J. of Shellfish Res. 20 (2), 653-657.

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Britz, P.J., 1996a. Effect of dietary protein level on growth performance of South African abalone, Haliotis midae, fed fishmeal-based semi-purified diets. Aquaculture 140, 55-

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Britz, P.J., 1996b. The suitability of selected protein sources for inclusion in formulated diets for the South African abalone, Haliotis midae Aquaculture 140, 63-73 Britz S.J., Hecht, T., 1997. Effect of dietary protein and energy levels on growth and body

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composition of South African abalone, Haliotis midae. Aquaculture 156, 195-210 Chiou, T.K., Lai, M.M., 2002. Comparison of taste components in cooked meats of small abalone fed different diets. Fish. Sci. 68, 388-394. Cho, C.Y., Slinger, S.J., Bayley, H.S., 1982. Bioenergetics of salmonid fishes: energy intake, expenditure and productivity. Comp. Biochem. Physiol. 73B, 25-41. Christie, W. W., 1982. Lipids Analysis. Christie, W. W. (Ed.). Pergamon Press, Oxford, p 1723, 51-61. Coote, T.A., Hone, P.W., van Barneveld R.J., Maguire, G.B., 2000. Optimal protein level in semi-purified diet for juvenile Haliotis laevigata. Aquaculture Nutrition 6, 213-220. Corazani, D., Illanes, J.E., 1998. Growth of juvenile abalone, Haliotis discus hannai Ino 1953 and Haliotis rufescens Swainson 1822, fed with different diets. J. Shellfish Res. 17 (3), 663-666.

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tissue of green abalone (Haliotis fulgens). Aquaculture 224, 257–270. Durazo-Beltrán E., Viana, M.T., D´Abramo L.R., Toro-Váquez J.F., 2004. Effect of starvation

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and dietary lipid on the lipid and fatty acid composition of muscle tissue of juvenile green abalone (Haliotis fulgens). Aquaculture 238, 329-341. Edwards, S., Cook, K., 1999. Rapid leaching of minerals from abalone dietary binders is not determined by ionic mobility and competition between cations. Dry matter loss is not a

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without fishmeal compared to a commercial diet. Aquaculture 165, 321-331. Hahn, K.O., 1989. Handbook of culture of abalone and other marine gastropods. CRC Press,

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Izquierdo, M. S., Watanabe, T., Takeuchi, T., Arakawa, Kitajima, C., 1989. Requirements of

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system of juvenile blacklip abalone (Haliotis rubra): Potential factors responsible for variable weaning success on artificial diets. Aquaculture 250, 341-355. Kemp, J.O.G., Britz P.J., 2012. Abalone nutrition from an aquaculture perspective: a review. Proceedings of the 8th International Abalone Symposium, May 2012, Hobart, Tasmania. pp 62. Kirkendale, L., Robertson-Andersson D.V., Winberg, P.C., 2010. Review on the use and production of algae and manufacturated diets as feed for sea-based abalone aquaculture in Victoria. Report by the University of Wollongon, Shoalhaven Marine and Freshwater Centre, Nowra, for the Department of Primary Industries, Fisheries Victoria, 198 p. Koike, Y., Flassch, J., Mazurier, J., 1979. Biological and ecological studies on the propagation of the ormer, Haliotis tuberculata Linnaeus. II: Influence of food and density on the growth of juveniles. La Mer (Bulletin de la Société Franco-Japonaise d'Océanog raphie) 17 (1), 43-52.

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Lopez, L.M., Viana, M.T., 1995. Determination of the quality of food elaborated from unheated

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protein level for growth. Aquaculture 136, 165-180. Mai, K., Mercer, J.P., Donlon J., 1996. Comparative studies on the nutrition of two species of abalone, Haliotis tuberculata L. and Haliotis discus hannai Ino. V. The role of polyunsaturated fatty acids of macroalgae in abalone nutrition. Aquaculture 139, 77-89.

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of the European Abalone Haliotis tuberculata Linnaeus 1758 (Gastropoda: Haliotidae). Biology and Environment: Proceedings of the Royal Irish Academy, Vol. 94B, No. 3. 285-304.

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2006. Can kelp extract (Kelpac) be useful in seaweed mariculture?. Journal of Applied

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update. Nutrition Abstracts and Reviews Series B74: 13N-21N.

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sources in diets for the South African abalone Haliotis midae L. J. of Shellfish Research, 20, (2), 637-645. Shpigel, M., Ragg, N.C., Lupatsch, I., Neori, A., 1999. Protein content determines the nutritional value of the seaweed Ulva lactuca for the abalone Haliotis tuberculata and H. discus hannai. J. Shellfish Res. 18, 227-223. SUDEVAB, 2007. Sustainable Development of European SMEs engaged in Abalone Aquaculture – SUDEVAB. FP 7-SME-2007-1/BSG-SME. Thongrod, S., Tamtin, M., Boonyaratpalin, M., 2003.Lipid to carbohydrate ratio in donkey´s ear abalone (Haliotis asinina, Linne) diets.Aquaculture 225, 165-174. Troell, M., Robertson-Andersson, D., Anderson, R.J., Bolton, J.J., Maneveldt, G., Halling C., Probyn, T., 2006. Abalone farming in South Africa: An overview with perspective on kelp resources, abalone feed, potential for on-farm seaweed production and socioeconomic importance. Aquaculture 257, 266-281.

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Scientific Fisheries 51(11), 1835-1839.

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Uki, N., Watanabe, T., 1986. Effect of heat-treatment of dietary protein sources on their protein

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quality for abalone. Bulletin of the Japanese Society of Scientific Fisheries 52(7), 11991204.

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Vandepeer, M. E., Hone, P.W., Van Barneveld, R.J., Havenhand, J.N., 1999. The utility of apparent digestibility coefficients for predicting comparative diet growth performance

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ACCEPTED MANUSCRIPT Walsh, M., Watson, L., 2011. A Market Analysis towards the Further Development of Seaweed Aquaculture in Ireland. Irish Sea Fisheries Board. Webber, H.H., 1970. Changes in metabolite composition during the reproductive cycle of the abalone Haliotis cracheroidii (Gastropoda: Prosobranchiata). Phisiol. 2, 213-231.

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World Wildlife Fund, Inc., 2010. Abalone Aquaculture Dialogue Standards.

ACCEPTED MANUSCRIPT Table 1 Proximate (% DW) and amino acid composition (% protein) of seaweed meals used in

U. lactuca

L. digitata

20.3

17.0

8.4

15.0

2.6

3.5

2.5

36.3

57.4

IP

2.8

65.7

67.5

Ash

40.7

22.1

23.6

14.7

Moisture

10.1

4.2

13.2

11.2

Alanine

5.8

3.7

6.1

Arginine

4.4

6.9

4.8

5.8

Aspartic

9.1

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11.5

5.6

13.2

10.5

Cystine

0.0

0.0

17.0

0.7

Glutamic

8.8

7.1

11.0

10.5

4.7

3.2

4.6

5.3

1.8

0.7

3.1

1.8

2.8

2.4

3.8

3.7

4.6

3.8

6.2

5.7

3.8

1.9

4.8

4.9

1.3

1.2

2.1

2.0

Phenylalanine

3.5

0.5

3.8

3.9

Proline

3.4

2.3

4.3

4.2

3.9

2.8

4.2

4.4

Threonine

3.6

2.4

4.4

3.9

Tryptophan

0.8

0.4

1.7

2.5

Tyrosine

2.2

1.6

3.0

3.5

Valine

4.2

3.1

4.9

5.3

Crude lipids Carbohydrates

*

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Crude protein

D

Histidine

Leucine Lysine

*

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Methionine

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Isoleucine

Serine

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Amino acids

Glycine

Calculated by difference (AOAC, 2005)

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G. cornea

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experimental feeds for abalone H. tuberculata coccinea P. palmata

ACCEPTED MANUSCRIPT Table 2 Ingredients composition (% DW) of the three experimental diets for abalone H. tuberculata

IP

T

coccinea

Experimental diets1

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Ingredients

UGL

UGP

16

16

15

15

12

-

-

12

2

2

22

22

22

18

18

18

4

4

4

2.5

2.5

2.5

1.5

1.5

1.5

1.35

1.35

1.35

0.65

0.65

0.65

5

5

5

G. cornea

16

U. lactuca

27

L. digitata

-

P. palmata

2

MA

Spirulina

2

Soybean meal3 4

Corn gluten 5

Starch

D

6

Vitamin mix

6

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Mineral mix 7

Lys

7

1

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Met

Na alginate

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UG

UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca + G. cornea + P. palmata. 50.8% protein, 9.2% lipid

3

48.2% protein, 3.6% lipid

4

78.3% protein, 6.3% lipid

5

8.4% protein, 4.7% lipid

6

Uki et al.. 1985a

7

Sigma (UK)

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2

ACCEPTED MANUSCRIPT Table 3 Proximate analysis of the fresh algae or experimental diets containing different algal species (% DW)

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Experimental diets2

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Fresh algae1

UGL

UGP

34.9

35.1

35.4

4

4.1

3.9

36.4

37.7

40.3

27.6±7.1

SC R

UG

24.7

23.1

20.4

83.3±3.0

20.6

16.2

19.3

GE (MJ kg )

14.9±1.8

15.9

16.3

16.7

Protein : energy ratio (mg kg-1) 5

13.9±2.5

20.9

21.5

22.3

91.9

65.7

72.7

57.8

Crude protein

21.0±5.8

Crude lipid Carbohydrates

5.3±1.5 3

46.0±3.5

NU

Ash Moisture

MA

-1 4

% Water stability6 1

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Fresh algae used: G. cornea and U. rigida reared in Grupo de Investigación en Acuicultura (GIA) integrated fish-seaweed culture system (n=4) 2 UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca + G. cornea + P. palmata. 3 Calculated by difference (AOAC, 2005) 4 Calculated gross energy (Cho et al., 1982). 5 Calculated metabolisable energy (Cho et al., 1982) 6 Water stability of fresh algae was calculated for a 3- days period and of pellets for 17 h of immersion (16:00 - 9:00 h)

ACCEPTED MANUSCRIPT Table 4 Fatty acid composition (% total fatty acids) of the fresh algae or experimental diets containing different algal species *

T

Experimental diets**

Fresh algae*

UGL

2.6

0.9

1.0

16:0

40.5

22.8

17:0

0.9

0.2

0.9

2.8

∑SFA*** 14:1n-5

44.9

26.6

2.2

0.3

16:1n-7

9.0

1.3

16:1n-5

0.6

18:1n-9

3.4

18:1n-7

6.6

20:1n-9

0.2

∑MUFA**** 16:2n-4

16:4n-3

2.7 27.5

0.2

0.2

1.1

1.0

0.1

0.1

0.2

0.3

12.7

12.6

2.9

1.8

2.0

0.5

0.5

0.5

22.0

17.8

16.6

16.5

0.8

0.2

0.1

0.2

1.5

-

-

-

-

0.6

0.3

0.4

MA

0.3

D

16:3n-1

2.3

25.0

21.5

12.5

TE

16:3n-3

1.3

23.4

NU

18:0

UGP

SC R

UG 14:0

IP

FA

0.6

0.1

-

0.1

2.7

44.9

46.3

45.6

18:2n-4

0.8

1.5

1.5

1.6

18:3n-3

3.7

4.0

4.0

3.9

18:4n-3

3.0

0.7

1.0

0.5

20:4n-6

1.2

0.1

0.2

0.1

20:3n-3

13.5

0.8

1.8

0.7

20:5n-3

2.4

0.5

1.1

0.7

22:5n-3

1.0

0.2

0.1

0.1

22:6n-3

0.3

0.6

0.3

0.5

∑PUFA***** Others******

31.5 1.6

54.2 1.4

56.7 1.7

54.3 1.7

∑n-3

25.9

6.9

8.3

6.4

∑n-6

3.9

45.0

46.5

45.8

n-3/n-6

6.7

0.2

0.2

0.1

AC

CE P

18:2n-6

*Fresh algae: G. cornea and U. rigida reared in GIA-IMTA; **UG: U. lactuca + G. cornea; UGL: U. lactuca+ G. cornea + L. digitata; UGP: U. lactuca+ G. cornea + P. palmata. ***SFA, saturated fatty acids. ****MUFA, monounsaturated fatty acids. *****PUFA, polyunsaturated fatty acids. ******Other includes all components <0.5%:15:0,16:(2n-6), 18:(1n-5), 20:0, 20:(2n-6), 20:(4n-3), 22:(1n-9). - not detectable.

ACCEPTED MANUSCRIPT

Table 5

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Survival and growth performance of abalone H. tuberculata coccinea fed algae (fresh algae) or experimental diets containing different algal species for 6

Final shell length (mm)

Fresh algae

97.8±3.8

33.1±0.1

44.2±0.9a

UG

98.3±2.4

33.1±0.1

37.9±0.1bA

UGL

98.3±2.4

33.1±0.0

36.9±0.4bB

UGP

95.0±2.4

33.1±0.1

38.7±0.1bA

Shell growth rate (μm d-1)

Initial weight (g)

Final weight (g)

SGR (% d-1)

Weight gain (%)

62.8±4.9a

4.7±0.2

12.6±0.8a

0.56±0.0a

168.8±15.4a

27.3±0.8bB

4.7±0.2

7.8±0.1bB

0.27±0.0bcAB

61.6±6.2bAB

21.1±1.5bC

4.7±0.2

6.9±0.1bC

0.22±0.0cB

48.4±0.8bB

31.9±0.7bA

4.7±0.0

8.5±0.1bA

0.34±0.1bA

83.8±7.2bA

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Initial shell length (mm)

MA N

Survival (%)

TE D

Treatment

CR

months * (Mean ± S.D.)

UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca + G. cornea + P. palmata

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formulated feeds, P< 0.05.

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* Low case letters indicate significant differences among feed treatments including fresh algae, whereas upper case letters indicate differences only among

ACCEPTED MANUSCRIPT

Table 6

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Consumption, feed efficiency and meat to shell ratio of abalone H. tuberculata coccinea fed algae (fresh algae) or experimental diets containing different algal

Feed intake (mg ind-1d-1) 86.4±4.1a

UG

68.2±0.9bB

UGL

73.9±0.0bA

UGP

65.2±2.5bB

TE D

Fresh algae

FCR

PER

SB/S

2±0.2c

2.4±0.2a

3.1±0.3a

4.1±0.4bB

0.7±0.1bAB

3.1±0.1aA

6.0±0.1aA

0.5±0.0bB

2.6±0.2bB

3.2±0.4bB

0.9±0.1bA

3.0±0.1aA

MA N

Treatment

US

CR

species for 6 months * (Mean ± S.D.)

CE P

Fresh algae: G. cornea and U. rigida reared in GIA-IMTA UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca + G. cornea + P. palmata.

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* Low case letters indicate significant differences among feed treatments including fresh algae, whereas upper case letters indicate differences only among formulated feeds, P< 0.05.

ACCEPTED MANUSCRIPT

Table 7

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Proximate composition of viscera and muscle of Haliotis tuberculata coccinea fed for 6 months algae (fresh algae) or experimental diets containing different

Moisture

Crude protein

Muscle

Viscera

Muscle

Fresh algae

70.0±0.9

71.9±0.8

57±1.4

74.6±0.2b

UG

70.2±0.1

73.1±0.4

56.8±2.4

73.7±0.5bc

UGL

72.9±0.6

72.8±0.1

57.1±2.2

UGP

71.1±2.2

72.4±0.9

58.3±2

Carbohydrate

Muscle

Viscera

Muscle

Viscera

Muscle

19.2±1.1

4.6±0.1c

15.8±1.2

15.1±0.7a

8±0.9

5.7±0.5b

19.8±0.0

6.2±0.0ab

15.3±2

13±0.2a

8.2±0.4

7±0.2a

72.3±0.6c

20.2±1.5

6.8±0.3a

12.9±2.8

14.5±0.8a

9.9±1.1

6.4±0.1ab

80.7±0.1a

20.4±2.4

5.7±0.2b

12.3±2.8

6.8±0.3b

9±1.7

6.7±0.1ab

CE P

Fresh algae: G. cornea and U. rigida reared in Grupo de Investigación en Acuicultura (GIA) - IMTA UG: U. lactuca + G. cornea; UGL: U. lactuca + G. cornea + L. digitata; UGP: U. lactuca + G. cornea + P. palmata.

AC

Ash

Viscera

TE D

Viscera

Crude lipid

MA N

Diets

US

CR

algal species*(g/100 g DW) (Mean ± S.D.) (Values in the same column with different letters are significantly different P< 0.05)

ACCEPTED MANUSCRIPT Table 8 Fatty acid composition (% total fatty acids) of the abalone tissues of Haliotis tuberculata coccinea fed

T

fresh algae or experimental diets containing different algal species for 6 months

Fresh algae*

UG

UGL

Viscera 4.6 0.5 32.3 4.0 3.2

∑SFA*** 16:1n-7 18:1n-9 18:1n-7 20:1n-9+n-7 20:1n-5 22:1n-11 22:1n-9

44.8 2.8 10.1 11.8 1.2 0.5 1.2 0.1

48.1 1.5 7.5 10.3 4.2 0.5 0.3 0.4

44.6 2.1 15.5 8.6 1.9 0.3 0.2 0.1

∑MUFA**** 18:2n-6 18:3n-3 18:4n-3 20:2n-9 20:2n-6 20:3n-6 20:4n-6 20:3n-3 20:4n-3 20:5n-3 22:4n-6 22:5n-3 22:6n-3 ∑PUFA***** Other****** ∑n-3 ∑n-6 n-3/n-6 ARA/EPA

27.6 2.7 4.6 2.2 0.3 0.6 0.3 5.2 0.6 1.3 5.1 0.2 3.1 0.5 26.8 0.8 17.4 9.1 1.92 1.0

24.8 3.3 2.9 1.4 0.1 0.2 0.1 8.2 0.1 0.3 3.2 0.3 4.6 0.2 24.9 2.3 12.6 12.1 1.04 2.6

D

28.8 13.0 1.0 0.6 5.0 2.8 0.2 1.0 1.3 0.6 25.9 0.7 2.8 22.6 0.12 2.8

TE

CE P

AC

Muscle 2.6 0.9 30.9 10.7 -

Viscera 4.1 30.1 4.1 3.2

45.1 1.7 11.6 5.8 3.2 1.8 0.4 0.5

25.0 9.2 0.8 0.1 3.3 0.5 8.1 0.2 0.1 1.7 1.4 2.3 0.6 28.4 1.5 5.8 22.6 0.26 4.8

NU

Muscle 2.0 1.7 34.4 10.0 -

MA

Viscera 5.3 0.9 31.0 3.6 4.0

SC R

FA 14:0 15:0 16:0 18:0 20:0

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Experimental diets**

UGP

Muscle 3.1 0.9 32.1 11.0 -

Viscera 4.7 29.5 4.6 3.0

Muscle 3.2 1.1 33.7 10.6 -

41.5 1.7 15.1 7.4 2.5 0.2 0.4 0.2

47.1 2.0 12.5 5.6 3.1 1.9 0.5 0.5

41.8 1.8 14.1 7.2 2.5 0.3 1.2 0.3

48.6 1.6 11.6 6.5 3.6 1.6 0.6 0.6

27.6 16.4 1.1 0.1 0.8 4.1 0.6 3.5 0.1 1.2 1.0 0.4 29.2 1.7 2.8 25.6 0.11 3.1

26.2 8.5 0.7 0.1 2.8 0.3 6.7 0.1 0.1 1.6 1.2 1.7 0.5 24.0 2.7 4.9 19.1 0.25 4.1

27.5 15.7 1.0 0.1 07 4.1 0.6 3.2 0.1 0.1 1.6 0.9 1.1 0.2 29.4 1.4 4.2 24.5 0.17 2.0

26.1 8.6 0.6 0.1 2.7 0.4 6.2 0.1 0.8 1.1 1.2 0.2 21.6 3.7 3.0 18.6 0.16 7.9

*Fresh algae: G. cornea and U. rigida reared in GIA-IMTA; **UG: U. lactuca + G. cornea; UGL: U. lactuca+ G. cornea + L. digitata; UGP: U. lactuca+ G. cornea + P. palmata. ***SFA, saturated fatty acids. ****MUFA, monounsaturated fatty acids. *****PUFA, polyunsaturated fatty acids. ******Other includes all components <0.5%: 14(1n-7), 14(1n-5), 16:0ISO, 16:(1n-5), 16:(2n-6), 17:00, 16:(3n-4), 16:(3n-3), 18:(1n-5), 18:(3n-6), 18:(3n-4), 20:(1n-5). – not detectable.

ACCEPTED MANUSCRIPT Statement of Relevance

AC

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MA

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Development of vegetable based diets for abalone.

ACCEPTED MANUSCRIPT Highlights  Feeding the fresh algae produced far better growth for H. tuberculata coccinea than all the compound diets, indicating the high dietary value of the macroalgae reared in the IMTA system.

T

 The inclusion of P. palmata was found to improve growth, condition index and

IP

dietary protein utilization, while the use of L. digitata markedly reduced the 

SC R

efficiency of dietary protein.

The elevated contents, relative to their feeds, of ARA in the abalone fed the experimental diets and EPA in abalone fed the fresh algae, denoted the presence of the respective elongases and Δ5 desaturases. However, the low content of DHA



NU

further suggested that this fatty acid is not essential in abalone tissues. Overall, feeding H. tuberculata coccinea with vegetable-based artificial diets

AC

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resulted in high survival and good dietary protein utilization.