ES2947096B2 - Semi-moist formulated feed for fattening common octopus (Octopus vulgaris) - Google Patents

Description

DESCRIPTION

I. TITLE

Semi-moist formulated feed for fattening common octopus (Octopus vulgaris)

II. TECHNICAL SECTOR

The present invention is included in the agri-food sector of animal production, specifically in the area of aquaculture. In particular, it refers to a semi-moist feed that allows for the fattening of sub-adults (600-800 g) and adults of common octopus in a profitable way, presenting itself as an alternative to feeding with natural diets based on fish or crustaceans.

III BACKGROUND

Cephalopods are an important fishery resource, with a catch volume ranging between 3.5 and 4.8 million tons per year (FAO, 2018). However, the production of cephalopods from aquaculture is merely symbolic (0.5-28 tons per year between 2000 and 2020 in Spain; MAPAMA, 2020), showing a significant lag compared to the evolution of the fish farming sector. Spain, together with Japan and Italy, is one of the largest consumers of cephalopods, the price of which has doubled in the last decade.

Among cephalopods, the rock octopus (Octopus vulgaris) is a species with great potential for aquaculture due to its biological characteristics: rapid growth, high efficiency in feed conversion, high fertility, high protein content in its nutritional composition, easy adaptation to life in captivity, as well as a high commercial and gastronomic value (Vaz-Pires et al., 2004).

To date, the two main drawbacks to the development of industrial octopus aquaculture have been the impossibility of obtaining juveniles on a massive scale in captivity (Iglesias and Fuentes, 2014), as well as the absence of formulated feeds with appropriate nutritional composition and acceptability. For these reasons, since its beginnings in 1995, the octopus production system has not shown great variations and has been carried out in an artisanal way: capture of sub-adults (700-800 g) from the natural environment; stabling in different types of cages; feeding with discards from fishing (fish, crustaceans and mollusks of low commercial value); and marketing of specimens of 3-3.5 kg after 3-4 months of fattening (Sánchez, Cerezo Valverde and García García, 2014). This system was originally established in Galicia, reaching a maximum production of 32 tonnes per year in 1998. In the Mediterranean, the introduction of this species is seen as a complementary activity to the gilthead and sea bass farms, since, due to its characteristics (production cycle conditioned by temperatures), it would contribute to improving the economic balance of the companies (García García et al., 2009). In any case, today, the industrial production of octopus is considered an unprofitable activity (García García and García García, 2011).

The most recent studies are helping to unblock this situation. In reference to larval reproduction and culture, the Spanish Institute of Oceanography in Vigo has developed a feeding protocol for octopus paralarvae that, added to appropriate zootechnics and environmental conditions, achieves high growth and survival rates, with production of benthic specimens between 65 and 75 days of life. This research represents a key milestone for octopus aquaculture and has led to a national invention patent (ES 2714 930; Tur et al., 2019). Previously, the University of Las Palmas developed an inert microdiet that allowed paralarvae to be fed from 30-40 days of life (ES 2599603; Estefanell et al., 2017).

However, for the future development of octopus aquaculture, feed will also be necessary for the fattening phase, as is evident from the success of other aquaculture species currently on the market, such as sea bream, sea bass, turbot or salmon. Furthermore, in the case of octopus, the availability of feed is of particular importance for several reasons:

-The main cost in octopus aquaculture facilities, as they are currently conceived, is due to the feed supplied (38-40% of the total; García García and García García, 2011). In fact, the best growth rates (18-20 g/day/octopus) and survival are obtained when the diet is composed exclusively of crustaceans or when they form an important part of the diet, which makes the production process more expensive (García García and Cerezo Valverde, 2006). Feeding only fish produces poorer growth (9-11 g/day/octopus; Aguado Giménez and García García, 2002). In general, fresh foods are subject to highly variable availability and price, however, the production of feed implies greater stability in supply, nutritional composition and price.

-The main environmental impact of octopus aquaculture comes from the inedible portion of the feed (50% in the case of crustaceans and 23% in the case of fish), and the solid and dissolved waste excreted (Mazón et al., 2007; García García et al., 2011). Octopus feed eliminates the impact derived from the inedible fraction and helps to reduce nutrient excretion rates due to its greater retention for growth.

-Feeds formulated with less moisture have other advantages: better conservation, lower cost per kg of animal produced, they are easier to store and distribute, minimal handling in the facilities and better stability in water.

Results from the INIA-RTA2012-00072 project (Ministry of Science and Innovation, Government of Spain) have shown that juvenile octopus can be fattened by supplying semi-moist feeds that generate growth rates similar to crustacean diets (16.8-20.7 g/day/octopus) with a high utilization of the diet (60-65% of the feed consumed incorporated in biomass), both in tanks in land-based facilities (Cerezo Valverde and García García, 2017) and in open-sea cages (Cerezo Valverde et al., 2019). This feed is considered the benchmark of the state of the art, and its base is made up of freeze-dried fish, mollusks and crustaceans. The main advantage of these ingredients is that their organoleptic and nutritional properties are preserved intact over the long term. This is achieved through a dehydration process that extracts water without heat treatment. On the other hand, freeze-drying is a process that is not widely implemented at an industrial level and is used for products with high added value, which ultimately increases the price of feed.

The development of this feed has been preceded by a long history of studies covering different formats of wet feed (Cerezo Valverde et al., 2008, 2013a; Quintana et al., 2008; Rosas et al., 2008; Estefanell et al., 2012), semi-moist (Castro et al., 1993; García-Garrido et al., 2010; Morillo-Velarde et al., 2012, 2015a, 2015b, 2015c; Celdrán Sáncho et al., 2015; Rodríguez-González et al., 2015, 2018a; Cerezo Valverde et al., 2017) and extruded dry feed (Domingues et al., 2007; Querol et al., 2014, (2015). Wet feeds (>70% water) had in common the use of a paste of fish, crustaceans or molluscs mixed with different types of binders (alginates or gelatins). Semi-moist feeds (40-60% moisture) were made with approximately equal parts of water and dry ingredients, and usually included gelatin as a binder due to its high acceptability and digestibility. The application of heat to these mixtures increased their stability in water, although it also caused a loss of their nutritional value. Finally, dry feeds (<20% moisture) have been made by prior kneading of their ingredients and a subsequent cooking-extrusion process.

Although the results were mixed, these studies revealed the problems with the design and formulation of feed for cephalopods. In most cases, granulated, extruded and semi-moist feeds as they were made for fish were not suitable, since they were broken down into small particles before being ingested, or were directly rejected. Feed for cephalopods had to be stable in water and have a firm and homogeneous texture, with a certain flexibility to avoid disintegration when handled. On the other hand, these animals have a high chemoreceptor capacity against various stimuli, including: acids, sugars, salts, crab extracts, amino acids, nucleotides and vitamin complexes. In this sense, any new ingredient or chemical substance could directly influence the acceptance or rejection of the feed. Feeds based on freeze-dried ingredients and powdered egg yolk showed better intake and growth rates compared to those that included heat-treated raw materials, whether flours of animal origin (fish or crustaceans) or vegetable origin (Morillo Velarde et al., 2015a).

The development of feeds with known composition, together with other metabolic and biochemical studies, allowed us to gain a deeper understanding of nutritional requirements. In general terms, cephalopods have a protein metabolism (García-Garrido et al., 2013), although they can also use carbohydrates from muscle tissue (Morillo-Velarde et al., 2011) or triglycerides from the digestive gland (Morillo-Velarde et al., 2013). Quantitatively, protein should be the main macronutrient, being used simultaneously for energy and growth purposes. A balanced amino acid profile should contain high proportions of arginine, leucine and lysine as essential amino acids, and glutamate and taurine as non-essential amino acids (Cerezo Valverde et al., 2013b). Glutamate is the most abundant amino acid in cephalopods and its supplementation in octopus feed improved growth and the productive value of the protein (Cerezo Valverde et al., 2013a). On the other hand, taurine plays a key role in the osmoregulation of mollusc and crustacean tissues, with a concentration three times higher than that observed in fish. Its supplementation was included in extruded feed by Querol et al. (2015) without causing rejection in the animals, although the absence of a control without taurine did not allow elucidating its effect on performance.

Although quantitatively lipid requirements appear to be small (<10% dry weight), qualitative composition could be an important factor. Phospholipids, cholesterol and polyunsaturated fatty acids were suggested as key nutrients for proper cephalopod development (Navarro and Villanueva, 2003; Cerezo Valverde et al., 2012). However, supplementation with soybean or marine lecithin did not lead to an improvement in feed performance (Rodríguez-González et al., 2018b, 2019).

The mineral content of cephalopod muscle tissue is twice that of fish, suggesting their high requirements. Specifically, the electrolytes Na and K play a major role in octopus, as well as in their preferred natural diets. Phosphorus, magnesium and calcium follow in order of importance. Copper is essential in the biosynthesis of hemocyanin, the oxygen-carrying pigment in cephalopods and crustaceans, however, its supplementation did not improve feed performance (Celdrán Sancho et al., 2015). Only crustaceans cover the range of concentrations observed in octopus for most of the minerals analyzed (Cerezo Valverde et al., 2015).

In the present invention, a reformulation of feed for fattening octopus has been carried out with the aim of making it economically viable. To do so, initially, a selection of new raw materials other than freeze-dried ones has been carried out to reduce production costs. This process has involved prior acceptability tests and the readjustment of the binders to the new formulation so that the feed could be ingested. In a second phase, it has been essential to correct the nutritional deficiencies of these raw materials, establishing the degrees of supplementation of some nutrients to improve growth rates and nutritional use.

References

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Castro, B.G., Dimarco, F.P., DeRusha, R.H., Lee, P.G. (1993). The effects of surimi and pelleted diets on the laboratory survival, growth, and feeding rate of the cuttlefishSepia officinalisL. Journal of Experimental Marine Biology and Ecology 170, 241-252.

Celdrán, M.M., Cerezo Valverde, J., Sáez Sironi, J., García García, B. (2015). Is copper supplementation required in formulated feeds forOctopus vulgans(Cuvier, 1797)?. Journal of Shellfish Research 34(2), 473-480.

Cerezo Valverde, J., Hernandez, M.D., Aguado-Giménez, F., García García, B. (2008) Growth, feed efficiency and condition of common octopus(Octopus vulgans) fed on two formulated moist diets. Aquaculture 275, 266-273.

Cerezo Valverde, J., Hernández, M.D., García-Garrido, S., Rodríguez, C., Estefanell, J., Gairín, Rodríguez, C.,Tomás, A., García García, B. (2012). Lipid classes from marine species and meals intended for cephalopod feeding. Aquaculture International 20, 71-89. Cerezo Valverde, J., Hernández, M.D., Aguado-Giménez, F., Morillo-Velarde, P.S., García García, B. (2013a). Performance of formulated diets with different levels of lipids and glutamate supplementation in Octopus vulgans. Aquaculture Research 44, 1952-1964. Cerezo Valverde, J., Martínez-Llorens, S., Tomas Vidal, A., Jover, M., Rodríguez, C., Estefanell, J., Gairín, J., Domíngues, P.M., Rodríguez, C.J., García García, B. (2013b).

Amino acids composition and protein quality evaluation of marine species and meals for feed formulations in cephalopods. Aquaculture International 21, 413-433.

Cerezo Valverde, J., Tomás Vidal, A., Martínez-Llorens, S., Pascual, M.C., Gairín, J.I., Estefanell, J., Garrido, D., Carrasco, J.F., Aguado-Giménez, F., García García, B. (2015). Selection of marine species and meals for cephalopod feeding based on their essential mineral composition. Aquaculture Nutrition 21, 726-739-Cerezo Valverde, J., Hernández, M.D., Aguado-Giménez, F., García García, B. (2017).

Development of low-lipid formulated feeds with different protein/energy ratios for Octopus vulganson growing. Aquaculture Nutrition 23, 681-691.

Cerezo Valverde, J, García García, B. (2017). High feeding and growth rates in common octopus(Octopus vulgans) fed formulated feeds with an improved amino acid profile and mixture of binders. Aquaculture Research 48, 3308-3319.

Cerezo Valverde, J, Rodríguez-González, T, Granero Fernández, M.D., Aguado-Giménez, F., García García, B. (2019). Successful rearing of common octopus(Octopus vulgans) fed a formulated feed in an offshore cage. Aquaculture Research 50, 968-972.

Domingues, P.M., López, N., Muñoz, J.A., Maldonado, T., Gaxiola, G., Rosas, C. (2007). Effects of a dry pelleted diet on growth and survival of the Yucatan octopus,Octopus maya. Aquaculture Nutrition 13, 273-280.

Estefanell, J., Roo, J., Guirao, R., Afonso, J. M., Fernández-Palacios, H., Izquierdo, M., Socorro, J. (2012). Efficient utilization of dietary lipids inOctopus vulgans(Cuvier 1797) fed fresh and agglutinated moist diets based on aquaculture by-products and low price trash species. Aquaculture Research 44, 93-105.

Estefanell, J., Mesa, A., Izquierdo, M.S., Ramírez, B. and Socorro, J. (2017). Microdiet for paralarvae of the common octopusOctopus vulgans(Cuvier, 1797) (ES 2 599 603). Spanish Patent and Trademark Office.

FAO (2018) Yearbook of fishery and aquaculture statistics. Fisheries and Aquaculture Department. Food and Agriculture of the United Nations, Rome. http://www.fao.org/fishery/static/Yearbook/YB2018_USBcard/index.htm.

García García, B., Cerezo Valverde, J. (2006). Optimal proportions of crabs and fish in diet for common octopus (Octopus vulgans) growing. Aquaculture 253, 502-511.

García García, B., Cerezo Valverde, J., Aguado-Giménez, F., García García, J. (2009). Growth and mortality of common octopusOctopus vulgans reared at different stocking densities in Mediterranean offshore cages. Aquaculture Research 40, 1202-1212.

García García, J., García García, B. (2011). Econometric model of viability/profitability of octopus (Octopus vulgans) growing in sea cages. Aquaculture International 19, 1177 1191.

García García, B., Cerezo Valverde, J., Gómez, E., Hernández, M.D., Aguado-Giménez, F. (2011). Ammonia excretion of octopus (Octopus vulgans) in relation to body weight and protein intake. Aquaculture 319, 162-167.

García-Garrido, S., Domingues, P.M., Navarro, J.C., Hachero-Cruzado, I., Garrido, D., Rosas, C. (2010). Growth, partial energy balance, mantle and digestive gland lipid composition of Octopus vulgans (Cuvier, 1797) fed with two artificial diets. Aquaculture Nutrition 17, 174 187.

García-Garrido S., Hachero-Cruzado I., Rosas C., Domingues P. (2013). Protein and amino acid composition from the mantle of juvenile Octopus vulgans exposed to prolonged starvation. Aquaculture Research 11, 1741-1751.

MAPAMA (2020). Aquaculture production data. https://www.mapa.gob.es/es/pesca/temas/acuicultura/produccion-de-acuicultura/ Iglesias J, Fuentes L. (2014). Octopus vulgans: Paralarval culture. In Iglesias J, Fuentes L, Villanueva R (eds), Cephalopod Culture, Springer Netherlands, p 427-450.

Mazón, M.J., Piedecausa, M.A., Hernández, M.D., García García B. (2007). Evaluation of environmental nitrogen and phosphorus contributions as a result of intensive ongrowing of common octopus(Octopus vulgans). Aquaculture 266, 226-235.

Morillo-Velarde, P.S., Cerezo Valverde, J., Serra Llinares, R.M., García García, B. (2011). Energetic contribution of carbohydrates during starvation in common octopus(Octopus vulgans). Journal of Molluscan Studies 77, 318-320.

Morillo-Velarde, P. S., Cerezo Valverde, J., Hernández, M. D., Aguado-Giménez, F., García García, B. (2012). Growth and digestibility of formulated diets based on dry and freeze-dried ingredients in the common octopus (Octopus vulgans). Aquaculture 368, 139-144. Morillo-Velarde, P.S., Cerezo Valverde J., Serra Llinares R.M., García García B. (2013) Changes in lipid composition of different tissues of common octopus(Octopus vulgans) during short-term starvation. Aquaculture Research 44, 1177-1189.

Morillo-Velarde, P.S., Cerezo Valverde J., García García B. (2015a). A simple format feed to test the acceptability of ingredients for common octopus(Octopus vulgansCuvier, 1797). Aquaculture Research 46, 994-1000.

Morillo-Velarde, P.S., Cerezo Valverde J., García-García B. (2015b). Utilization of diets with different fish oil content in common octopus(Octopus vulgansCuvier,1797) and resulting changes in its biochemical composition. Aquaculture Research 46, 2871-2884.

Morillo-Velarde, P.S., Cerezo Valverde J., Aguado-Giménez F., Hernández M.D., García García B. (2015c). Effective use of glucose rather than starch in formulated semi-moist diets of common octopus(Octopus vulgans). Aquaculture Nutrition 21, 206-213.

Navarro J.C., Villanueva, R. (2003). The fatty acid composition of Octopus vulgaris paralarvae reared with live and inert food: deviation from their natural fatty acid profile. Aquaculture 219:613-631

Querol, P., Morillo-Velarde, P.S., Cerezo Valverde, J., Martínez-Llorens, S., Moñino, A.V., Jover, M., Tomás, A. (2014). Inclusion of fish and krill meal in extruded diets for Octopus vulgaris (Cuvier, 1797): assessment of acceptance. Aquaculture Research 45, 1421-1424. Querol, P., Gairín, J. I., Guerao, G., Jover, M., Tomás, A. (2015). Growth and feed efficiency of Octopus vulgaris fed on dry pelleted. Aquaculture Research 46, 1132-1138.

Quintana, D., Domingues, P.M., García, S. (2008). Effect of two artificial wet diets agglutinated with gelatin on feed and growth performance of common octopus (Octopus vulgaris) subadults. Aquaculture 280, 161-164.

Rodríguez-González, T., Cerezo Valverde, J., Sykes, A. V., García García, B. (2015). Performance of raw material thermal treatment on formulated feeds for common octopus (Octopus vulgaris) growing. Aquaculture 442, 37-43.

Rodríguez-González, T., Cerezo Valverde, J., Sykes, A.V., García García, B. (2018a). Common octopus(Octopus vulgaris)performance when including fasting on feeding schemes: preliminary data regarding a formulated feed. Advances in Research 13(4), 1 11.

Rodríguez-González, T., Cerezo Valverde, J., García García, B. (2018b). Soybean lecithin dietary supplementation inOctopus vulgarisformulated feeds: Growth, feed efficiency, digestibility and nutritional composition. Aquaculture Research 49, 3777-3791.

Rodríguez-González, T., Cerezo Valverde, J., García García, B. (2019). Performance of marine lecithin supplemented feeds for the common octopus (Octopus vulgaris) growing: changes in proximate composition and lipid classes’ profile. Fishes 4 (47), 1-17.

Rosas, C., Tut, J., Baeza, J., Sánchez, A., Sosa, V., Pascual, C., Arena, L., Domingues, P.M., Cuzon, G. (2008). Effect of type of binder on growth, digestibility, and energetic balance of Octopus maya. Aquaculture 275, 291-297.

Sánchez, F.J., Cerezo Valverde J., García García B. (2014).Octopus vulgaris:Ongrowing. In Iglesias J, Fuentes L, Villanueva R (eds.), Cephalopod Culture, Springer Netherlands, p 451-466.

Tur, R., Rodrígues, P.M., Almansa, E., Lago, M.J., García, P., Pérez, E. (2019). Procedure for the cultivation of paralarvae of the common octopusOctopus vulgaris(ES 2 714 930). Spanish Patent and Trademark Office.

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IV DESCRIPTION OF THE INVENTION

Due to the particular way the octopus feeds, it has been difficult to develop feeds that offer comparable results to natural diets. It is clear that freeze-dried feeds offer the best intake and growth rates, however, their high price and laborious manufacturing process make their application at an industrial level difficult.

The present invention consists of a semi-moist feed for fattening octopus that allows the replacement of feed based on freeze-dried products and natural diets, presenting the following advantages:

A) Regarding the state of the art (Cerezo Valverde and García García, 2017; Cerezo Valverde et al., 2019):

-Minimum amount or absence of freeze-dried products in its composition, improving profitability and the manufacturing process.

-Lower protein percentages (<60% dry matter) compared to other cephalopod feeds (>65% dry matter). Since protein is the most expensive component in the formulation, greater profitability is also expected.

-More than 50% of the protein in the feed developed is of plant, terrestrial animal or supplemental origin, and not of marine origin. These selected proteins are economical and highly available.

-Reduction of cooking time from 2 hours to 15-25 minutes to achieve a stable feed format, minimising the risk of loss of nutritional value and the energy cost of manufacturing.

-Instead of feeding 4-5 days a week, animals can be fed on alternate days without any decrease in performance. This allows more time for other tasks and better management in a facility.

B) Regarding natural diets:

-Intermediate growth rates (12-16 g/day/octopus) between fish and crustacean diets, but with better nutritional use (50-66% of feed incorporated in growth).

-100% of the feed is edible, avoiding the accumulation of waste generated by natural diets and causing less environmental impact.

C) Other general advantages (common to the state of the art):

-Due to the raw materials used, its manufacture does not compete with the high demand for fishmeal and fish oil used in fish feed.

-The feed is preserved by vacuum packaging and in a cold chamber (6-8°C), preserving its organoleptic properties and prolonging its shelf life for at least two months. -High stability in sea water for 24 h with low disintegration rates.

The feed is made up of a wet part and a dry part. The wet part is based on sawdust from hake and swordfish, by-products of low commercial value generated by the frozen fish industry. The optimal proportions of these ingredients have been obtained in preliminary tests.

In the dry part, two essential nutrients are included to correct the deficiencies of fish sawdust. On the one hand, sodium glutamate was used, more as a source of this electrolyte than as an amino acid. Specifically, the supplementation levels have been increased from 0.5-2% used by Cerezo Valverde et al. (2013a) and Cerezo Valverde and García García (2017) to 4-5%, in order to cover the high concentrations of sodium in the muscle tissue of the octopus. On the other hand, taurine supplementation was also necessary, taking as a reference the content in crustaceans and cephalopods. A 2% supplementation would cover 100% of the concentration observed in crustaceans and 70% of that observed in the octopus. The high growth rates of the octopus also suggest high requirements for this nutrient.

Another novelty is the incorporation of inexpensive vegetable protein sources that have been accepted by the octopus, such as soy protein isolate and wheat gluten. Both ingredients complement each other in their essential amino acids (high levels of lysine and low levels of methionine in soy, and low levels of lysine and moderate levels of methionine in gluten) and should be added in similar percentages to achieve optimal performance.

In common with the state of the art is the inclusion of spray-dried egg yolk. It has been used as a source of cholesterol, phospholipids and sulphur-containing amino acids (methionine and cysteine), as well as for its attractive effect and improvement in feed palatability. Likewise, a minimum amount of freeze-dried crab or krill meal has been included to stimulate intake. Unlike what happened with freeze-dried crab, the increase in the krill meal content had a negative effect on feed acceptability.

As the ingredients have been varied with respect to the state of the art, the percentages of binders have necessarily had to be readjusted to achieve an appropriate texture. The most obvious change has been the reduction from 10% of gelatin to 5-6% in the proposed formulation. This has the advantage of replacing this relatively expensive protein source, which is deficient in essential amino acids, with other, better-balanced sources.

Finally, starch has been used as a filling ingredient to adjust the protein content to the reference value observed in crustaceans (58-61% dry matter according to Cerezo Valverde et al., 2013b).

The developed formulas are presented below, showing the weight composition of their ingredients expressed as a % of the total mixture.

SP4 feed

The wet part is composed of the following raw materials:

-Water: 10%

-Hake sawdust (Productos del Mar Ancavico, S.L., Pol. Ind. Els Algars, Cocentaina, Alicante, Spain): 30%

-Swordfish sawdust (Productos del Mar Ancavico, S.L., Pol. Ind. Els Algars, Cocentaina, Alicante, Spain): 20%

The dry part is composed of the following raw materials:

-Soy protein isolate (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 5 %

-Wheat gluten (Distriver Hernández, S.L., Pol. Ind. Oeste, Alcantarilla, Murcia, Spain): 5 % -Egg yolk powder (Avícola San Isidro, S.L., Los Belones, Murcia, Spain). 5 % -Freeze-dried blue crab (Callinectes sapidus) (Acuicex, Los Alcázares, Murcia): 2 % -Antarctic krill flour (CC Moore & Co Ltd, Henstridge, United Kingdom): 2 %

-Soy lecithin (Especias El Reloj, S.L., Pol. Ind. San Luis, Malaga, Spain): 0.5% -Monosodium glutamate (Especias El Reloj, S.L., Pol. Ind. San Luis, Malaga, Spain): 4% -L-Taurine (Guinama, S.L.U., Alboraya, Valencia, Spain): 1%

-Bloom 220 gelatin (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 5% -Pork collagen (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 0.5% -Potato starch (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 10%

I think SP7T2

The wet part is composed of the following raw materials:

-Water: 5 %

-Hake sawdust (Productos del Mar Ancavico, S.L., Pol. Ind. Els Algars, Cocentaina, Alicante, Spain): 40%

-Swordfish sawdust (Productos del Mar Ancavico, S.L., Pol. Ind. Els Algars, Cocentaina, Alicante, Spain): 20%

The dry part is composed of the following raw materials:

-Soy protein isolate (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 5 %

-Wheat gluten (Distriver Hernández, S.L., Pol. Ind. Oeste, Alcantarilla, Murcia, Spain): 5 % -Egg yolk powder (Avícola San Isidro, S.L., Los Belones, Murcia, Spain). 2 % -Freeze-dried blue crab (Callinectes sapidus) (Acuicex, Los Alcázares, Murcia): 2 % -Antarctic krill flour (CC Moore & Co Ltd, Henstridge, United Kingdom): 2 %

-Monosodium glutamate (Especias El Reloj, S.L., Pol. Ind. San Luis, Málaga, Spain): 4% -L-Taurine (Guinama, S.L.U., Alboraya, Valencia, Spain): 2%

-Bloom 220 gelatin (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 5% -Pork collagen (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 1% -Acetyl distarch phosphate (E-1414. Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 1%

-Potato starch (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 6 %

I think SP8GM5

The wet part is composed of the following raw materials:

-Hake sawdust (Productos del Mar Ancavico, S.L., Pol. Ind. Els Algars, Cocentaina, Alicante, Spain): 45%

-Swordfish sawdust (Productos del Mar Ancavico, S.L., Pol. Ind. Els Algars, Cocentaina, Alicante, Spain): 20%

The dry part is composed of the following raw materials:

-Soy protein isolate (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 2 %

-Wheat gluten (Distriver Hernández, S.L., Pol. Ind. Oeste, Alcantarilla, Murcia, Spain): 2 % -Egg yolk powder (Avícola San Isidro, S.L., Los Belones, Murcia, Spain): 2 % -Freeze-dried blue crab (Callinectes sapidus) (Acuicex, Los Alcázares, Murcia): 2 % -Monosodium glutamate (Especias El Reloj, S.L., Pol. Ind. San Luis, Málaga, Spain): 5 % -L-Taurine (Guinama, S.L.U., Alboraya, Valencia, Spain): 2 %

-Bloom 220 gelatin (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 6 % -Pork collagen (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 1 % -Acetyl distarch phosphate (E-1414. Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 1 %

-Potato starch (Productos Sur, S.A., Pol. Ind. Oeste, San Ginés, Murcia, Spain): 12% SP8C0 feed

Composition in ingredients same as the previous feed (SP8GM5) but replacing the 2% of freeze-dried blue crab (Callinectes sapidus) with starch.

Nutritional composition (Values expressed as % of dry matter)

SP4 feed: Humidity = 49.82%; Protein = 59.10%; Lipids = 14.49%

SP7T2 feed: Humidity = 52.04%; Protein = 66.26%; Lipids = 11.53%

SP8GM5 feed: Humidity = 51.41%; Protein = 58.36%; Lipids = 10.50%

SP8C0 feed: Humidity = 51.63%; Protein = 56.86%; Lipids = 10.20%

Manufacturing process

1) Fish sawdust is supplied in the form of frozen blocks or cubes. Before use, the product is defrosted at room temperature in vats or trays, where the released juices are collected (Figure 1A). In the same container, the entire product is mixed and homogenized before being weighed.

2) The quantities of hake and swordfish sawdust are weighed.

3) Weigh the water and soy lecithin in a glass. Dissolve the lecithin completely, avoiding the formation of bubbles (SP4 feed only).

4) In a separate container, weigh and mix the remaining dry ingredients (Figure 1B). 5) Mix the wet and dry parts and knead until a homogeneous mixture is obtained (Figure 1C).

6) Vacuum packing the dough in the form of rectangular sheets 20 to 25 mm thick (Figure 1D).

6) Cooking in a water bath at 50°C for 25 minutes (SP4 and SP7 feeds) or 45°C for 15 minutes (SP8 feeds) (Figure 1E). It is especially important not to exceed 50°C during this process to avoid protein denaturation processes.

7) Place in a cold chamber (6-8°C) for a minimum of 12 hours. After this time, the feed is ready to be used (Figure 1 F).

Supply and feeding method

The feed is removed from the chamber just before feeding the animals. It does not need to be tempered. The bag is opened and cut into cubes (approx. 4x2.5x3 cm) according to the size of the animals (Figures 1G and 1H). To this end, equations have been developed that estimate the absolute feeding rates per animal based on weight (AAR in g/day/octopus) for the feeds that have offered the best results (see Figure 2A-C):

SP4 feed. Weight: 600-1500 g; Temperature: 19-21°C;

Eq. (1):TAA (g/day/octopus) = 0.6651*Weight0.6367; R2 = 70.34 %

SP8GM5 feed; Weights 800-1700; Temperature: 17.5-20.5°C

Eq. (2):TAA (g/day/octopus) = 3.5167*Weight0.3778; R2 = 45.17 %

SP8C0 feed. Weight 900-1900 g; Temperature: 17-19°C

Eq. (3): TAA (g/day/octopus) = 1.0354*Weight0.5193; R2 = 54.64 %

Alternatively, a ration of 5% of body weight or tank biomass can be fed initially, and the ration adjusted based on demand. Animals are fed on alternate days, with a fasting day between feedings. If animals are to be fed on two consecutive days, the estimated ration on the second day will be 50% of that estimated by the equations to avoid feed wastage.

When the animals are small (<800 g) a single portion is provided per animal, ensuring that all animals catch it. This is especially relevant when there are dominant individuals that tend to catch more feed than their share, leaving uneaten remains and causing others to go without food. It is also important to provide the feed simultaneously to all animals to avoid theft and disputes between them. When the animals are large, the ration is provided in two or three portions per animal and not in a single portion.

V. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 (A-H). Stages in the feed production process: (A) Defrosting of fish sawdust; (B) Weighing of the dry part ingredients and mixing; (C) Mixing of the wet and dry parts. Kneading; (D) Vacuum packing; (E) Cooking at low temperature; (F) Final format in plates after 12 h in cold storage (6-8°C); (G) Cutting into portions; (H) Feeding.

Figure 2(A-C). Absolute feeding rates (AFR in g/day/octopus) as a function of octopus weight for the selected feeds: (A) SP4 feed (Weights 600-1500 g; Temperature 19-21°C; AFR = 0.6651*Weight0.6367), (B) SP8GM5 feed (Weights 800-1700; Temperature 19-21°C; AFR = 0.6651*Weight0.6367), (C) SP8GM5 feed (Weights 800-1700; Temperature 19-21°C; AFR = 0.6651*Weight0.6367), (D) SP8GM5 feed (Weights 800-1700; Temperature 19-21°C; AFR = 0.6651*Weight0.6367), (E) SP8GM5 feed (Weights 800-1700; Temperature 19-21°C; AFR = 0.6651*Weight0.6367), (F) SP8GM5 feed (Weights 800-1700; Temperature 19-21°C; AFR = 0.6651*Weight0.6367), (G) SP8GM5 feed (Weights 800-1700; Temperature 19-21°C; AFR = 0.6651*Weight0.6367), (H) SP8GM5 feed (Weights 800-1700; Temperature 19-21°C; AFR = 0.6651*Weight0.6367), (I) SP8GM5 feed (Weights 800-1700; Temperature 19-21°C; AFR = 0.6651*Weight0.6367), (J) SP8GM

17.5-20.5°C; TAA = 3.5167*Weight0.3778) and (C) SP8C0 feed (Weights: 900-1900 g;

Temperature: 17-19°C; TAA = 1.0354*Weight0.5193).

Figure 3. Economic conversion index (ECI or cost of raw material to produce 1 kg of octopus, in €/kg) with the tested feeds (SP4, SP7T0, SP7T2, SP8GM5 and SP8C0) and the reference diets (freeze-dried feed PL, crab, bogue, and mixed diet of crab and bogue). The prices stipulated for the feeds were 2.8, 2.4, 2.7, 2.6, 2.1 and 7.3 €/kg for SP4, SP7T0, SP7T2, SP8GM5, SP8C0 and PL, respectively (raw materials purchased at retail and excluding the cost of manufacturing the feed). For the natural diets, prices of 1.5, 0.75 and 1.1 €/kg were set for crab, fish (bogue) and the mixed diet (3 days bogue-1 crab), respectively.

Figure 4. Time required to reach a commercial size of 1.5 kg from sub-adult specimens of 700-800 g with the developed feeds (SP4, SP7T0, SP7T2, SP8GM5 and SP8C0) and the reference diets (Feed with PL freeze-dried fish, crab, boga, and mixed diet of crab and boga).

VI. PREFERRED EMBODIMENT OF THE INVENTION

Several octopus fattening trials with the developed feeds are presented below as examples of preferred implementation. In order to demonstrate the effectiveness of the selected nutrients and supplementation levels, both feeds with industrial applications (SP4, SP8GM5 and SP8C0) and those with worse results have been included. All the trials have been carried out at the Experimental Cephalopod Aquaculture facilities (ACUICEX. Pol. Ind. de Los Alcázares, 30710 Los Alcázares, Murcia. REGA: ES309020140027).

General conditions common to all tests

The animals came from the Mediterranean Sea and were caught by professional fishermen using trawling. The animals in good condition were kept on board in 500-litre nursery tanks, with continuous water renewal until the port of destination. The specimens were then transported by road to the ACUICEX experimental facilities.

Upon arrival, the animals were housed in 2000-litre circular tanks maintained in recirculation with temperature control (17-20°C) and oxygen levels above 80% saturation. The water circuits were equipped with the necessary equipment to ensure optimal conditions for water quality and animal welfare, including: mechanical and biological filtration, aeration, skimmer, UV lamps and PVC pipes for shelter. Other conditions included: ammoniacal nitrogen concentration < 0.1 mg/l, nitrites < 0.4 mg/L nitrates < 50 mg/l, pH 7.5-8, KH = 5-8°, Salinity 36-38 %o and natural photoperiod. After appropriate acclimatisation (7-10 days) and checking that they regularly accepted natural diets based on crab and fish, the experiments were started under the following conditions:

-The animals were weighed at the beginning and at the end of the test; 5 specimens per tank (3 females and 2 males)

-Trial duration between 30 and 50 days

-Feeding to satiety on alternate days (3-4 days per week), supplying the feed first thing in the morning (8-9 am) and collecting and weighing the surplus at the end (13-14 h).

-The following indices were obtained from the data obtained:Pi= Initial average weight (g);Pf= Final average weight (g);IP= Weight increase (g);TAA= Absolute Feed Rate (g/day/octopus);TAR= Relative Feed Rate (%Weight/day);TAAP= Absolute protein feed rate (g/day/octopus);TCA= Absolute Growth Rate (g/day/octopus);

TEC= Specific daily growth rate (% weight/day);IEA= Feed efficiency index (%) = (TCA/TAA)*100;IC= Conversion index (TAA/TCA);PER= Protein efficiency ratio (TCA/TAAP)

Example 1. Fattening of subadults and adults of common octopus fed with a reference formulated feed (SP4) in 300 or 2000 L tanks.

Fifteen octopus specimens were fed the same reference feed (SP4) for 30 days (19.8±0.7°C) under different housing conditions: Group SP4I (5 subadult specimens housed individually in 5 300 L tanks; Pi = 757±94 g), Group SP4GS (5 subadult specimens housed in a group in a 2000 L tank; Pi = 628±78 g) and Group SP4GA (5 adult octopuses housed in a group in a 2000 L tank; Pi = 1038±60 g). At the end of the trial, survival was 100 % in all groups. The worst results were obtained in the SP4I group, with lower intake rates (TAA = 17.76 g/day/octopus; TAR = 2.02 %P/day), growth (Pf = 1000±110 g; IP = 243 g; TCA = 8.10 g/day; TEC = 0.93 %P/day) and diet utilization (IC = 2.21; IEA = 45.61 %; PER = 1.54). Although the specimens of the SP4GS group were smaller, the results of intake (TAA = 20.69 g/day/octopus; TAR = 2.63 %P/day), growth (Pf = 945±98; IP = 316 g; TCA = 10.55 g/day; TEC = 1.36 %P/day) and diet utilization (IC = 1.96; IEA = 51.07 %; PER = 1.72) were better than those of the SP4I group. The best results were obtained in the SP4GA group, both for intake (TAA = 32.52 g/day/octopus; TAR = 2.54 %P/day) and growth (Pf = 1523 g; IP = 485 g; TCA = 16.17 g/day; TEC = 1.28 %P/day). The nutritional utilization indices were similar (IC = 2.01; IEA = 49.72% and PER = 1.68).

The results show that space availability is a limiting factor for the growth of the species and that group housing stimulates competition for feed capture, improving intake and growth. The best performance with the reference feed SP4 is obtained in adult specimens (>900 g) and in large tanks (2000 L), with growth close to half a Kg per month per individual.

Example 2. Fattening of common octopus subadults with feed formulated with different degrees of taurine supplementation (SP7T0 and SP7T2).

Three groups of 5 subadult octopuses each were housed in 2000 L tanks. Two of the groups were fed for 30 days (19.8 ± 0.7 °C) with the formulated feed SP7, but varying the taurine content: Group SP7T0 (0 % L-Taurine supplementation; Pi = 834 ± 31 g;) and Group SP7T2 (2 % L-Taurine; Pi = 859 ± 97 g). The 2 % Taurine supplementation was performed replacing 2 % starch in the formulation. A third control group (Group C; Pi = 875 ± 94 g) was fed exclusively with fresh or thawed blue crab (Callinectes sapidus). Taurine supplementation slightly improved intake rates (TAA = 25.11 g/day/octopus and TAR = 2.46 %P/day) compared to the unsupplemented group (TAA = 21.98 g/day/octopus and TAR = 2.23 %P/day). Likewise, growth rates were slightly higher in the SP7T2 group (Pf = 1181 ± 91; IP = 322 g; TCA = 10.73 g/day/octopus; TEC = 1.06 %P/day) compared to the SP7T0 group (Pf = 1133 ± 77 g; IP = 299 g; TCA = 9.97 g/day/octopus; TEC = 1.02 %P/day). L-Taurine supplementation did not affect diet utilization (Group SP7T0: IC = 2.20; IEA = 45.35 %; PER = 1.52; and Group SP7T2: IC = 2.34; IEA = 42.74 %; PER = 1.34). The best results in terms of intake (TAA = 75.03 g/day) and growth (Pf = 1381 ± 158; IP = 506 g; TCA = 18.05 g/day; TEC = 1.52 %P/day) were obtained with the crab diet, although at the expense of a worse diet utilization (IC = 4;16; IEA = 24.1 %; PER = 2.07). Considering that these indices have been calculated based on the food ingested, the incorporation of the edible fraction (50% in the case of crab) in the calculation would penalize these values even more (IC = 8.32 and IEA = 12.0%).

The results suggest that improvements with taurine supplementation occur by stimulating intake, and therefore growth, but without modifying the utilization of the feed consumed.

Example 3: Fattening of common octopus with feed formulated with different degrees of monosodium glutamate supplementation (SP8GM2 and SP8GM5 feeds).

Two groups of 5 octopuses were fed for 45 days (17.9±0.3°C) with two formulated feeds with different MSG contents: group SP8GM2 (2% MSG; Pi =1270±108 g;) and group SP8GM5 (5% MSG; Pi =1103±130 g). The supplementation of 3% additional MSG in SP8GM5 feed was compensated by removing 3% potato starch. A third control group (Pi = 1175±110 g) was fed exclusively with fresh or thawed blue crab (Callinectes sapidus). The feeds were accepted by all animals, with 100% survival rates in both groups. Feeding rates were similar for both feeds (Group SP8GM2: TAA = 23.50 g/day/octopus and TAR = 1.54 %P/day; Group SP8GM5: TAA =22.91 g/day/octopus and TAR = 1.59 %P/day), however, octopuses fed with SP8GM5 feed showed higher growth (Pf = 1783 ± 184; IP = 680 g; TCA = 15.11 g/day/octopus; TEC = 1.07 %P/day) compared to those fed with SP8GM2 feed (Pf = 1790 ± 60; IP = 520 g; TCA =11.56 g/day/octopus; TEC = 0.76 %P/day). Likewise, feed utilization was better with SP8GM5 feed (IC = 1.52; IEA = 65.95 %; PER = 2.33) compared to SP8GM2 feed (IC = 2.03; IEA = 49.18 %; PER = 1.94). The best results in terms of intake and growth were obtained with diet C (Pf = 2039 ± 213; IP = 864 g; TCA = 19.19 g/day; TEC = 1.22 %P/day), at the expense of a worse conversion (IC = 4.28; IEA = 23.30 %; PER = 2.01).

According to these results, improvements with monosodium glutamate supplementation do not occur by stimulating intake, but through an increase in feed conversion.

Example 4: Fattening of common octopus with feed formulated without raw materials derived from crustaceans (SP8C0 Feed).

The aim was to test the performance of an inexpensive and easy-to-produce feed, avoiding the laborious process of freeze-drying crustaceans. In addition, the levels of taurine (2%) and monosodium glutamate (5%) supplementation that had offered the best results in examples 2 and 3 were incorporated. Therefore, the composition of the feed was the same as that of SP8GM5 tested in example 3, but replacing the 2% of crab freeze-dried food with starch (SP8C0 feed). Two groups of 5 octopuses of different sizes, Subadult group (Pi = 971±148 g; T =) and Adult group (Pi = 1370±130 g) were fed for 45 days (17.9±0.3°C) with SP8C0 feed. The feed was accepted by all animals with 100% survival rates. Absolute feeding rates were moderate in both groups (19.7 and 22.2 g/day for subadults and adults, respectively). Despite differences in animal weight, growth rates were similar (Subadult Group: Pf = 1498±290 g; IP = 527 g; TCA = 11.7 g/day; TEC = 0.96 %P/day; Adult Group: Pf = 1911±266 g; IP = 541 g; TCA = 12.0 g/day; TEC = 0.74 %P/day). Diet utilization was better in subadult individuals (IC = 1.68; IEA = 59.5 %; PER = 2.16) than in subadults (IC = 1.84; IEA = 54.2 %; and PER = 1.97).

The main advantage of SP8C0 feed is its price and ease of production compared to other feeds developed, although further testing is required to achieve high growth rates by incorporating crustaceans into the formulation.

VII INDUSTRIAL APPLICATION

The direct application is feeding octopus during the fattening phase in aquaculture facilities (producers). On the other hand, the frozen fish processing industry would benefit from the use and increase in the value of its by-products. Likewise, through this procedure, the usefulness of other by-products other than those tested could be tested, as well as their application for feeding other species. It is noteworthy that the use of fishing by-products is in line with the criteria of sustainable development, circular economy and blue growth that are being promoted in the EU. Finally, feed manufacturers would also be involved, potentially increasing the supply for a new aquaculture sector such as cephalopod farming.

In order to make a comparison between the performance of the developed feeds and those preceding the state of the art (feeding with fish or crustaceans of low commercial value or with feed based on PL freeze-dried ingredients; Cerezo Valverde et al., 2019), the values of the ICE (Economic Conversion Index or cost of the raw material to produce a Kg of octopus, in €/Kg; Figure 3) and the time required to reach a commercial size of 1.5 Kg from sub-adult specimens (700-800 g) with the different diets are shown below (Figure 4). This is the weight range selected because it is the closest to the tests carried out.

Of all the feeds developed, the best ICE would be obtained with the SP8C0 feed (3.5 €/Kg), followed by SP8GM5 (3.9 €/Kg), SP7T0 (5.2 €/Kg), SP4 (5.6 €/Kg), SP7T2 (6.4 €/Kg) and PL (10.4 €/Kg). As for natural diets, a crustacean diet would have an ICE of 12.8 €/Kg, a fish diet 2.6 €/Kg, and a mixed fish and crustacean diet 7.3 €/Kg. However, the time needed to reach 1.5 Kg varies greatly depending on the diet selected. With a crustacean diet or PL feed, octopuses would double their weight in just 40 days. At the other extreme, with a fish-based diet it would take 74 days. The SP4 and SP8GM5 feeds would have similar values (46-49 days) and close to the optimal diets. With the cheapest feed (SP8C0) it would take 64 days to reach the weight of 1.5 kg. Therefore, the cheapest diet does not have to be the most profitable, as it takes longer to reach a certain commercial size, and increases the operating costs of a facility. Based on this analysis, the SP8GM5 and SP8C0 feeds would be the most appropriate for an industrial facility. In any case, the high selling price of octopus to the final consumer (10-15 €/kg, depending on weight) would leave a sufficient profit margin to make an octopus fattening farm profitable with the feeds developed.

Claims (3)

1. Semi-moist formulated feed for fattening octopus characterized by the following composition: -Water: 0-10% -Hake sawdust: 30-45% -Swordfish sawdust: 20% -Soy protein isolate: 2-5% -Wheat gluten: 2-5% -Egg yolk powder: 2-5% - Freeze-dried blue crab (Callinectes sapidus): 0-2% -Antarctic Krill Meal: 0-2% -Soy lecithin: 0-0.5% -Monosodium glutamate: 4-5% -L-Taurine: 1-2 % -Gelatin Bloom 220: 5-6% -Pork collagen: 0.5-1% -Distarch acetylated phosphate (E-1414): 0-1% -Potato starch: 6-15% 2. Process for the production of formulated feed according to claim 1 comprising the following steps: -Defrosting, weighing and mixing of wet ingredients -Dissolving soy lecithin in water -Weighing and mixing dry ingredients -Mixing and kneading all the above ingredients -Vacuum packaging of the resulting mass -Cooking in a bain-marie at temperatures between 45-50° for 15-25 minutes -Cooled in a chamber (6-8°C) for 12 hours 3. Procedure for fattening octopus with a semi-moist feed of composition and preparation described in claims 1 and 2, respectively, using a feeding protocol based on alternate days and where the animals are housed in groups.