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Aquaculture 275 (2008) 266 273 www.elsevier.com/locate/aqua-online

Growth, feed efficiency and condition of common octopus (Octopus vulgaris) fed on two formulated moist diets
Jess Cerezo Valverde , Mara Dolores Hernndez, Felipe Aguado-Gimnez, Benjamn Garca Garca
IMIDA-Acuicultura, Consejera de Agricultura y Agua de la Regin de Murcia, Puerto de San Pedro del Pinatar, Apdo. 65, 30740 San Pedro del Pinatar, Murcia, Spain Received 28 November 2007; received in revised form 10 January 2008; accepted 10 January 2008

Abstract In the present study, the growth, feed efficiency and condition of Octopus vulgaris (0.58 to 1.02 kg; 17 to 21 C) fed two formulated moist diets based on fish and prawn mixed with alginate (group A) or gelatin (group G) as binders, and a control diet based on a crab diet (group C) are compared. The animals showed a suitable degree of acceptance of both formulated diets, grew and produced faeces, with 100% survival in all groups. The best results were obtained with group C, where the animals showed a specific growth rate, specific feeding rate and feed efficiency of 1.61% BW/day, 4.67% BW/day and 33.95%, respectively. They were followed by the animals of group A (0.71% BW/day, 3.10% BW/day and 22.51%) and group G (0.22% BW/day, 1.31% BW/day and 16.06%). No significant difference was observed between groups C and A with regard to feed efficiency or protein productive value (PPV values of 49.01 and 28.49%, respectively), although differences were significant (P b 0.05) between these groups for lipid productive value (23.65 and 1.59%, respectively). The animals of group C showed the highest digestive gland index and greater lipid content in this organ (15.01%) compared with groups A (6.41%) and G (1.27%), although there were no difference in protein or mineral content. A similar pattern was observed for overall body composition. The results suggest that the differences in growth could have been due to lower feed intake of animals fed either formulated diet and indicates the importance of including taste enhancers. The excellent use of the lipids obtained from the crab emphasises the importance of both the quantity and type of lipid supplied. The stability of these diets in water, their texture, nutritive composition and manufacture are discussed. Other formulated feeds with known nutritional composition could be developed based on the format proposed in this work to improve our knowledge of the nutritional requirements of octopus. 2008 Elsevier B.V. All rights reserved.
Keywords: Formulated diet; Growth; Nutrition; Feed efficiency; Condition; Octopus vulgaris

1. Introduction The industrial development of common octopus (Octopus vulgaris) rearing is limited by the impossibility of obtaining benthic juveniles on a commercial scale in captivity (Navarro and Villanueva, 2000, 2003; Iglesias et al., 2004, 2007), and the lack of formulated feeds of appropriate nutritional composition that are acceptable to the animals (Lee, 1994; Vaz-Pires et al., 2004). At the moment, the production system is based on the capture of subadults weighing 700 to 800 g in the wild. These
Corresponding author. Tel./fax: +34 968184518. E-mail address: jesus.cerezo@carm.es (J. Cerezo Valverde). 0044-8486/$ - see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2008.01.012

are then reared in different types of cages, some suspended and others self-floating, and fed different kinds of, otherwise undesirable, low cost species from trawls, to reach a markable weight of 3 to 5 kg after 3 to 4 months (Chapela et al., 2006; Rodrguez et al., 2006). However, the profitability of this activity is low at present (Garca Garca et al., 2004). In any case, the successful commercial development of the ongrowing of any species depends on a formulated diet because of the enormous advantages that such a diet will have over natural diets (O'Dor and Wells, 1987; Lee, 1994). Although various formulated diets have been tried experimentally for feeding subadult cephalopods, including cuttlefish and octopus, the results have always been inferior to those

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obtained with natural diets. In most studies, growth has been negative or zero (Lee et al., 1991; Domngues et al., 2007) and, even when significant, always below that achieved with natural diets (Castro et al., 1993; Castro and Lee, 1994; Domngues et al., 2005). This has often been attributed to the lack of palatability of the diets and, if ingested at all, to their deficient nutritional composition. In this sense, the levels of copper (Castro et al., 1993; Villanueva and Bustamante, 2006), quantity and type of lipid (Navarro and Villanueva, 2000, 2003; Koueta et al., 2002; Garca Garca and Aguado, 2002; Moltschaniwskyj and Johnston, 2006; Petza et al., 2006), and balance of amino acids (Lee, 1994; Villanueva et al., 2004; Domngues et al., 2005; Garca Garca and Cerezo Valverde, 2006) should be borne in mind when designing a feed for cephalopods. Whatever the composition and balance in the diet produced, it will be of no benefit if the animals find it unacceptable. Both the moisture content and the chemical agents used to make the diet more palatable could influence the degree to which it will be found acceptable and ingested (Lee et al., 1991; Domngues et al., 2007). Indeed, the chemoreceptive capacity of octopus is well developed and has been demonstrated in assays involving acids, sugars, salts, crab extracts, amino acids and nucleotides (Wells, 1963; Wells et al., 1965; Boyle, 1983; Chase and Wells, 1986; Lee, 1992). Therefore, the first problem in designing a feed for common octopus is its presentation so that the animal finds it acceptable and ingests it completely (Lee, 1994; Garca Garca and Cerezo Valverde, 2006). Granulated, extruded and semimoist feeds such as those designed for fish are not suitable for common octopus since they disintegrate before they are consumed when manipulated by the animal. From our previous experience using different mixtures and binders and information available in the literature (Guerra, 1978; Lee et al., 1991; Castro et al., 1993), any formulated feed for octopus must have particular physical characteristics and be suited to the animal's method feeding. For example, the feed is often not consumed immediately after being supplied so that it must remain stable for long periods in the water. On the other hand, the animal sometimes cuts the feed into pieces immediately with its jaw and swallows it (Altman and Nixon, 1970; Guerra, 1978), although an external pre-digestion stage cannot be excluded (Nixon, 1984). Any feed, therefore, must be stable and have a firm but not granular texture; the particles of the components must be small and cohesive. The feed must also be attractive because octopuses are quite capable of not accepting it even though it may have spent several days without food. We have recently achieved a gummy texture by using mixtures of several binders including alginate or gelatin with triturated fish or prawn. The growth, feed efficiency and condition of common octopus fed on formulated diets based on these binders, and a control diet based on crab, are compared in the present work.
2. Materials and methods 2.1. Experimental animals and maintenance
The octopus (O. vulgaris) were caught at sea (Murcia, S.E. Spain) and kept in 2000 l tanks in the laboratory. The experiment began when the animals

had acclimated (2 weeks) and were observed to be feeding on the appropriate amount of feed calculated according to Aguado and Garca Garca (2002) and Garca Garca and Cerezo Valverde (2006). Defrozen bogue (Boops boops) and crab (Carcinus mediterranus) were supplied on alternate days prior to starting the feeding trial. For the feeding trial, 14 octopus weighing 581 to 1018 g were placed in individual 216 l tanks, containing PVC tubes as shelters and connected to a recirculating seawater system. The photoperiod was 12L/12D, the water temperature varied between 17 and 21 C (mean 18.9 1.2 C), which is within the optimal range of temperatures for this species (Aguado and Garca Garca, 2002), while salinity remained constant at 37 and dissolved oxygen was maintained at above 90% saturation, so that this factor was not limiting (Cerezo and Garca Garca, 2005).

2.2. Preparation and water stability of the formulated diets

Two formulated diets were prepared using gelatin (feed G) or alginate (feed A) as binders (Table 1). For the first diet, gelatin was dissolved in cold distilled water using a magnetic stirrer. Fish (B. boops) and prawns (Hymenopenaeus muelleri), previously cleaned of bones or shell, were triturated in a blender and the resultant mixture was added to the gelatin solution and heated in a water bath to 80 C with occasional stirring. The mixture was poured into an aluminium mould and placed in a refrigerator for 24 h at 4 C to gel. To prepare diet A, a puree of alginate and the fish/prawn mixture was made with a blender. A calcium solution was then added to help harden the resultant mixture, which was then treated in the same way as the first feed (refrigerated for 24 h at 4 C). Finally both diets were cut into 3 cm cubes and kept in the freezer until use. The water stability of both feeds was determined by weighing 5 samples of each and then soaked in water for 4 and 24 h. With the data obtained, the mean values of the following indices were calculated: VW (%) = (Wf Wi) / Wi 100, which expresses the variation in weight of the diet after soaking in water, where Wi and Wf are the initial and final weights, respectively. F = Wi / Wf, which represents a correction factor. The weight of uneaten diet was multiplied by this correction factor to account for water absorption or disintegrating in order to estimate the change in weight.

2.3. Experimental design

The experimental diets were assayed in a feeding trial lasting 4 weeks (May 2007): group G (gelatin, N = 5) and group A (alginate, N = 5). A third diet based on crab served as control (group C, N = 4). All diets were fed ad libitum at 09.00 h (6 days per week) and the uneaten food was collected at 13.0013.30 h in the case of both formulated diets and the following morning in the case of diet C because of the greater time required to extract the crab meat. The uneaten food was dried with absorbent paper and weighed to calculate daily intake.

2.4. Analytical methods

The proximate composition (moisture, crude protein, crude fat, ash and nitrogen-free extract) was analysed in five octopuses of similar weight (initial control) to those used in the experiment and in all the octopuses at the end of the experiment. To do this, animals excluding digestive gland (EDG) and the digestive gland (DG) were ground and mixed separately three times

Table 1 Composition in wet weight (%) of the experimental diets based on gelatin (G) or alginate (A) as binders Diet/ingredient Gelatin a Water Alginate b Calcium c Bogue d Prawn d G A 10 40 30 10 40 50 10 10

Supplied by Productos Sur, S.A. (Pol. Ind. Oeste, San Gins, Murcia). a Gelatin Bloom 220. b Puree of alginate contains 62.5 g of POKEL MERL in 1 l of distilled water. c Calcium solution contains 1 g of POKEL CALS in 10 ml distilled water. d Edible portion.

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J. Cerezo Valverde et al. / Aquaculture 275 (2008) 266273 Table 3 Composition of each diet (percent wet substance) Diet a Moisture Crude Crude Ash NFE b Gross energy P/E c (%) protein (%) lipid (%) (%) (%) (kJ/100 g) (g/MJ) G A C
a b c

consecutively until a homogenous sample was obtained. Subsequently, EDG and DG analyses were carried out. Total body proximate composition (TBC) was calculated from EDG and DG values according to the following equation: TBC (%) = (EDGW (EDG / 100) + DGW (DG / 100)) / (BW) 100, where TBC, EDG and DG are the percentage of macronutrients in the whole animal, the animal excluding the digestive gland and the digestive gland, respectively; BW, EDGW, and DGW are the weights of the different fractions, respectively. The composition of the formulated feeds and edible portion of crab was also analysed. A 1 g sample (in the case of moisture, crude protein and ash) or 2 g sample (in the case of crude lipid) was used for the analyses, which were carried out in triplicate. Crude protein was determined by the Kjeldhal method using a conversion factor of 6.25. The crude lipid was obtained by ether extraction (SOXTEC System-HTC). Moisture was obtained by drying (105 1 C, 24 h) to constant weight in a drying chamber (KOWELL, D2 NOVA), and ash by incineration (loss in weight: at 450 1 C, 24 h) in a MUFLA oven (HOBERSAL, HD230). Gross energy and the protein energy ratio (P/E in g/MJ) were estimated using the Miglavs and Jobling (1989) energy coefficients: protein 23.6 kJ/g, lipid 38.9 kJ/g and carbohydrate 16.7 kJ/g.

71.33 74.28 80.94

21.29 11.31 11.92

6.46 9.47 2.49

0.80 0.12 2.62 2.32 3.43 1.22

756 674 399

28.17 16.78 29.91

G = gelatin; A = alginate; C = crab. Nitrogen-free extract. P/E = protein/energy ratio.

2.5. Parameters calculated and data analysis

All samples were weighed at the beginning and end of the feeding trial. The following indices were calculated: Weight gain: WI = Wf Wi; Absolute feeding rate: AFR = IF / t; Absolute protein feeding rate: APFR = IP / t; Absolute lipid feeding rate: ALFR = IL / t; Specific feeding rate: SFR = AFR100 / Wa; Absolute growth rate: AGR = (Wf Wi) / t; Specific growth rate: SGR = (LnWf LnWi)100 / t; Feed efficiency: FE = (Wf Wi)100 / IF; Feed conversion ratio: FCR = IF / (Wf Wi); Protein productive value: PPV = 100(Retained protein / IP); Lipid productive value: LPV = 100 (Retained lipid / IL); Digestive gland index: DGI = (DGW / Wf) 100; where Wf = final weight in g; Wi = initial weight in g; Wa = average weight between sampling in g; t = time in days; IF ingested food in g (food supplied food uneaten F, where F values were 1.14, 0.99 and 1 for diets G, A and C, respectively); IP ingested protein in g; IL ingested lipid in g; DGW = digestive gland weight. To analyse the variation in daily ingestion and throughout the experiment, the instantaneous feeding rate (IFR) was calculated on a daily basis. IFR (%) was obtained as g of feed consumed per octopus and day using the body weigh estimated from the SGR for this animal. The effect of the different diets on the above indices was analysed by one way ANOVA and the significance of the differences in mean values was analysed by Tukey's HSD test for unequal number of samples. A Neperian logarithmic transformation of the indices and percentages was made before the ANOVA.

from the way in which they were made, being the greater protein and energy content and P/E ratio of diet G (Table 3). Compared with the crab, both formulated diets had a higher energy content and particularly lipid, which reached almost 10% of weight of diet A. The percentage of protein in diet G (21.29%) was double that of the other two diets. At the beginning of the feeding trial, there was no significant difference between the mean weights of the different groups. The animals of groups G and A accepted the diets well, grew and produced a significant amount of faeces, with a survival of 100%. However, animals fed crab showed significantly better growth indices (WG, AGR and SGR), and better ingestion rates, significantly in the case of AFR and APFR with respect to both groups A and G, but also in the case of SFR with respect to group G (Table 4). Group A showed double the ALFR (2.56 g/day) of group C (1.19 g/day) and triple that of group G (0.73 g/day). The crab-fed group also showed the best feed efficiency indices (FE, FCR, PPV and LPV), incorporating 33.95% of the ingested food into body weight, followed by group A (22.51%) and finally group G (16.06%). Between groups C and G, the differences in the four feed efficiency indices (FE, FCR, PPV and LPV) were statistically significant, while only LPV showed significant difference between groups C and A. Unlike groups C and A, which showed positive PPV values (49.01 and 28.49%, respectively) and LPV (23.65% and 1.59%, respectively), the animals in group G showed negative values for both indices (PPV = 5.32%; LPV = 7.57%). Similarly DGI values were lower in group G (3.42%) than in C (4.97%) and A (4.19%), although these differences were not significant (Table 4).
Table 4 Mean values S.D. for every index of each experimental group Diet Crab 4 819 149a 1257 150a 438 38a 47.96 2.82a 5.72 0.33a 1.19 0.07a 4.67 0.52a 16.23 1.40a 1.61 0.25a 33.95 3.85a 2.97 0.35a 49.01 6.74a 23.65 3.53a 4.97 0.25a Gelatin 5 852 105a 903 92b 51 28b 11.32 2.62b 2.41 0.56b 0.73 0.17a 1.31 0.35b 1.87 1.02b 0.22 0.12b 16.06 7.40b 7.15 2.60b 5.32 20.76b 7.57 8.33b 3.42 1.14a Alginate 5 810 157a 979 165b 169 68c 27.09 5.38c 3.06 0.61b 2.56 0.51b 3.10 0.75a 6.26 2.52c 0.71 0.30c 22.51 5.01ab 4.65 1.16ab 28.49 9.27a 1.59 1.37c 4.19 0.52a ANOVA n.s. () () () () () () () () () () () () n.s.

3. Results Both formulated diets had a firm texture before being put into water. However, diet G lost 11.8% of its weight and diet A gained 0.37% after 4 h in water, demonstrating the greater stability of the diet containing alginate as binder; this also made it more acceptable to the octopus. The difference between the two diets was even more pronounced after 24 h in water, with diet G losing approximately 19% of its weight and becoming so soft that the animals found it difficult to manipulate, while diet A conserved its initial firmness (Table 2). Both diets showed a moisture content of 70 to 75%, so that they can be regarded as moist diets, the main difference between them, apart
Table 2 Results of the water stability tests for diets based on gelatin (G) or alginate (A) as binders Diet 4 h in water VP (%) a G (N = 5) A (N = 5)
a b

N Wi (g) Wf (g) WG (g) AFR (g/day) APFR (g/day) ALFR (g/day) SFR (% BW/day) AGR (g/day) SGR (% BW/day) FE (%) FCR PPV (%) LPV (%) DGI (%)

24 h in water Fb 1.14 0.99 VP (%) a 18.75 2.08 Fb 1.25 0.98

11.88 0.37

Mean variation in weight of diet (% alter after immersion in water). Correction factor for calculating real intake.

Values on the same line and different superscripts are significantly different (n.s. = not significant (P N 0.05); *P b 0.05; **P b 0.01; ***P b 0.001). Wi, initial weight; Wf, final weight; WG = weight gain; AFR, absolute feeding rate; APFR, absolute protein feeding rate; ALFR, absolute lipid feeding rate; SFR, specific feeding rate; AGR, absolute growth rate; SGR, specific growth rate; FE, feed efficiency; FCR, feed conversion ratio; PPV, protein productive value; LPV, lipid productive value; DGI = digestive gland index.

J. Cerezo Valverde et al. / Aquaculture 275 (2008) 266273 Table 5 Wet body composition of whole O. vulgaris (TBC), excluding digestive gland (EDG) and digestive gland (DG) of each experimental group Diet TBC 1 Moisture Crude protein Crude lipid Ash NFE 2 EDG Moisture Crude protein Crude lipid Ash NFE 2 DG Moisture Crude protein Crude lipid Ash NFE 2 Initial (N = 5) Crab (N = 4) Gelatin (N = 5) Alginate (N = 5) ANOVA

269

79.86 0.47a 78.31 0.77b 80.84 0.85a 79.78 0.53a () 15.79 0.63a 16.27 0.85a 14.78 1.05a 15.61 0.47a n.s. 0.45 0.09 ac 0.90 0.10b 2.36 0.07a 1.52 0.54a 1.99 0.11b 2.53 0.65a 0.29 0.09a 0.49 0.09c ()

2.02 0.13b 2.14 0.10ab () 2.06 0.82a 1.98 0.40a n.s.

80.69 0.50ab 79.41 0.75a 81.11 0.80b 80.38 0.50ab () 15.52 0.70a 16.07 0.87a 14.68 1.09a 15.46 0.52a n.s. 0.16 0.05a 2.39 0.07a 1.23 0.57a 0.16 0.12a 2.00 0.11b 2.35 0.64a 0.26 0.08a 0.23 0.05a 2.02 0.14b 2.14 0.10b 1.93 0.88a 1.78 0.43a n.s. () n.s.

the values of the initial control and of group A, both in the digestive gland (15.01% compared with 7.03 and 6.41%, respectively) and TBC (0.90% compared with 0.45 and 0.49%, respectively). The animals from group G showed the lowest levels of lipid in both fractions (1.27% for the digestive gland and 0.29% for TBC). There were no significant differences in the lipid percentage in the EDG fraction (P N 0.05). The percentage of protein in the different fractions analysed varied from 14.78 to 16.27% in TBC, from 14.68 to 16.07% for the EDG fraction and from 18.12 to 21.85% in GD. There were no differences in the protein percentages in the case of TBC and EDG, while the level in the digestive gland of animals fed diet G showed significantly lower values with respect to the initial control (P b 0.05), but no differences compared with the other groups. The percentage of minerals in the TBC varied from 1.99% in group C to 2.36% in the initial control, with significant differences between this last and groups C and G. There were no differences in the percentage of minerals or MELN among the different experimental groups in any of the fractions analysed.

61.09 1.67a 57.20 0.69b 73.00 1.58c 66.03 2.45d () 21.85 1.72a 20.24 0.77ab 18.12 1.54b 18.96 2.00ab () 7.03 2.08
a

4. Discussion The present study describes the elaboration of two formulated moist diets for octopus, with which significant growth and feed efficiency were obtained, unlike in previous studies, in which the difficulty of getting cephalopods to accept such diets was emphasised (Lee et al., 1991; Castro et al., 1993), including common octopus (O'Dor et al., 1984). According to previous studies (see Table 6), even when the diet was found acceptable, the low ingestion rates compared with control animals consuming a natural diet, the poor or zero growth recorded and even high mortality (Castro et al., 1993) made the feeds unsuitable for commercial purposes. However, research in this area is limited, especially compared with research carried out on fish, and investigations into the palatability and composition of feeds for octopus is still in its infancy. Fig. 1 compares the AGR, AFR and FE obtained with the three diets provided in this study with the values obtained by

15.01 1.41 1.74 0.16a 5.81 1.59a

1.27 0.38

6.41 2.36

() n.s. n.s.

1.75 0.25a 8.27 0.76a

2.11 0.34a 2.04 0.28a 5.50 2.15a 6.56 1.41a

Values on the same line and different superscripts are significantly different (n.s. = not significant (P N 0.05); *P b 0.05; **P b 0.01; ***P b 0.001). 1 TBC was calculated from EDG and DG values. 2 Nitrogen-free extract.

The main differences in the nutritional composition of the experimental diets were related to the moisture and lipid contents of the octopuses (Table 5). The moisture content of the animals ranged from 78.3 to 80.8%, with significantly lower values (P b 0.001) in the group fed crab. This was particularly true in the case of the digestive gland, where the values ranged from 57.3 in group C to 73.0% in group G. The opposite was true in the case of the lipid content, animals fed crab showed double

Fig. 1. Comparison of TCA, TAA and IEA obtained for diets in present study. (C = crab; G = gelatin; A = alginate) and for other diets (S = sardine; B = bogue; Cr = crustaceans) in similar experimental conditions (body weight = 1 kg; temperature = 18.9 C) according to Garca Garca and Aguado (2002) and Garca Garca and Aguado (2002).

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J. Cerezo Valverde et al. / Aquaculture 275 (2008) 266273

Garca Garca and Aguado (2002) and Aguado and Garca Garca (2002) for other natural diets fed under similar experimental conditions. The degree of acceptability of diet A was similar to that obtained when fish (sardine or bogue) were fed (23 to 27 g/day), while the results obtained with crab or crustacean-based diets doubled or tripled these values (48 to 69 g/day), which was translated into a greater ingestion of protein (5.7 g/day for group C and 3.1 g/day for group A according to our results). Furthermore, the growth of animals fed crustaceans (16 to 17 g/day) doubled that obtained in the group fed diet A (6 to 7 g/day) or with fish (8 to 9 g/day). This cannot be attributed to the poor feed efficiency of the last two groups since there were no significant difference in FE between animals fed crustaceans (25 to 34%) and those fed diet A (23%), the diet low in lipid (B. boops) showed the best results (41%). Protein retention did not seem to be responsible for the different growth rates either: PPV values of 28 to 49% with crustaceans, 33% with a mixed diet of fish and crab, 36% with fish and 28% with diet A (PPV values according to Garca Garca and Cerezo

Valverde, 2006). It seems, therefore, that the greater difference is due to the quantity of food ingested, depending on whether crustaceans or other type of food was provided, which agrees with the observations made in previous studies (Cagnetta and Sublimi, 2000; Aguado and Garca Garca, 2002; Garca Garca and Cerezo Valverde, 2006). The role of moisture content, texture, chemical substances/attractants, nutritional composition and even the way of manufacture, among numerous questions, remains to be clarified. According to our results, the moisture content should not have influenced the ingestion level since both diets and the crab diet showed similar levels of around 70 to 80%. Even so, it is possible that this factor may have influenced the palatability of the formulated diets in cephalopods. Octopus bimaculoides rejected a diet with a 10% moisture content (dry mysid pellets) and partially ingested a diet of 40% moisture (penaeid pellets), while a diet with a moisture content close to that of natural diets was accepted (Lee et al., 1991). Castro et al. (1993) obtained ingestion rates of less than 3% with a pellet-based diet of 47%

Fig. 2. (AB). Absolute growth rate (AGR) as function of absolute feeding rate (AFR) and absolute protein feeding rate (APFR) for the three diets.

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moisture, and a rate of 89% BW/day with a surimi diet with a moisture content of 82% (similar to that of the natural diet). Differences may also be species-related, high ingestion levels being obtained in Octopus maya even with dry diets (Domingues et al., 2007). This is the example to follow because a dry diet has numerous advantages over moist diets. For fish, such dry feeds are readily available and are of constant quality; they are easily stored and transported, involve minimal handling in the installation and reduce infectious diseases (because of the temperatures involved in their manufacture); they are highly digestible and store well (because of the low percentage of water), and, importantly, have a minimal environmental impact in the fish farms. This last aspect is especially important in the case of octopus ongrowing where the main environmental impact derives from the uneaten portion of the food provided (Mazn et al., 2007). With regard to the different textures assayed, homogeneous and pliable textures have been suggested to be more suitable than fibroid (Castro et al., 1993). Feeds of unsuitable texture tend to break into pieces and be dispersed by the water current from the siphon (Lee et al., 1991; Domingues et al., 2005; personal observation). Both formulated diets prepared in the present study had uniform and flexible texture, with no evident signs of disintegration upon manipulation by the octopus. Only some samples of diet G were disaggregated, perhaps because of its lower stability in water. Another aspect to be considered is the inclusion of attractant/ taste-enhancing substances to improve the palatability and organoleptic properties of the feeds. As can be seen from Fig. 2 (AB), the octopus growth rate showed a linear relation with the AFR or APFR regardless of the diet fed, suggesting that growth is linked not only to the nutritional properties of the food supplied but also to the preference for one food or another. In this respect, it is necessary to differentiate between substances that favour the capture of prey and those that favour its ingestion. Lee et al. (1991) observed how two different types of surimi that included natural or artificial extracts of crab were rapidly captured by O. bimaculoides, but were rejected immediately. In the case of O. vulgaris glutamic acid, glycine and AMP have been seen to act as attractants (Chase and Wells, 1986), but it remains to be demonstrated that they also encourage ingestion. The nutritional composition and even the way in which the feeds are made may have had an effect on their long term ingestion. The octopuses showed higher IFR the day after fasting than the other days (Fig. 3), a compensatory response that enabled them to show similar growth rates to animals that had not fasted on any day of the week (Garca Garca and Cerezo, 2004). However, in the case of the two formulated diets, two clearly differentiated responses were observed after fasting. Despite the fact that IFR of up to 4 to 5% BW/day were achieved in the case of diet G, ingestion rates were not maintained and fell sharply until the feed was rejected on subsequent days. In contrast, the ingestion of diet A usually remained constant, as in the case of the crab diet, suggesting that there was a difference between the two diets. A priori, diet G would seem more suitable from a nutritional point of view, presenting a

similar energy/protein ratio and moisture content to crab, together with lower fat levels which may have favoured protein digestibility (Garca Garca and Cerezo Valverde, 2006; Mazn et al., 2007). The texture of both diets was similar and both were initially taken eagerly, so that the main difference may lie in the way in which they were prepared. For example, it was necessary to apply heat in the preparation of diet G for it to gel, which may have affected its nutritional quality. The destruction of vitamins, breakdown of proteins and release of amino acids (Domngues et al., 2005) or even Maillard reactions (Deng et al., 2005) may have lessened its nutritional qualities for octopus. In a similar way, Castro et al. (1993) attributed the long term decrease in surimi consumption by Sepia officinalis to a change in behaviour brought about by a nutritionally deficient diet. Another aspect that needs to be looked at is the influence of diet on the nutritional state of the octopuses. For example, the differences observed in the lipid content of the animals can be attributed entirely to the digestive gland since the values observed in EDG fraction were similar. The lipid content of the GD of the crab-fed animals doubled that of the animals from the initial control and group A. According to LPV values, this is reflected in the overall retention of 23.6% of the lipid ingested in the crab-fed group and only 1.6% in group A. This finding points to the greater retention of the lipid obtained from crab, perhaps because of their greater digestibility (98%) compared with the lipid from fish (93%) (Mazn et al., 2007), or because
Table 6 Summary of body weight (BW), specific feeding rate (SFR) and specific growth rates (SGR) in formulated diet assays for cephalopods Species Octopus bimaculoides Octopus bimaculoides Octopus bimaculoides Sepia officinalis Octopus vulgaris Sepia officinalis Sepia officinalis Sepia officinalis Food Shrimp pellets Surimi (crab flavour) Chicken pellets Penaeid pellets Penaeid pellets BW (g) 125 125 125 420 N 250 75 75 106 SFR SGR Ref. a (% BW/day) (% BW/day) 5.3 0.2 2.7 13 Palatable 39 1.03.0 2.45.3 10.5 0.5 6.8 0.00.6 No growth 0.54 0.33 (1) (1) (1) (1) (1) (2) (2)

Surimi (fish based) Pellets (shrimp based) Surimi (semi-purified diet) Sepia Supplemented officinalis surimi diet Octopus maya Dry pelleted diet Octopus maya Pellets (with squid paste) Octopus Moist pellets vulgaris (type A) Octopus Moist pellets vulgaris (type B)

0.260.33 (3) 0.020.30 (4) 0.0 0.08 0.7 0.2 (5) (6) (7) (7)

322451 2.62.8 472 486 810 852 2.8 Palatable 3.1 1.3

a (1) Lee et al. (1991); (2) Castro et al. (1993); (3) Castro and Lee (1994); (4) Domingues et al. (2005); (5) Domingues et al. (2007); (6) Rosas et al. (2007); (7) Present study.

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Fig. 3. Variation in mean instantaneous feeding rate (IFR) in the three experimental groups of octopus. Arrows indicate the day following fasting.

of the difference existing in the different kinds of lipid in these foods (Snchez-Hernndez, personal communication). Similar results were obtained by Garca Garca and Cerezo Valverde (2006), who found LPV values of 12.2 and 3.3% for crab and bogue, respectively, even though this may have been partially due to the lower lipid content of the former, which would have favoured its digestibility (Hernndez and Garca Garca, 2004). Rosa et al. (2004) in O. vulgaris observed that it is in the digestive gland that the highest lipid, cholesterol and fatty acid levels occur. In a subsequent study, it was shown that both the content of lipid in the DG and their distribution into different classes varied with species and normally reflected the feeding strategy (Rosa et al., 2005), it being improbable that dietary lipids are modified before being deposited (Phillips et al., 2001). Moltschaniwskyj and Johnston (2006) in Eupryma tasmanica observed that most of the lipids present in the DG were structural components of the membranes (polar lipid) or digestionderived products, such as sterols or free fatty acids, with low levels of storage lipids (i.e. triacylglycerols, diacylglyceryl ethers or wax esters). Furthermore, the animals of this same species which grew most also showed low DG sterol levels, suggesting that sterols may be released to aid growth. Therefore, in the future design of formulated diets, both the quantity and type of lipids should be taken into account. Due to their low capacity to catabolize lipids (Mommsen and Hochachka, 1981; O'Dor et al., 1984), essential fatty acids, cholesterol and phospholipids could be more important in cephalopods (Rosa et al., 2005). The behaviour of proteins was similar, with difference being confined to the DG. The animals fed diet G were the only group to show lower protein levels than the initial control, together with negative LPV values. A deficient diet may well have the same effect as fasting situations, where lipids and amino acids from the digestive gland are mobilized (O'Dor et al., 1984; O'Dor and Wells, 1987; Castro et al., 1992). Previous results (Domngues et al., 2005) also demonstrated a lower concentration of amino acids, particularly those considered essential, in

the DG of Sepia sp. fed a formulated diet than in animals fed a natural diet, emphasising the importance of a suitable amino acid balance. Minerals also play an important role in formulated diets. Although we observed no difference in the respective DG mineral contents, the initial control animals had higher EDG levels than the other groups. Similar results were observed by Garca Garca and Cerezo Valverde (2006), the initial control animals had higher mineral content (2.41%) than groups fed crab or bogue (2.23%), findings that could be attributed to the higher beak/total body weight ratio in the initial controls, which were smaller than animals at the end of experimental period. Whatever the reason, the mineral needs of cephalopods are not well known, although there is some evidence concerning the need for copper (Castro et al., 1993), strontium (Hanlon et al., 1989), calcium and sulphur (Villanueva and Bustamante, 2006). The animals fed crab also showed higher DGI values than the groups fed the formulated diets. As in Sepia sp. (Castro and Lee, 1994), the DGI might be useful as an index of octopus condition. Indeed, in our results, as in the results of the above author, the DGI and growth rate were positively correlated. Castro and Lee (1994) also observed that the index increased when a natural diet replaced the prepared diet. Moltschaniwskyj and Johnston (2006) emphasised the importance of treating this index with caution and of taking the species into account. Our results reveal a wide range of questions, the answers to which may hold the key to increasing the acceptability of formulated feeds. The type of feed proposed in this study could be used to produce other feeds of known composition and to increase our knowledge of the nutritional requirements of octopus. Acknowledgements This project was financed by JACUMAR Spanish National Plans for Aquaculture. We thank Productos Sur S.A. for their advice and for providing the binders used in the study.

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