Growth, survival and physiological condition of Octopus maya when fed a successful formulated diet

Aquaculture 426–427 (2014) 310–317

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Growth, survival and physiological condition of Octopus maya when fed a successful formulated diet Rosario Martínez a, Pedro Gallardo b, Cristina Pascual b, Jorge Navarro c, Ariadna Sánchez b, Claudia Caamal-Monsreal b, Carlos Rosas b,⁎ a Departamento de Agronomía, División de Ciencias de la Vida, Campus Irapuato-Salamanca, Universidad de Guanajuato, Carretera Salamanca, Valle de Santiago km 3.5, Palo Alto, Salamanca, Gto., Mexico b Unidad Multidisciplinaria de Docencia e Investigación, Facultad de Ciencias, UNAM, Puerto de abrigo s/n, Sisal, Yucatán, Mexico c Posgrado Institucional en Ciencias Agropecuarias y Manejo de Recursos Naturales Tropicales, Campus de Ciencias Biológicas y Agropecuarias, Universidad Autónoma de Yucatán (UADY), Mérida, Yucatán, Mexico

a r t i c l e

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Article history: Received 10 August 2013 Received in revised form 4 February 2014 Accepted 6 February 2014 Available online 18 February 2014 Keywords: Formulated diets Octopus maya Growth Metabolites Silage Energy budget

a b s t r a c t A formulated diet for Octopus maya that provoked a growth rate and survival higher than that observed when animals were fed diet based only on freeze-dried crab is reported for first time. Early juveniles (0.37 g wet weight; ww) of O. maya were used. Diets were made by mixing freeze-dried ingredients (crab paste; Callinectes spp. meal; (Cr), squid meal; Dosidicus gigas (Sq) and silages (Si)) all enriched with a mix of vitamins and minerals, and bound with gelatin, forming a paste. At the end of the experiment, Crab–Squid (CrSq) was the only diet that showed 100% survival, followed by Crab (Cr) (55%). The highest specific growth rate (SGR, % day−1) was obtained in animals fed CrSq (3.04% day−1) followed by animals fed Cr (1.96% day−1) and Squid (Sq) (1.09% day−1). Marginal (0.36% day−1) and negative (−0.73% day−1) SGR values were observed in animals fed Silage–Crab– Squid (SiCrSq) and Silage–Squid (SiSq). Principal component analysis (PCA) showed that muscle glycogen and DG protein contents had the strongest influence on the separation of groups in a canonical analysis and were positively related with growth indicating that CrSq diets have the best nutritional characteristics to satisfy growth requirements. These results were confirmed when energy balance was measured. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Dependence on natural diets for feeding cephalopods has slowed down the development of its cultivation on a commercial scale (Lee et al., 1991; O'Dor and Wellls, 1987; Uriarte et al., 2011). The lack of a balanced feed that can also be stored and used easily remains as the decisive factor in large-scale growing of different species, such as Sepia officinalis (Domingues et al., 2005) or Octopus maya (Voss and Solis Ramirez, 1966), which have direct embryonic development. Feeds that cover nutritional requirement of octopus are a basic requirement, and although O. maya accepts natural food in captivity, habitually the specimens are fed with fresh frozen crab pieces, making their culture costly and impractical (Rosas et al., 2013). To achieve its aquaculture potential, it is necessary to replace the natural diets with specifically designed artificial feed. Domingues et al. (2005) mentioned that the use of prepared feed to replace living or natural diets may decrease production costs by 40%, with a potential reduction of up to 80% of the initial cost.

⁎ Corresponding author. E-mail address: [email protected] (C. Rosas).

http://dx.doi.org/10.1016/j.aquaculture.2014.02.005 0044-8486/© 2014 Elsevier B.V. All rights reserved.

There have been many attempts to obtain formulated foods for cephalopods, mainly for S. officinalis, Octopus vulgaris and O. maya. For examples see Aguado-Giménez and García-García, (2002), Domingues et al. (2005) and Rosas et al. (2007). Unfortunately, until now, growth and feeding rates obtained with elaborated diets have not been obtained as when animals fed fresh, frozen or freeze dried crab, fish or squid (some examples in Cerezo-Valverde et al., in press; Querol et al., 2012). In O. vulgaris, one of the most studied species of cephalopods in the world, to date there are no balanced foods for this species that can be used as a substitute for natural diets (Estefanell et al., 2013). A dry pellet diet made with fish meal did not promote O. maya growth, but animals did not lose weight and, more importantly, regularly ate all the food supplied, with feeding rates higher than reported in the literature for prepared diets (Aguila et al., 2007; Domingues et al., 2007; Rosas et al., 2007). During that experiment, higher growth rates and assimilated energy were obtained when feeding O. maya with frozen crabs compared to the dry pellet, which had a high lipid content (21%). In other studies, the use of alginate as a binder for the artificial diets could reduce digestibility (Rosas et al., 2008), since O. maya does not have the capacity to hydrolyse carbohydrates (unpublished data). Different protein sources have been used to feed octopuses, and

R. Martínez et al. / Aquaculture 426–427 (2014) 310–317

among them, fresh squid paste and crab have induced positive growth in O. maya (Quintana et al., 2011; Rosas et al., 2013). The protein requirement of O. maya may be around 60%, which is the average protein content commonly found in squid meal (Rosas et al., 2011). This suggests that if high-quality protein sources are used, with gelatine as a binder, a formulated diet could be elaborated for O. maya, based mainly on squid and crab paste (Rosas et al., 2013). Nutritional status is considered as one of the important factors that determine the ability of animals to use the ingested nutrients. In a previous study, we determined the metabolites in blood, the digestive gland (DG) and the arm muscles in an attempt to relate diet quality with the nutritional condition of O. maya (Aguila et al., 2007). The results demonstrated that blood, DG and muscle metabolites are related to the quality of the diet, which helped us to understand what kind of metabolic route is used when octopus are fed with a particular type of diet. In this sense, Cerezo-Valverde et al. (in press) suggested that digestive gland analysis could be useful to understand how a particular diet is metabolized. Recently, freeze-dried protein sources were recommended to formulate diets for octopus species (Querol et al., 2012; Rosas et al., 2013) because those ingredients enhance digestibility and consequently the growth. The present study aimed to evaluate the effects of several formulated diets made with freeze-dried protein sources on growth, survival, metabolite concentration in tissues and energy balance of early juvenile O. maya in an attempt to obtain a formula that covers the nutritional requirements of this octopod species. 2. Material and methods 2.1. Animals The study was carried out in the Experimental Cephalopod Production Unit (EPHAPU) at the UMDI-UNAM, Sisal, Yucatan, Mexico, following the procedures of Rosas et al. (2008) and Moguel et al. (2010) for collecting and maintenance of egg-laying females, as well as rearing, maintenance, feeding and growth of O. maya post-hatchlings. The octopuses were obtained from spawning of females copulated in controlled conditions. Hatchings (100 ± 16 mg living weight) were grown in tanks (54 × 35 cm, 35 L, 30 octopuses per tank) during the first 15 days after hatching. During that time, animals were fed ad libitum with live Artemia salina adults without any enriched. Those A. salina adults were obtained from Artemia farm located in Celestun, Yucatán, México. Afterwards, octopuses were individualized in containers of 500 mL and fed ad libitum with fresh crab broken pereiopods and chelae (Callinectes sapidus) for 5 weeks. Every day the food was added opening the cap of the container without disturbing the animals. A clean Megalongena corona bispinosa shell was placed in each container as a refuge. The containers were provided with windows covered with mesh and placed into 40-L tanks connected to a flow-through seawater system, and coupled to a skimmer and anthracite earth filter. Fifty per cent of the seawater in the flow-through system was renewed daily. Six 500-mL containers were placed in each tank. Seawater in the experimental containers was maintained at dissolved oxygen N5.0 mg/L, pH N 8, temperature 24 ± 1 °C, and 38 UPS. All the octopuses and diet combinations were randomly distributed between octopus containers. Twenty cultured O. maya were used for each experimental diet (five diets; N = 100; initial weight 0.39 ± 0.02 g; age 35 days after hatching). The octopuses were individually maintained for 55 experimental days.

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was mixed into the 20-L tank for 3 days. The silage pH was recorded 5 and 12 h after starting the process and every 24 h until the end of the silage time. The silage pH was maintained constant at 3.8 using 2 mL kg− 1 formic acid after 5 h of the process. Once the silage time was concluded, silages were dried at 80 °C until constant weight. Besides the silage described, frozen fresh mantle of squid (Dosidicus gigas) and wild fresh crab (Callinectes sp.) were used as ingredients, which were freeze-dried to constant weight at −43 °C and 0.110 mBar vacuum, then ground and sieved (500 μm). All the ingredients were conserved at 4 °C until use. With these ingredients, five experimental diets were produced: Crab paste (Cr), Squid paste (Sq), Crab–Squid paste (1:1, CrSq), Silage–Squid paste (1:4, SiSq) and Silage–Crab–Squid paste (1:2:2, SiCrSq) (Table 1). The ingredients used for each semi humid paste were ground in a laboratory mill (Thomas Willey Mod. 4, USA) to particles b 0.5 mm in diameter, and their moisture, total protein, total lipids, and ash contents were determined following methods established by A.O.A.C. (protocols 934.01, 976.05, 920.39 and 942.05, respectively). The diets were prepared by thoroughly mixing ingredients with purified water (200 ml to crab paste, squid paste, crab–squid paste and 125 ml to silage squid paste and silage–crab–squid paste) until a semi-moist diet resulted, with gelatin used as agglutinant (4.5%). Gelatine was first pre-gelatinised with cold water and after added to the dry ingredients during mixed with the rest of water. The semi humid paste was prepared on a 2-week basis and stored at − 10 °C. Each diet's energy content was measured in a calorimetric pump (Parr@) calibrated with benzoic acid (Table 1). Octopuses were fed twice a day (9:00 and 14:00 h) with 30% living weight day−1 for all diets (Quintana et al., 2011). Diets were separated into 200 mg portions and stored at −10 °C until use. All the portions were placed over bivalve conchs that were offered to the octopus as a plate. 2.3. Growth, feed intake and absorption efficiency Specific growth rate was determined as: SGR (% wet body weight day ) = [(LnW2 − LnW1) / t] ∗ 100, where W2 and W1 are octopuses' final and initial wet weights, Ln is the natural logarithm and t is the number of experimental days. Growth in mg per day was calculated as: growth (mg animal−1 day−1) = (W2 − W1) / t, where W2 and W1 are octopuses' final and initial wet weights and t is the number of experimental days. Survival was calculated as the difference between the number of animals at the beginning and at the end of the experiment. The ingestion rate (I) was calculated as the difference between the delivered feed and that which remained 2 h after being offered to the animals, and corrected by the percentage of leached nutrients. The leaching percentage of the pastes were measured using the shaking method (Obaldo et al., 2002). For this, 10 samples of 2 g of each diet were placed in 250-mL flasks placed in a horizontal shaker for 2 h. After that time, all the water was passed through a paper filter to separate the remaining paste from the leached water. The leached and original 10 feed samples of each diet were dried in a convection oven at 60 °C for 48 h until constant weight, and then cooled in a desiccator. Dried feed samples were weighed and analysed for dry matter retention. Ingestion rate was calculated according to the following equation: −1

I ¼ ½offered food–recovered food  ½1–ðlixiviated foodÞ

2.2. Silage elaboration and experimental diets Grouper (Ephinephelus morio) fresh scraps without skin and head and octopus viscera (O. maya) were obtained directly from a processing plant and stored at −40 °C until the silage process. After mixing, scraps and viscera (1:1 w/w) were liquefied until particles reached b1-mm diameter. Formic acid (90%) was added at a ratio of 20 mL kg−1. The meal

where offered and recovered food are expressed as dry weight (60 °C, 48 h), and lixiviated food as % of the food lost during shaking procedure (Obaldo et al., 2002). The crumbs were evaluated as non-ingested food. After, ingestion rate (I) was expressed as mg ingested food (wet weight) considering the water content obtained after dry sampling at 60 °C for 48 h.

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Table 1 Ingredients (g/100 g dry matter) and chemical characteristics of experimental diets (mean ± SEM) (dry base) used to feed O. maya juveniles. Cr: Crab; Sq: Squid; CrSq: Crab–Squid (1:1); SiSq: Silage–Squid (1:4); SiCrSq: Silage–Crab–Squid (1:2:2). Ingredients

Cr

Crab lyophilized meal Squid lyophilized meal Silage lyophilized meal Vitamins and minerals mixa Vitamin Cb Gelatine Total, g Dry matter, g/100 g Crude protein, g/100 g Carbohydrates, g/100 g Total lipids, g/100 g Ash, g/100 g Dry matter lixiviation, % DM Digestibility, % Gross energy, kJ g−1 dw Digestible energy, kJ g−1

94.0

a b

0.5 1.0 4.5 100.0 27.5 ± 0.83 91.3 ± 2.74 2.4 ± 0.07 2.4 ± 0.07 3.87 ± 0.12 3.9 ± 0.12 90.1 ± 2.70 18.6 ± 0.56 16.8 ± 0.84

Sq

CrSq

94.0

47.0 47.0

0.5 1.0 4.5 100.0 29 ± 0.87 95.7 ± 2.87 3.4 ± 0.10 0.1 ± 0.00 0.75 ± 0.02 3.5 ± 0.11 93.2 ± 2.80 22.6 ± 0.68 19.3 ± 0.97

0.5 1.0 4.5 100.0 28.5 ± 0.86 92.7 ± 2.78 3.1 ± 0.09 1 ± 0.03 3.2 ± 0.10 3.6 ± 0.11 93.2 ± 2.80 22.8 ± 0.68 21.3 ± 1.07

SiSq

SiCrSq

75.2 18.8 0.5 1.0 4.5 100.0 30.5 ± 0.92 76.5 ± 2.30 4.8 ± 0.14 14.5 ± 0.44 4.21 ± 0.13 5.1 ± 0.15 89.9 ± 2.70 18.8 ± 0.56 16.9 ± 0.85

37.6 37.6 18.8 0.5 1.0 4.5 100.0 35 ± 1.05 63.7 ± 1.91 4.1 ± 0.12 27.8 ± 0.83 4.5 ± 0.14 8.9 ± 0.27 90.2 ± 2.71 19 ± 0.57 16.9 ± 0.85

Vitamin and Mineral premix without vitamin C by DSM. Ascorbyl phosphate (Stay C-35%; DSM). Cr: Crab; Sq: Squid: CrCq: Crab–Squid (1:1:); SiSq: Silage–Squid (1:4); SiCrSq: Silage–Crab–Squid (1:2:2).

2.4. Digestive gland (DG) and muscle metabolites

2.6. Oxygen consumption and nitrogen excretion

Before sampling, octopuses (24 h fasting animals) were anaesthetised in a cold seawater bath (15 °C) for 1 to 2 min (Moltschaniwskyj et al., 2007). Once anaesthetised the brain of octopus was cute and after samples of DG and arms were obtained. DG samples and two arms of each sampled animal were frozen in liquid nitrogen and then stored at −80 °C until analysis. From these samples, soluble protein, acyglycerides, cholesterol, glucose and glycogen were measured in the DG. Muscle glycogen was evaluated in the octopus arms. Total soluble protein was evaluated with the Coomassie blue dye method according to the Bradford (1976) technique adapted to a microplate method using a commercial chromogen reagent (BioRad, Cat. 500–0006) and bovine serum albumin as the standard. Commercial kits were used for glucose (Bayer Sera Pak Plus B01 4509–01), acylglycerol (Bayer Sera Pak Plus B01 455101) and cholesterol (Bayer Sera Pak Plus B01 4507–01) measurements. Determinations were adapted to a microplate using 10 mL DG extract and gastric juice (GJ) (dilution 1:10) and 200 μL chromogen reagent. Absorbance was recorded in a microplate reader (BioRad 550) and the concentrations were calculated from a standard substrate solution. Glycogen was determined using the method described by Carroll et al. (1956). Glycogen in the DG and arms were extracted with trichloroacetic acid 5% (TCA) and determined through the reaction with sulphuric acid and phenol. Sections of DG and arms were weighed (20–30 mg) and homogenized in TCA for 2 min at 4550 g. One hundred microlitres of supernatant was pipetted into a tube and mixed with 500 μL ethanol 95%. Tubes were placed in an oven at 37 °C for 3 h. After precipitation, the tubes were centrifuged at 4550 g for 15 min. The supernatant was discarded leaving the glycogen as a pellet. By adding 1 mL concentrated sulphuric acid and 200 μL phenol 5%, glycogen was dissolved. From the mix, 200 μL was transferred to a microplate and read at 490 nm in an ELISA reader (BioRad 550). Total weight of the digestive gland was also recorded.

The effect of the type of diet on oxygen consumption was measured in individuals from each experimental diet. Oxygen consumption (VO2) was measured using a continuous flow respirometer where respirometric chambers were connected to a well-aerated re-circulating seawater system (Rosas et al., 2008). Juveniles were placed in 90-mL chambers with an approximate flow rate of 0.1 L min−1. All animals were allowed to acclimatize to the chambers for 1–1.5 h before measurements were made. Animals were offered a Melongena corona bispinosa shell as a shelter. A chamber without an octopus (with a shelter) was used as a control both during routine metabolism and feeding metabolism measurements. To do that, the control chamber was fed with a similar ration used to feed each octopus. Measurements of dissolved oxygen (DO) were recorded for each chamber (entrance and exit) every minute using oxygen sensors attached to flow-cells that were connected by optical fibre to an Oxy 10 mini-amplifier (PreSens©, Germany). The sensors were calibrated for each experimental temperature using saturated seawater (100% DO) and a 5% sodium sulphate solution (0% DO). Fasting metabolism was obtained from measurements taken every minute for 60 min after the conditioning period. Afterwards, octopuses were fed, taking into consideration that half of the ration (15% ww day−1) was given two times a day to complete a total ration of 30% ww day−1. Oxygen consumption measurements during the feeding phase were taken every minute until the oxygen consumption returned to pre-feeding values. At the end of the experiment, octopuses were weighed. Oxygen consumption was calculated as the difference in dissolved oxygen concentrations between the input and output of each chamber, with the water flow being timed. Routine metabolism (Rrout) was estimated from the VO2 (mg g− 1 ww h−1) of fasting octopuses. The apparent heat increase (RAHI; J g−1 h−1) was estimated from the difference between the VO2 of fasting octopuses and the maximum value attained after feeding, taking into account the time needed to reach the oxygen consumption peak after feeding. Partial energy budget was estimated using the following equation (Lucas, 1993):

2.5. Energetic balance Ingested food (I) was calculated as: I ¼ Ir  Gef where Ir is the ingested food rate (g dry weight day−1 g ww−1) and Gef is the diet's gross energy content (kJ g−1).

As ¼ Rtot þ Pg where As is assimilated energy, Rtot indicates respiration (Rtot = Rrout + RAHI), and Pg is the energy invested in growth, all of them expressed as kJ g−1 ww day−1. Energy produced (Pg) was obtained using the actual growth rate of all octopuses during the experimental time (55 days). The value of 10.1 kJ g−1 was used to transform the growth data into production units (Pg; J g−1 day− 1 live weight; Rosas et al., 2007, 2008).

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Assimilated, respiratory and production gross efficiencies were calculated as As/I × 100, R/I × 100 and Pg/I × 100, respectively. Respiratory (R) and production net efficiencies (Pg E) were calculated as R/As × 100 and Pg/As × 100, respectively. 2.7. Statistical analysis One-way ANOVA followed by Duncan's multi-comparison test was applied to weight, growth, ingested food, oxygen consumption and energy balance data. Prior to ANOVA, homogeneity of variances was verified by the Cochran test (Montgomery, 2004). Discriminant canonical analysis (DCA) was applied to DG metabolites, and glycogen in DG and arm muscle, followed by lineal regressions (Johnson and Wichern, 2002). Prior to DCA, homogeneity of the variances–covariances matrix was verified by the M of Box method (Pardo and Ruíz, 2001). Statistical analysis was carried out with the computer programme Statgraphics 5.1. Differences are reported as statistically significant when P b 0.05.

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proteins and muscle glycogen (coefficients higher than 0.6; Table 4). The percentage of cases correctly classified in discriminant analysis was 83.72%, and showed that diets grouped by concentrations of metabolites were classified correctly as 71.43, 66.67, 100, 66.67 and 75% for Cr, Sq, CrSq, SiSq and SiCrSq, respectively. The experimental diet dispersion diagram shows the dispersion of allowances in relation to the first two discriminating function diagrams (Fig. 1). This chart allows four welldefined groups to be identified. The first group was formed by individuals that were fed with CrSq, the second by SiSq, the third by Cr and Sq, and the fourth by SiCrSq. Taking into consideration that DG proteins and muscle glycogen were the two metabolites that had the strongest influence on the separation of groups in canonical analysis, regression analysis between both metabolites and growth rate were performed (Fig. 2). These analyses showed that muscle glycogen and digestive concentration are positively related to growth. Animals with high values of SGR% day−1 showed high values of DG proteins and muscle glycogen (CrSq diet) while low values of SGR% day−1 were related with low values of both metabolites in animals fed silage diets (Fig. 2).

3. Results 3.3. Oxygen consumption

3.1. Proximate composition of the formulated diets All diets were accepted and consumed by juveniles of O. maya. Proximal data analysis showed that experimental diets made with crab (Cr), squid (Sq) and crab–squid mix (CrSq) had protein levels (mean value of 93.2% CP) higher than observed in the diets SiSq (76.5%) and SiCrSq (63.7%) (Table 1). An inverse relationship between CP and total lipids (TL) was observed with high values of TL in diets made with silages and low TL in diets made with raw ingredients (Table 1). 3.2. Weights, growth, ingestion rate and survival The highest final weight was observed by octopuses consuming the CrSq diet, followed by animals fed the Cr diet. Negative weights were obtained from octopuses fed the SiSq diet (Table 2). The specific growth rate (SGR% day−1) showed differences between the experimental diets (Table 2; P = 0.0000) with high values for animals fed CrSq (3.04 ± 0.39% day−1) followed by animals fed Cr and Sq, and low values for animals fed SiCrSq. Negative values were obtained from animals fed SiSq (P b 0.0001; Table 2). At the end of the experiment, CrSq was the only diet that showed 100% survival, followed by Cr (55%) and Sq (50%) (Table 2). Survival of animals fed silages showed survivals lower than 50% (Table 2). Digestive gland and muscle metabolites varied with the experimental diets (Table 3). To make a more integrative analysis of the data, a discriminant canonical analysis was applied (Table 4). The Box M test showed that the independent variables observed in the variance– covariance matrix were equal in all groups (P = 0.174). The discriminant functions were 1 (P = 0.0000) and 2 (P = 0.0087). Test of Wilks' Lambda (95% confidence level) showed that both functions were statistically significant. According to the canonical discriminant analysis data, three groups were separated due to the influence of DG

Oxygen consumption was affected by the type of diet (Fig. 3). Lower routine oxygen consumption was observed in animals fed Cr and CrSq (mean value of 0.35 mg O2 h−1 g ww) than obtained in octopus fed Sq or silage diets (1.19 mean value of 0.34 mg O2 h−1 g ww). After feeding, the oxygen consumption of animals increased reaching its maximum value 1.16, 1.1, 2.08, 0.9 and 0.15 h after Cr, Sq, CrSq, SiSq and SiCrSq diets, respectively. Oxygen consumption calculated as RAHI was affected by the type of diet, with low values in animals fed Cr, Sq and CrSq diets, intermediate in animals fed SiCrSq and high values in animals fed SiSq (Fig. 2; P b 0.001). 3.4. Energy balance Ingested energy (I) was affected by the type of diet, with higher values in animals fed Sq, silage diets and CrSq (mean value of 484 kJ day − 1 g − 1 ww) than observed in octopus fed Cr (270 kJ day−1 g−1 ww; P b 0.05; Table 5). Total respiratory metabolism (Rtot = Rrout + RIHA) was also affected by type of diet with higher values obtained in animals fed Sq and silage diets than observed in animals fed Cr and CrSq (P b 0.05; Table 2). Energy channelled towards biomass production of animals fed CrSq was 2.5 times higher than observed in the control diet (Cr) and 4.5 and 18 times higher than observed in animals fed Sq and SiCrSq diets (Table 5). Of the total ingested energy, 85 to 93% was channelled towards assimilation (AS) in all the experimental animals. Of the ingested energy of animals fed CrSq, only 25% was channelled towards Rtot while 68% was channelled towards biomass production. In contrast, animals fed Cr channelled 41% of ingested energy towards Rtot and 49% towards Pg, while animals fed Sq or silage diets channelled 73 to 92% towards Rtot and only 4 to 12% towards Pg (Table 2).

Table 2 Initial and final weight (g), specific growth rate (% day−1) and growth rate coefficient (DGC % day−1) and survival (%) of early O. maya juveniles fed five experimental diets for 55 days. Values are mean ± SD; the number of individuals (n) is indicated in each case.

Initial weight, g Final weight, g SGR,% day−1 Growth rate, mg day−1 n Time, days Survival, %

Cr

Sq

0.37 ± 0.02a 1.09 ± 0.11d 1.96 ± 0.22d 13.09 ± 1.05d 20 55 55

0.42 0.81 1.19 7.09 20 55 50

± ± ± ±

0.03a 0.08c 0.10c 0.85c

CrSq

SiSq

SiCrSq

0.41 ± 0.03a 2.18 ± 0.21e 3.04 ± 0.39e 32.18 ± 5.15e 20 55 100

0.35 ± 0.02a 0.31 ± 0.03a −0.22 ± −0.03a −0.73 ± −0.11a 20 55 20

0.41 0.50 0.36 1.64 20 55 30

Different letters means statistical differences P b 0.05. Cr: Crab; Sq: Squid; CrSq: Crab–Squid (1:1); SiSq: Silage–Squid (1:4); SiCrSq: Silage–Crab–Squid (1:2:2).

± ± ± ±

0.03a 0.05b 0.05b 0.23b

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Table 3 Digestive gland (DG) metabolites and muscle glycogen of O. maya juveniles fed experimental diets for 55 days. Values as mean ± SE. mg/g tissue

Experimental diets Cr

Protein DG Acylglycerol DG Cholesterol DG Glucose DG Glycogen DG Muscle glycogen

Sq

13.85 0.39 7.26 0.39 2.18 0.71

± ± ± ± ± ±

1.32 0.21 2.26 0.06 0.32 0.04

CrSq

11.47 0.34 0.80 0.32 1.59 0.64

± ± ± ± ± ±

2.05 0.14 0.80 0.05 0.07 0.05

26.19 0.28 1.63 0.37 3.14 1.55

SiSq ± ± ± ± ± ±

1.55 0.08 0.70 0.04 0.24 0.11

11.04 0.24 9.64 0.38 1.24 1.24

SiCrSq ± ± ± ± ± ±

1.62 0.14 4.82 0.07 0.03 0.11

5.42 0.20 2.54 0.29 1.28 0.45

± ± ± ± ± ±

1.60 0.10 1.47 0.01 0.30 0.02

Cr: Crab; Sq: Squid; CrSq: Crab–Squid (1:1); SiSq: Silage–Squid (1:4); SiCrSq: Silage–Crab–Squid (1:2:2).

4. Discussion In the present study, a formulated diet for O. maya that provoked a growth rate and survival higher than that observed when animals were fed live or frozen crab is reported for first time. This diet was made with freeze-dried crab and squid meal, supporting the idea that using native protein it is possible to successfully feed octopuses. In this study different protein sources to formulate elaborated diets for O. maya were used, with the purpose of identifying which of them is the most appropriate for feeding this octopus species. As Callinectes sp. is used as the basic diet for this species, the results obtained with the crab diet (Cr) were taken as the reference point to make the comparison between experimental diets (Rosas et al., 2008, 2013). As expected, the source of dietary protein affected the animal's growth rate. Our results show that the CrSq diet produced the highest SGR (% day−1), with a mean value of 3.04% day−1. Also, it was observed that octopuses fed the CrSq diet had the highest survival (100%) while animals fed Cr or Sq had only 55% and 50% survival, respectively. Since the percentage of raw protein is similar between Cr, Sq and CrSq, we can propose that when Cr and Sq are combined, this diet provokes a synergy between its components providing enough elements to achieve higher octopus growth rate than those fed with only crab or squid. It is interesting to note that after 46 nutritional tests made with O. maya early juveniles, pre-adults and adults, marine crustacean raw protein always provoked positive growth rates higher than 1.5% day−1; when other types of protein (fresh clam, squid, fish or any other cooked protein, or silage in the present study) were used, marginal or negative growth rates were observed (for examples see Aguila et al., 2007; Rosas et al., 2008, 2013). Similar results were obtained in O. vulgaris (Aguado-Giménez and García-García, 2002; Cerezo-Valverde et al., 2008; Domingues et al., 2010; Estefanell et al., 2013; García-García and Cerezo-Valverde, 2004; García-Garrido et al., 2011; Prato et al., 2010; Quintana et al., 2008). All these data confirm that only some marine ingredients in their raw form have the specific characteristics that cover nutritional requirements for growth of octopods. A question arises: what kind of ingredients could be so specific as to promote or limit growth of O. vulgaris or O. maya? According to Cerezo-Valverde et al. (in press), Arginine (Arg) and Leucine (Leu) are the limiting amino acids (AA) for O. vulgaris, with bivalves (Mytilus galloprovincialis), squid (L. gahi) and crustaceans Table 4 Standardized canonical variable coefficients obtained from the PCA of tissue metabolites of O. maya fed experimental diets. CVC Variables

1

2

Proteins DG Acylglycerol DG Cholesterol DG Glucose DG Glycogen DG Glycogen muscle % of variance explained Canonic correlation

0.329894 −0.0903416 −0.208463 −0.391352 0.350237 0.885211 77.87 0.91198

−0.761546 −0.243306 0.260407 0.0740876 −0.328298 0.625478 15.39 0.70288

DG: Digestive Gland.

(Carcinus maenas, Penaeus spp. and P. clarkii) being the food groups that had the highest chemical scores. Fish, fish meal and vegetable meals had lower chemical scores explaining, at least in part, the worse result that those ingredients provoked in O. vulgaris. Using the criterion of Cerezo-Valverde et al. (in press) and Callinectes sapidus crab, jumbo squid D. gigas and O. maya amino acids data obtained by Aguila et al. (2007) and Cordova-Murueta et al. (2003), we found that in the crab, practically there are no limiting AA ((Crab Arg / octopus Arg) × 100 = 158%), while in the squid, Arg appears as the limiting AA for O. maya ((Squid Arg / octopus Arg) × 100 = 16%). That condition was almost reversed when crab and squid were mixed, reaching values close to 87%. According to these results, we can now explain why jumbo squid alone as a diet for O. maya provokes marginal growth rates (Rosas et al., 2013; present study), and when it is mixed, with crab, provokes even better results than when only crab is used alone as a food. According to the present results, DG proteins and muscle glycogen were the metabolites that better explain the separation of groups obtained in the canonical analysis. The relationship between both metabolites and growth was evident suggesting that the CrSq diet has nutritional components that facilitate the accumulation of proteins in DG and the gluconeogenesis pathway in muscle. Muscle growth involves two processes: (i) hyperplasia, the generation of new muscle fibres; and (ii) hypertrophy, the increase in size of those fibres already in existence (Semmens et al., 2011). In their study, Semmens et al. (2011) observed the hyperplasia process in an octopus species (Octopus pallidus) through the histological analysis of mantle muscle fibres. Poor and rich mitochondrial muscle fibres were observed even in octopus adults, demonstrating why octopus species maintain nonasymptotic growth during their life cycle. Although hyperplasia was not demonstrated in O. maya, we can assume that a similar growth process occurs in this species and that this process has a high demand for nutrients and energy for protein synthesis. In cephalopods, as in other invertebrates, glycogen is mainly derived from dietary proteins, via amino acid metabolism (Miliou et al., 2005; Rosas et al., 2002). In this sense and according to the present results, we can hypothesize that the CrSq diet used in the present study has nutritional characteristics (i.e. AA content) that improve the use of protein and their metabolic pathways to improve the muscle synthesis and hyperplasia mechanisms involved, allowing a higher growth rate than observed in the rest of the treatments. The use of protein as a main source of protein and energy has also been observed in octopods and squids demonstrating that this is a generalized characteristic between cephalopods (George-Zamora et al., 2011; Houlihan et al., 1990; Katsanevakis et al., 2005; Miliou et al., 2005; Sakurai et al., 1993; Storey and Storey, 1978). In fact, from an energetic point of view, the main metabolic energy used by cephalopods comes from protein metabolism (O'Dor and Wellls, 1987). In this sense, the results obtained in the present study demonstrate that protein characteristics have a strong influence on the destination of energy ingested in O. maya. Animals fed experimental diets ingested more energy than the octopuses fed the reference diet (Cr) suggesting that there are some components in experimental diets that stimulated an increment of the ingestion energy. Previous studies have shown that deficient food provoked an increment of ingested energy when that diet did

R. Martínez et al. / Aquaculture 426–427 (2014) 310–317

315

Fig. 1. Discriminant function analysis. Cr: Crab; Sq: Squid; CrSq: Crab–Squid (1:1); SiSq: Silage–Squid (1:4); SiCrSq: Silage–Crab–Squid (1:2:2).

4

y = 0.10x + 0.31 R² = 0.54; p < 0.001

SGR, % day-1

3.5 3 2.5

Cr

2

Sq

1.5

CrSq

1

SiSq SiCrSq

0.5

the attraction of O. maya, we can assume that the increase in values of ingested energy in animals fed CrSq was at least in part stimulated by the use of gelatin in the diet, which has a high Gly content. As in the case of ingested energy, there was an inverse relationship between energy derived from respiratory metabolism and the diet that provoked high and low growth rates; animals fed Sq or silage diets invested more energy in respiratory metabolism than animals

Oxygen consumption, mgO2 h-1 g-1 ww

not satisfy the energy requirement of O. maya (Rosas et al., 2007). This is the case of diets made with Sq and silage, which we can suppose does not cover the energetic requirements of octopus, thus stimulating its ingestion in an attempt to compensate for the deficiency. The case of the CrSq diet is different — animals had a higher growth rate than the rest of the treatments suggesting that such a high ingested energy is more a consequence of stimuli provoked by the ingredients, which, besides being nutritionally adequate, were highly attractive to O. maya. Previous studies have demonstrated that, besides its nutritional role, Gly is one of the attractant amino acids used in diets for fish (Hughes, 1985). Although there are no formal studies that demonstrate the role of Gly in

1.8

b

1.6

b

b

SiSq

SiCrSq

1.4 1.2 1 0.8 0.6

a

a

0.4 0.2 0 Cr

Sq

CrSq

Experimental diet 0

5

10

15

20

25

30

35

Digestive gland proteins, mg/g 4.5

y = 0.001x + 0.65 R² = 0.47, p < 0.005

4 3.5 3

Cr

2.5

Sq

2

CrSq

1.5

SiSq

1

SiCrSq

0.5 0 0

500

1000

1500

2000

2500

3000

Muscle glycogen, mg/g Fig. 2. Digestive gland proteins and muscle glycogen (mg/g tissue) and specific growth rate (SGR, % day−1) relationship of O. maya juveniles fed experimental diets. Cr: Crab; Sq: Squid; CrSq: Crab–Squid (1:1); SiSq: Silage–Squid (1:4); SiCrSq: Silage–Crab–Squid (1:2:2).

Oxygen consumption, mgO2 h-1 g-1 ww

0

SGR, % day-1

Routine

4.5

AHI

c

4 3.5 3 2.5 2

b

1.5 1

a a

0.5

a

0 Cr

Sq

CrSq

SiSq

SiCrSq

Experimental diet Fig. 3. Routine oxygen consumption of O. maya used to calculate apparent heat increment of animals fed different experimental diets. Different letters means statistical differences P b 0.05. Means ± SE. Cr: Crab; Sq: Squid; CrSq: Crab–Squid (1:1); SiSq: Silage–Squid (1:4); SiCrSq: Silage–Crab–Squid (1:2:2).

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Table 5 Effect of diets on energetic balance of O. maya juveniles. Values as kJ day− 1 g − 1 WW. Mean ± SE. Different letters means statistical differences, P b 0.05. Cr Ingestion (I) 270 ± 22a Respiratory metabolism, R R routine 94 ± 50a RAHI 17 ± 5b Rtot 111 ± 33a Production (Pg) 132 ± 17d AS = R + Pg 244 AS/I% 90 Rtot/I,% 41 Pg/I,% 49 Rtot/As,% 46 P/AS,% 54

Sq

CrSq

SiSq

SiCrSq

573 ± 63b

476 ± 43b

441 ± 53b

446 ± 67b

409 ± 53b 8 ± 5a 417 ± 29b 72 ± 15c 489 85 73 12 85 15

103 ± 24a 16 ± 5b 119 ± 14a 325 ± 35e 444 93 25 68 27 73

311 ± 136b 93 ± 10c 404 ± 73b −7 ± 2.9a 397 90 92 −2 102 −2

380 ± 100b 4 ± 1a 384 ± 51b 18 ± 7b 402 90 86 4 95 5

Cr: Crab; Sq: Squid; CrSq: Crab–Squid (1:1); SiSq: Silage–Squid (1:4); SiCrSq: Silage– Crab–Squid (1:2:2).

fed Cr and CrSq suggesting that those worse diets were nutritionally unbalanced provoking an increase in catabolism and, consequently, loss of energy. Routine metabolism includes energy derived from catabolism and anabolism, basal metabolism being a component involved in the catabolic process that produces energy to maintain the vital functions of organisms (Lucas, 1993). For this reason, an increase in Rrout related to a diet that provokes a low or negative growth indicates that a big part of the assimilated energy was lost as catabolic energy (between 85 and 95%) while diets with low Rrout values provoked high growth rates indicating that assimilated energy was mainly channelled towards biomass production (between 54 and 73%). High Rrout values were observed in O. maya pre-adults when fed diets elaborated with fish meal and hydrolysed fish protein (low growth rates) and low Rrout values were obtained when animals were fed crab (high growth rates) (Aguila et al., 2007). In the present study, with the exception of the SiSq diet, the other experimental diets did not provoke significant differences in the absolute AHI of animals, showing that the energetic costs of the feeding process were not affected by these types of diets. In contrast, animals fed SiSq had high AHI values, indicating that with such a diet, the energy requirement of all the behavioural, physiological and biochemical processes involved in feeding was strongly affected. The effects of high AHI values observed in animals fed SiSq were negative for the octopus energetics, provoking, among other consequences, weight loss during the experiment. The present study shows for the first time an elaborated diet that was better than the reference diet used conventionally to feed O. maya juveniles. This diet is now being successfully used in a pilot-scale octopus production at the National Autonomous University of México (Uriarte et al., 2011). Although this diet enhances the growth of octopus and facilitates the food management in laboratory conditions, it is necessary to reduce its cost so that its production could be economically feasible at a commercial scale. Improving this diet and its economic aspects, along with other aspects, will be the next steps in the O. maya culture. Acknowledgments Thanks are given to CONACYT for scholarship number 48221 to Martinez R. The present study was partially financed by DGAPAUNAM project IN212012 and CONACYT-CB201001 project No. 150810 to Rosas C, and PAPITT IN207809-3 project to Gallardo P. Thanks are also given to Maite Mascaró for their statistical suggestions. References Aguado-Giménez, F., García-García, B., 2002. Growth and food intake models in Octopus vulgaris Cuvier (1797): influence of body weight, temperature, sex and diet. Aquac. Int. 10, 361–377.

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