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    • 专利摘要:
    Procedure for the culture of paralarvae of the common octopus Octopus vulgaris until its settlement (benthic phase) based on a diet of prey comprising the caprolids Phtisica marina and Caprella equilibra and/or gammarids of the genus Jassa spp. The conditions of light, water renewal, temperature among other factors are optimized to obtain the maximum survival of the paralarvae. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2714930A1
    申请号:ES201731369
    申请日:2017-11-29
    公开日:2019-05-30
    发明作者:Estrada Ricardo Tur；Dos Santos Domingues Pedro Miguel Rodrigues；Berro Eduardo Almansa；ROUCO Mª JESÚS LAGO；Fernández Pablo García；Rial Evaristo Pérez
    申请人:INST ESPANOL DE OCEANOGRAFIA；
    IPC主号:
    专利说明:

    [0001]
    [0002] Procedure for the culture of paralarvae of the common octopus Octopus vulgaris
    [0003]
    [0004] The present invention relates to a method for the larval culture of common octopus (Octopus vulgaris). In particular, it refers to a culture protocol that allows reaching the settlement phase of the paralarvae of this species. The present invention therefore belongs to the aquaculture sector.
    [0005]
    [0006] BACKGROUND OF THE INVENTION
    [0007] The common octopus (Octopus vulgaris) is a species with high potential for diversification in aquaculture due to its rapid growth, high fecundity, easy adaptation to captivity, high market price and high demand. Octopus industrial fattening began in 1996 in Galicia, northwest of Spain. This interest arises from results obtained by researchers from the Spanish Institute of Oceanography, IEO (Iglesias, J. and Fuentes, L. (2014) "Octopus vulgaris paralarval culture," in Cephalopod Culture, eds J. Iglesias, L. Fuentes, and R. Villanueva (New York, NY, Heidelberg, Dordrecht, London: Springer), 427 450.), in pilot experiences in which it was found that juveniles of 750 g reached commercial size (of 2.5-3 kg) in only 4 months. Following the advances marked by these initial results, during 1997 a production of 12 metric tons (t) was obtained, located mainly in the Galician Rias Bajas (García-García, J. et al (2004) Cost analysis of octopus ongrowing installation in Galicia. Spanish Journal of Agricultural Research, 2: 531-537).
    [0008]
    [0009] However, the potential of this type of production is very limited, because the supply of individuals for fattening is totally dependent on juveniles caught in the wild. Due to these reasons, this type of production is reduced to only 4.5 t in 2012 and without significant data at present. Another aspect that limits this production is the absence of an artificial diet for adults, currently dependent on frozen dams of low commercial value (eg crustaceans, fish, etc.). However, the latest studies in this field are improving the results obtained with artificial diets (Cerezo Valverde, J., & Garcia, BG (2017) High feeding and growth rates in common octopus (Octopus vulgaris) fed formulated feeds with an improved amino acid profile and mixture of binders. Aquaculture Research, 48 (7), 3308-3319). The solution to all these problems involves the production of juveniles through cultivation techniques, instead of capturing them from the natural environment. However, the high mortality (practically 100%) in the first days of life prevents the production of juveniles at a commercial level (Iglesias and Fuentes, 2014).
    [0010]
    [0011] For all this, the viability of octopus aquaculture at a commercial level depends mainly on increasing the survival of individuals during the life phase known as paralarva. This phase is characterized because individuals live in the water column (they are planktonic). The duration of this phase depends a lot on the temperature and the diet, being able to last about 65-70 days at 20 ° C (Iglesias y Fuentes, 2014). Initially these individuals have a characteristic physiognomy (Figure 1), weighing between 0.20-0.30 mg of dry weight and presenting 3 suckers in each arm. When the octopus exceeds 9 mg of dry weight and shows about 18-20 suckers, they acquire the typical shape of a small adult octopus and migrate to the bottom (they become benthic), entering the juvenile phase. At this point, it is important to clarify that part of the literature on common octopus culture has been produced in Japan (Okumura, S., et al. (2005) Improved survival and growth in Octopus vulgaris paralarvae by feeding large type Artemia Sp. and Pacific Sandeel, Ammodytes Personatus, Aquaculture 244, 147-157). However, recently, it has been shown that the population of Japan is a different species now called Octopus sinensis (Amor, MD et al. (2017).) Morphological assessment of the Octopus vulgaris species complex in light of molecular-based phylogenetic inferences. Zoologica Scripta, 46 (3), 275-288), so any comparison should be made with caution, as the results have been different and not very reproducible in the European population (Iglesias y Fuentes, 2014).
    [0012]
    [0013] Mortality in the paralarva phase seems to be related to zootechnical and / or nutritional aspects. In the first stages of life, the best results have been obtained with live prey, being the best accepted to date the larvae (zoeas) of crustacean decapods and especially of the spider crab (Maja sp). The first time the O. vulgaris cycle was closed under captive conditions was in 2001 using king crab zoeas as prey, but in that trial only two octopi reached adulthood. Other authors have obtained similar results, but never exceeding 5% survival in the settlement (Iglesias y Fuentes, 2014).
    [0014] It should also be added that these experiences have not been replicable, since subsequent results have shown great variability (Garrido, D., et al. (2016).) Meta-analysis approach to the effects of live on the growth of Octopus vulgaris paralarvae under culture conditions Reviews in Aquaculture doi: 10.1111 / raq.12142). To all this, it is added that in all of them, crustacean zoeas such as spider crab that are not viable on a commercial scale have been used given the cost and difficulty of obtaining them on a large scale.
    [0015]
    [0016] To this day, the only commercially viable prey is Artemia sp., A small crustacean easily available and widely used in aquaculture to feed fish larvae. However, the tests carried out with this Artemia sp. they have not achieved the growth or survival necessary to achieve the settlement of the paralarvae, despite numerous attempts to enrich their nutritional profile. Another alternative to the use of these dams has been the design of artificial inert microdiets (0.5-2 mm in diameter) with a nutritional profile suitable for the development of the larva. Several studies have tried this route but until now there have been no significant improvements in survival and growth, possibly due to a sum of causes such as low acceptance, low buoyancy or the lack of an adequate nutritional profile. Recently, a patent has been requested (ES 2 599 603) proposing a new type of inert diet, improving previous results. However, the results obtained by this diet (2.5 mg of dry weight at 73 days) are still far from the 9.5 mg of dry weight at 45 days obtained in previous studies with king crab zoea. For all this, it is necessary to identify alternative dams that can be produced under captivity at a reasonable cost or that are abundant and easy to obtain in the natural environment.
    [0017]
    [0018] Finally, another essential aspect is the need to have a standardized culture procedure or protocol that has proven effective. In this regard, studies have been numerous, as is evident in the aforementioned review by Iglesias and Fuentes (2014), although the results are still insufficient. In this line, recent studies (Garrido, D., et al. (2017), Assessment of stress and nutritional biomarkers in cultured Octopus vulgaris paralarvae: effects of geographical origin and dietary regime Aquaculture, 468, 558 568) proposed a standardized protocol comparing their results in different octopus populations (Atlantic and Mediterranean). This protocol has served as the basis to different tests carried out within the OCTOWELF project (AGL2013-49101-C2-1-R, MINECO, Government of Spain).
    [0019]
    [0020] The invention presented below makes use of a new dam, which is easy to collect from the natural environment due to its abundance in certain marine niches and also achieves growth values in paralarvae superior to those of Artemia sp. to those obtained with zoa of crustacean decapods, allowing their settlement when exceeding 9 mg of dry weight at 65-75 days of age. An improved culture protocol is also proposed based on previous studies that have allowed significant increases in the survival of the paralarvae throughout the entire planktonic period and up to the time of settlement.
    [0021]
    [0022] DESCRIPTION OF THE INVENTION
    [0023]
    [0024] The procedure for the larval culture of the common octopus Octopus vulgaris of the present invention has allowed to obtain a significant improvement in the viability of the first stages of life of the octopus (called paralarva), overcoming the bottleneck that has been the survival until now of the paralarvae for the commercial cultivation of this species.
    [0025]
    [0026] The inventors have found that the use of novel parameters of culture and, especially, of new dams (food) has allowed to obtain high rates of survival and growth in paralarvae of octopus, with respect to the previous crops of this species.
    [0027]
    [0028] The term paralarva in the present invention refers to the state of development of the common octopus Octopus vulgaris from the moment it has left the egg covers (hatching) to its total settlement on the seabed or tank (benthic phase). You can not talk about the larval period since they do not undergo a metamorphosis properly speaking, so the term paralarva is used to differentiate it from the juvenile-adult phase. The paralarva lives in the water column, which is why it is also called the "plantonic phase." The duration of this phase depends a lot on the cultivation conditions (water temperature, light, volume of culture, etc.) and the diet As an example, it can be said that at 20 ° C and a suitable diet, the duration of This phase is 65-75 days (Iglesias y Fuentes, 2014). Initially these individuals have a characteristic physiognomy (Figure 1), weighing between 0.2-0.3 mg (dry weight). When the octopus exceeds 9 mg of dry weight, it is considered that they have reached the settlement phase (normally this occurs between 65-75 days of age). At that moment they begin to acquire the typical form of a small adult octopus (Figure 6) and migrate to the bottom (they become benthic). From this moment they eat mainly at the bottom of the tank (and not in the water column) and tend to look for a shelter (some kind of hollow), showing preference for areas with low lighting.
    [0029]
    [0030] Prior to settlement, there is an intermediate phase that we can call pre-settlement that begins when the paralarva has a dry weight between 6 and 9 mg, which usually occurs from 50-70 days of life and lasts until the time of settlement. This pre-settlement phase is characterized by the fact that the paralarva begins to migrate to the bottom without fully settling and culminates when reaching the settlement phase.
    [0031]
    [0032] Once settled, the juvenile phase begins (benthic). In this phase, individuals will feed on crustaceans such as crabs and small shrimp (about 1 cm in length) and can also start supplying an inert diet based on these same frozen crustaceans or some type of specific feed for cephalopods (Churches and Fuentes, 2014). However, the present invention relates to the process of cultivation until the settlement of the paralarvae. For the present invention, in particular, the gammaids of the genus Jassa have been identified . spp., preferably Jassa falcata and Jassa marmorata, and two genera of caprolids : Phtisica spp. and Caprella spp., preferably Phtisica marina and Caprella equilibrate as new prey for the culture of octopus paralarvae.
    [0033]
    [0034] Both gammarids and caprolids belong to the group of amphipods.
    [0035]
    [0036] Then, the present invention relates to a process for the culture of paralarvae of the common octopus Octopus vulgaris emplaced in a culture tank containing water, characterized in that said method comprises the addition of selected prey of:
    [0037] - gammarids of the genus Jassa spp.,
    [0038] - caprélidos of the genera Phtisica spp. and / or Caprella spp. or
    [0039] - combinations of them
    [0040] to said culture tank.
    [0041]
    [0042] Preferably, the gammarids of the genus Jassa spp. they are selected from Jassa falcata and Jassa marmorata.
    [0043]
    [0044] Preferably, the catellites of the genus Phtisica spp. they are Phthisica marina and the caprélidos of the genus Caprella spp. they are Caprella equilibra.
    [0045]
    [0046] These prey can be added as sole food or in combination with Artemia sp. or other crustacean zoeas (for example when the number of prey obtained is not enough or the paralarvae at birth do not have a size that allows them to capture and ingest the prey). In the case of both dams, the most common densities described in the state of the art can be used and can range between 0.1 and 1 individuals / mL (preferably around 0.5 mL). As indicated in the state of the art, it should be given in several doses, preferably between 3 and 5 doses per day.
    [0047]
    [0048] In the case of adding combinations (mixture) of gammarids of the genus Jassa spp. and caprélidos of the genera Phtisica spp. and / or Caprella spp. preferably between 1 and 10% of the total number of prey in the form of caprolids of the genera Phtisica spp. and / or Caprella spp. and between 90 and 99% of gammarids of the genus Jassa spp . More preferably, 5% of caprolids of the genera Phtisica spp. and / or Caprella spp. and 95% of gammarids of the genus Jassa spp.
    [0049]
    [0050] In a preferred embodiment, the gammarids are added from a day comprised between 1-10 days of life of the paralarva until the settlement thereof, while the caprolids are added from 20-30 days of life and until the settlement of the same.
    [0051]
    [0052] When the gammaids are administered together with the caprolids, the proportions used of both are preferably those indicated above. The beginning of the feeding with gammaridos of the sort Jassa spp can vary between on day 1 (day of hatching of the egg) and 10 days of age as mentioned. This beginning of feeding can vary between said days of life taking into account the size of the paralarvae at birth and their capacity to attack and ingest the prey, something that the expert in the matter can easily see by observing the behavior of the paralarvae.
    [0053]
    [0054] In another preferred embodiment of the invention, the gammaids of the genus Jassa spp. from the first day of life of the paralarvae and until their settlement and the caprélidos of the genera Phtisica spp. and / or Caprella spp. they begin to be administered from day 10 of life until their settlement. Preferably, the ratio between the gammarids of the genus Jassa spp. and caprélidos of the genera Phtisica spp. and / or Caprella spp. when they are administered together, it is as indicated above.
    [0055]
    [0056] In another preferred embodiment of the invention, both gammaids of the genus Jassa spp. as caprélidos of the genera Phtisica spp. and / or Caprella spp. from day 30 of life of the paralarvae and until its settlement. Preferably, the ratio between the gammarids of the genus Jassa spp. and caprélidos of the genera Phtisica spp. and / or Caprella spp. when they are administered together, it is as indicated above.
    [0057]
    [0058] In a preferred embodiment, the density of paralarvae in the culture tank is between 3-7 paralarvae / liter in order to optimize the total number of prey with a good ratio of prey / paralarva, as well as to avoid the stress of the individuals. In the case of settlement tanks (see details below), the density of individuals will be reduced, with a density of 0.1 and 0.5 paralarvae per liter being preferable.
    [0059]
    [0060] In a preferred embodiment, a total of about 3-5 individuals of these preys are supplied to the culture tank (either gammarids of the genus Jassa spp., Caprelids of the genera Phtisica spp., And / or Caprella spp., Or combinations thereof). by paralarva / day, distributing the intakes so that there are always prey available in the tank. To achieve this, the first shot is adjusted by making an approximation of the number of prey remaining in the tank of the previous day, with the total number of shots per day preferably ranging from 1 to 4. As an example. If there are 300 paralarvae in the tank, the number of prey items to add per day would be 900 (300x3) in case 3 dams were supplied per paravava / day. These 900 dams would be added distributed between 1 to 4 takes preferably.
    [0061]
    [0062] Preferably, the size of the prey supplied will vary according to the size / age of the paralarvae. In a preferred embodiment, in a first phase, from day 1 of life to 20-30 days of age, gammarids of the genus Jassa spp. between 1-4 mm of total length (distance between the telson and half of the eyes). From this point (20-30 days of age) and until 50-70 days, gammarids between 2 and 8 mm in length and caprelids of the genera Phtisica spp. and / or Caprella spp. between 4 and 30 mm in length. Finally, between 50 and 70 days of life and until settlement, the diet is again restricted to gammarids with a size between 1 and 4 mm in total length. This last change is due to observations made in newly settled paralarvae, which apparently become more vulnerable during the settlement period, and may be attacked at the bottom of the tank by the larger gammarids.
    [0063]
    [0064] In a preferred embodiment, the culture tanks used between the first day of life and the pre-settlement phase (between 6-9 mg of dry weight and about 50-70 days of life) are preferably frustoconical and black with a volume that can preferably be between 100 and 1000L. In a more preferred embodiment, those paralarvae that reach 6 mg of dry weight (usually occurs between 50 and 70 days) will be transferred individually to another culture tank (hereinafter referred to as a settlement tank). This settlement tank will have a flat bottom and will preferably have a light color (eg Gray) and a rectangular or quadrangular shape, more preferably it will have between 200 and 400 L of volume, height between 40-60 cm, and it will be partially covered with a mesh or canvas. These settlement tanks will be prepared previously, filling them with water from the original tank (matured water) and will preferably include dens (eg PVC pipes 1-2 cm in diameter) and small stones to enrich the habitat. All this will minimize the stress associated with changing the tank.
    [0065]
    [0066] Another important aspect in the protocol of this invention is that the bottom of the tanks is maintained without siphoning (that is, without sucking the remains of food and larvae that may be in the bottom) throughout the cultivation of the paralarvae. In this way, the surviving prey is allowed to colonize and keep it clean, since by being Detritivores consume the organic matter deposited in it, avoiding the proliferation of pathogens. This in turn improves the welfare of the paralarvae by eliminating the possible stress caused by the siphoning process.
    [0067]
    [0068] In a preferred embodiment of the method of the invention, during the cultivation of the paralarva the tank is artificially illuminated by a lamp or light placed on the edge of the tank (instead of a central position), causing a change in the angle of the tank. incidence of light on the surface and resulting in heterogeneous light conditions within the water column, due to changes in the reflection and refraction of light. Preferably, a fluorescent lamp (cold white) will be used.
    [0069]
    [0070] Preferably, the intensity levels of the supplied light vary between 40 and 800 lux (69-1436 W / m2). More preferably, the levels used range from 600 to 800 lux (1077-1436 W / m2) for the first 10-15 days of life of the paralarvae and between 300 and 600 lux (517-1077 W / m2) from the 10-15 days of life. At the moment that the first paralarvae are observed in pre-settlement (between 6-9 mg and at 50-70 days of life), the light is reduced to a range between 40 and 300 lux (69-517 W / m2) and it stays that way until all the paralarvae have settled or transferred to the settlement tank.
    [0071]
    [0072] Once the paralarvae have been transferred to the settlement tanks, the same position of the lamp and the last intensity used in the frustoconical tanks will be maintained (40 to 300 lux, that is, 69-517 W / m2). The photoperiod throughout the development of the paralarva (from the first day of life) until the settlement will have between 10 and 14 hours of light, preferably from 8:00 to 22:00.
    [0073]
    [0074] Regarding the rest of the conditions of the water of crop, the percentage of renewal and the use of green water constitute also an important aspect. In both cases, numerous variants of the protocols can be found in the bibliography, but there are no trials that allow to see differences between them. For all this, in the present invention a protocol is proposed that varies both aspects (renewal and green water) depending on the development of the paralarva.
    [0075]
    [0076] Preferably, during the first two days there is no type of water renewal in the tanks. Subsequently, it begins to renew with a preferable flow rate that renews between 4 and 10% of the total volume of the tank every hour. The renewal time is increased gradually, so that it is renewed 20% daily until the 5 days of life of the paralarvae and from there it is increased until reaching 100% daily at 10-15 days. This renewal is maintained at 100% until 30 days and from there, the renewal is left open permanently (24 hours) which represents a daily renewal of approximately 200%. This percentage of renewal will be maintained until the paralarvae settle or are transferred to the settlement tanks (reaching between 6 and 9 mg of dry weight and about 50-70 days of life). This percentage of renewal (around 200% per day) will remain the same in the settlement tanks.
    [0077]
    [0078] The beginning of the renovation will preferably be done at noon (around 12:00) after the routine maintenance tasks (measurement of oxygen, temperature, etc.) and the first food intake, unless there is an oxygen fall (below 5.8 mg / L), in which case the flow will be opened as soon as possible and the expected time will be maintained, and may be extended if the oxygen levels do not exceed 5.8 mg / L.
    [0079]
    [0080] The recirculation of the water in the culture tank can be carried out by means of a system that can operate both in a closed circuit (by recirculating the water) and in an open circuit. In the latter case, a filtration system should be used to prevent the entry of sediment or any living organism, especially those that may be potentially pathogenic. The water outlet of the tank will be made through an outlet filter preferably a tube in the central position and with a mesh light between 300-400 microns, preferably 300 microns.
    [0081]
    [0082] In turn, and preferably, the method makes use of the green water technique, technique consisting of the addition of a mixture of microalgae (phytoplankton), preferably Isochrysis spp and Nannochioropsis spp.
    [0083] The addition of microalgae is preferably done once a day (from the first day of life of the paralarvae) if necessary and the amount added is necessary to obtain a concentration between 0.7 and 1.5 x 106 cls / mL being preferable 1 x 106cls / mL Preferably, the addition of algae is done at the same time each day (once the renewal is closed) and only if necessary to reach the concentration level of them mentioned above. After 30 days the addition of microalgae is eliminated and they will not be added to the settlement tank either.
    [0084]
    [0085] In a preferred embodiment, the concentration of oxygen in the culture water is always maintained higher than 5.5 mg / L (and preferably higher than 6 mg / L). At the most you can reach up to 100% saturation, by means of an aerator with moderate flow, otherwise, there is danger of anoxia and massive mortality of the paralarvae.
    [0086]
    [0087] In another preferred embodiment, the salinity of the water will be the natural one of the water of the sea (around 35-36 g / L) avoiding sudden falls of can cause massive mortalities.
    [0088]
    [0089] In another preferred embodiment, the temperature of the culture water is always maintained between 18-22 ° C.
    [0090]
    [0091] To determine the dry weight of the paralarvae, which is used as a reference for the different phases of the crop, a sample of paralarvae is taken from the tank (preferably between 10-30 paralarvae) that will be anesthetized (Cl2Mg 1.5% in seawater ) and slaughtered (Cl2Mg 3.5% in seawater) following the indications of Fiorito et al., (2015). Guidelines for the Care and Welfare of Cephalopods in Research-A consensus based on an initiative by CephRes, FELASA and the Boyd Group. Laboratory animals, 49 (2_suppl), 1-90. These larvae will be dehydrated in an oven (100 ° C for 20h) and weighed in precision scales.
    [0092]
    [0093] The method or culture protocol of the present invention is advantageous with respect to the protocols already known. In relation to the dams used, its great advantage over other previous ones (eg Zoea de centolla) is that they are very easy to obtain, since they are produced naturally and massively in the rafts, cetáreas and purification of mussels from the Galician estuaries ( Camacho, AP, Gonzalez, R., & Fuentes, J. (1991), Mussel culture in Galicia (NW Spain), Aquaculture, 94 (2-3), 263 278). It is also a species of wide geographical distribution and with seasonal abundance variations whose maximum peak coincides with the natural period of hatching of the octopus, having described up to 30 individuals per cm2 in the Mediterranean (Scinto, A., Benvenuto, C., Cerrano, C., & Mori, M. (2007). cycle of Jassa marmorata Holmes, 1903 (Amphipoda) in the Ligurian Sea (Mediterranean, Italy). Journal of Crustacean Biology, 27 (2), 212-216).
    [0094]
    [0095] Both groups of amphipods (gammarids and caprelids) are easily obtained from the ropes used in the rafts of the mussel culture located in the Galician estuaries. On the other hand, gammarids can also be obtained in the output channels of cetareas for the maintenance of crustaceans and mussel sewage treatment plants. To obtain this amphipod, simply submerge the strings or masses of mussels in a tank with water (25-50 L) and shake them gently, which causes most to separate from them and go to the walls of the tank. These tanks will be transported to the culture facilities using preferably a small aerator to avoid oxygen drops.
    [0096]
    [0097] Once in the culture facilities, the amphipods show an easy adaptation to the captivity and do not need special maintenance conditions. Preferably they will be kept in culture tanks similar to those of the settlement, although not covered and with a major renovation (about 10-12 renovations a day). They can be fed with fish feed or fish meat or molluscs supplied ad libitum, avoiding that there are too many remains without consuming at the bottom.
    [0098]
    [0099] The collection of the dams of the storage tank of the same can be carried out by different procedures. The first is by siphoning the walls and bottom of the storage tank of the dams. The amphipods will be concentrated in a mesh whose light can vary according to the size of prey that is being sought. This system will be preferably used for larger dams. The second collection system consists of a concentrator that uses the water outlet tube of the culture tank itself. In the walls of this tube is placed a mesh that allows to enter the dams (and whose mesh size can vary according to the desired size of prey) and in the bottom (outlet) of the tube is placed a smaller mesh (preferably 200 microns ) that holds them inside the tube. Once the dams are in the inner part of the tube, this will be removed and the dams will be transferred to a container to be added to the paralarvae tank. In both cases (siphoning and concentrating) an attempt will be made to prevent the prey from coming into contact with the air, as this may cause air bubbles to accumulate in the exoskeleton, which would prevent them from submerging, making it difficult for them to catch them. the paralarvae
    [0100] By means of the method of the present invention, survival data of> 90% have been achieved at 40 days of age,> 80% at 50 days and> 65% at 60 days of age. These results are superior, even to the best obtained so far with spider crab and, therefore, superior to those obtained with Artemia sp. (the only commercially viable diet) whose paralarvae rarely exceed 30 days of age. In relation to growth, the proposed invention protocol obtains weight increases between 5 and 6% of its dry weight per day. These values are below 7-8% obtained by the previous authors with king crab zoea, but they are superior to many of those obtained with Artemia sp. (3-4% in most cases) and the studies confirm that they are sufficient to achieve settlement, although with a delay of about 10-20 days compared to those fed with king crab zoea. Despite this difference, the ease of obtaining the proposed prey (amphipods) against the difficulty of obtaining zoeas of spider crab, place the amphipods with a clear advantage to advance their cultivation on a commercial scale.
    [0101]
    [0102] The natural high availability of the dams used in the process of the present invention would allow an industrial production (at least at a small or medium scale). There is also the possibility of carrying out a multitrophic production associated with rafts and cetareas (a joint crop of different species where the surplus or waste of one species is used to feed others, reducing the impact and increasing profitability).
    [0103]
    [0104] Throughout the description and the claims the word "comprises" and its variants do not intend to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0105]
    [0106] BRIEF DESCRIPTION OF THE FIGURES
    [0107]
    [0108] Fig. 1: Shows a photo of a recently hatched paralarva of common octopus O. vulgaris where its characteristic physiognomy can be appreciated.
    [0109]
    [0110] Fig. 2: Shows photos of adult specimens of Jassa falcata (A) and Jassa marmorata (B).
    [0111] Fig. 3: Shows photos of adult Phthisica marina, male and female (A) and Caprella equilibra, male and female (B).
    [0112]
    [0113] Fig. 4: Graphs showing the growth in mg of dry weight of octopus paralarvae in different experiments: 4A: A control group with paralarvae fed exclusively with Artemia sp. until day 40 of life and an experimental group fed with Artemia sp. and zoea until day 30 and later with amphipods exclusively from day 31 to 50. The bars indicate the standard deviation. 4B: Paralarvae fed until day 25 with a control treatment (Artemia sp.) And an experimental treatment with Artemia sp. up to 8 days and amphipods from 9 to 25 days old.
    [0114]
    [0115] Fig. 5: Scheme showing the culture tanks used in the invention. 5A: Trunk cone used from the first day of age until the larvae enter the settling phase (6-9 mg of dry weight, which usually occurs between 50-70 days of life). The diagram shows the water inlet (1) and the light source (2), the central aeration (3), the central tube (4) fitted with a filter and the external drain that allows to regulate the height of the water ( 5). 5B: Settlement tank where the larvae are transferred until they reach the settling phase (above 9 mg of dry weight). The diagram shows the water inlet (1) and the light source (2), the dens, both suspended and deposited in the bottom (3), the central tube provided with a filter (4) and the external drain that allows to regulate the height of the water (5).
    [0116]
    [0117] Fig. 6: Paralarvae in the settlement phase. In B) PVC pipes used as shelters are also observed.
    [0118]
    [0119] EXAMPLES
    [0120]
    [0121] The invention will now be illustrated by means of tests carried out by the inventors, which highlights the effectiveness of the product of the invention.
    [0122]
    [0123] Example 1: Larval octopus culture assay known in the state of the art (Garrido, D. et al. (2017) , Aquaculture, 468, 558-568):
    [0124] Experiment carried out in frustoconical tanks of 500L volume, walls and black bottom, soft-moderate aeration in central position. Use of green water (phytoplankton) and a 150% day renewal throughout the experiment (30 days) with water filtered at 1 micron using cartridge filters. For the light, a cold white 36W fluorescent light was used located in the upper central part of the tank. A single intensity of 700 lux was used throughout the experiment. The feeding was based on Artemia sp. enriched with microalgae versus another enriched with a marine phospholipid. Artemia sp. It was delivered in 3 separate shots throughout the day. Survival at 30 days of age was between 0.14 and 3.77%, while the growth rate showed an increase in dry weight between 3.9 and 6.4% per day.
    [0125]
    [0126] Example 2: Control test: common octopus larval culture (1000L tank) using Artemia sp. exclusively as prey from day 1 to day 40 of life of the paralarvas
    [0127]
    [0128] Experiment carried out in a truncoconical tank of 1000L volume, walls and black bottom, ventilation in central and moderate position avoiding that the current created by the air bubbles prevent the paralarvae from moving freely through the tank (Figure 5A). A density of 5 paralarvae / liter and with values for dissolved oxygen that ranged between 5.5-6.7mg / L for a temperature range of 18.5-21.3 ° C and a salinity of 35 g / L (during the entire experimental period). Green water was used until 30 days, with a concentration of 106 cls / mL of Nannochloropsis sp. and Isochrysis aff. galbana at the time of the addition of the microalgae. This procedure was the same as in the previous test (state of the art test), but modifications in the renewal in this second test affected its concentration. These differences consisted in keeping the tank closed the first two days, taking place what could be called a "maturation" of the tank (1. unlike the procedure of the state of the art). After this period begins to renew (with filtered water at 1 micron with cartridge filters), starting with a renewal rate of 15-20% of the volume of the tank (10mL / s for 5 hours) during the following 5 days, increasing the time gradually until reaching 100% of the renewal at 15 days. This percentage is maintained until the 30th day, after which a continuous renewal is carried out (24h a day) which represents a renovation of around 200% at the day and that will remain until the closing of the tank at 40 days of life (2nd difference). The water outlet was made through a central tube with a mesh of 300 microns and the water level is maintained thanks to an outer tube.
    [0129]
    [0130] For the light, a cold 35W white light fluorescent lamp was used located on the edge of the tank (instead of a central position) causing a change in the angle of incidence of light on the surface and giving rise to heterogeneous light conditions within the water column, (3rd difference). The intensity values used during the test would be encompassed in three levels of intensity. The levels used range from 600 to 800 lux (1077-1436 W / m2) for the first 15 days of life and between 300 and 600 lux (517-1077 W / m2) for the interval between 15 and 40 days of life. (4th difference). He had a photoperiod of 14:10 (light: dark), between 8:00 and 22:00 from the first day of life until the end of the trial.
    [0131]
    [0132] Feeding during the whole trial (40 days) was based on Artemia sp. fattened for 7 days with Isochrysis aff. galbana and supplied to the paralarvae with a density of 0.5 individuals / mL, distributing the intakes so that there are always prey available in the tank, which gave a range between 1-4 shots / day. The tank was decided to close at 40 days due to the fact that the growth rate (CI) did not increase more than 4% per day, maintaining between 1 and 1.5 mg of dry weight (See Figure 4A). The survival at that age was 65%.
    [0133]
    [0134] Example 3: Common octopus larval culture test (1000L tank) using Artemia sp. and crab zoeas from day 1 to day 30 and with the amphipods of the present invention from day 31.
    [0135]
    [0136] This test was carried out simultaneously with the previous test and using the same culture conditions, in order to compare the effect of the different preys. In this case the Artemia sp. It was supplemented with zoeas of spider crab (0.1 individuals / mL) until 30 days of age (however, given the difficulty to obtain the zoeas, these could only be added to the tank in 10 of the first 30 days, which reduced the expected growth). From then on, amphipods were exclusively supplied. On the one hand, the gammarids of the genus Jassa spp. (preferably J. falcata and J. marmorata) representing 95% of the total number of individuals and the caprolids of the genera Phtisica spp. and / or Caprella spp. (preferably Phtisica marine and Caprella equilibra) that represented 5% of the total number of individuals. The gammarids reached between 2 and 8 mm in length and the caprelids between 4 and 30 mm in length. In the case of the amphipods, the feeding protocol consisted in the supply of 3-5 prey per paravelar / day, distributing the intakes so that there are always prey available in the tank, which gave a range between 1-4 shots / day (5.a difference from the state of the art).
    [0137]
    [0138] After 55 days of age, paralarvae that exceeded 6 mg of dry weight began to migrate towards the bottom. These individuals were transferred to a new tank called Settlement Tank (Figure 5B) of 400L, quadrangular shape, height of 50 cm, flat bottom, gray color and covered with a shading mesh in% parts of its surface. This tank was filled with water from the original tank and lairs (1-2 cm PVC tubes) were included in the bottom of the tank or hanging from vertical threads, as well as small stones to enrich the habitat. The density of individuals was 0.1 paralarvas / L. At this point, it has been seen that the paralarvae become vulnerable (at least temporarily) and can be attacked by the larger prey, for which they are only supplied with gammarids of 4 mm maximum in length (6th difference).
    [0139]
    [0140] In this settlement tank, a renewal percentage similar to that in the initial tank was maintained after 30 days of age of the paralarvae, that is, a continuous renovation (24h) that renews approximately 200% of the total volume of the tank every day. The water outlet was made through a central tube with a mesh of 300 microns. With regard to lighting, this was reduced in the initial tank to a range between 40 and 300 lux (69-517 W / m2) at the time when the first paralarvae were observed in pre-settlement (at 55 days and above 6 mg dry weight). Once transferred to the settlement tank, the intensity remained around 40 lux (69 W / m2) with the same photoperiod of the initial tank (14:10) (7th difference).
    [0141]
    [0142] In none of the described tests, the bottom of the paralarva tank is siphoned. The objective is to reduce the stress of the paralarvae and also allow the amphipods to colonize the bottom of the tank, since being detritivores help to keep the bottom of organic remains clean avoiding the proliferation of possible pathogens (8th difference).
    [0143] The amphipods (gammarids and caprelids) were obtained from the rafts, and sewage treatment plants used in the mussel culture of the Galician estuaries. They were kept in 1000L tanks similar to those in Figure 5B (Settlement Tanks), but without coverage and with 10 renewals of their total volume per day. The food was based on fish feed and remains of molluscs ad libitum. To feed the paralarvae, the amphipods were captured by two systems. The larger ones were obtained by siphoning the walls and bottom of the tank and concentrating on a mesh. The second collection system consisted of a concentrator that uses the water outlet pipe of the tank where the dams are stored. On the walls of this tube, a 500 micron mesh was placed to allow the dams to enter and at the bottom of the tube (outlet) a 200 micron mesh was placed to hold them inside the tube. Once the dams are in the inner part of the tube, it is removed from the tank and the prey is concentrated in a container before adding them to the tank of the paralarvae by plastic jars of 1-2 L. (9.a difference).
    [0144]
    [0145] The data from this trial showed survival of more than 90% at 40 days of age and around 65% at 60 days of age. The Growth Index showed an increase in weight between 5 and 6% per day, reaching 5 mg of dry weight at 50 days of age (Figure 4A) and subsequently obtaining paralarvae with dry weights greater than 9 mg among the 65 and 75 days of age.
    [0146]
    [0147] Example 4: Control test: common octopus larval culture (100L tank) using Artemia sp. exclusively as prey from day 1 to day 25 of life of the paralarvae:
    [0148]
    [0149] This test was carried out with the same culture conditions described in the two previous examples, with the difference that the tanks volume was 100 L, instead of 1000 L and three replicas were used instead of one. The trial lasted 25 days. As in Example 2, a control treatment was used, only with Artemia sp. fattened for 7 days with Isochrysis aff. galbana. The growth results of the paralarvae were similar to those of example 2, as can be seen in Figure 4B with the dry weight around 1 mg at 25 days of age.
    [0150] Example 5: Common octopus larval culture test (100L tank) using Artemia sp. and crab zoeas from day 1 to day 8 and with the amphipods of the present invention between days 9 and 25 days of life:
    [0151]
    [0152] This test was carried out in parallel and with the same culture conditions described in Example 4, in order to compare the Artemia sp. with an experimental treatment that used the same amphipods of the genus Jassa spp. of example 3. The difference is that on this occasion, these amphipods began to be supplied from day 8 of life, because it was when it was observed that a majority of larvae ingested this type of prey (this can easily be seen by the expert in the matter observing the behavior of the paralarva or doing the protocol explained in the following example in order to know when to start supplying the prey). In this case, feeding during the first 8 days consisted only of Artemia sp. (of the same type as example 4) and between 9-10 days were fed with a mixture of Artemia sp. (75% of all individuals) and amphipods (25%) of the genus Jassa spp. between 1-3 mm in length. After 10 days, the larvae began to feed exclusively on these amphipods under the same conditions of example 3 (3-5 prey / paralarva / day), until the closure of the crop. The growth results of the paralarvae in this example were similar to those of Example 3 as can be seen in Figure 4B with a growth rate of 6.7% and a survival of 70% at 25 days.
    [0153]
    [0154] Example 6: Assay of capture and ingestion of amphipods at different ages
    [0155]
    [0156] Parallel to the rest of the experiments, a new experiment was carried out in order to be able to determine more accurately the age at which the paralarvae are able to capture and ingest the amphipods of the genus Jassa spp. and if there can be differences between sunsets. For this purpose, paralarvae obtained from two different females were compared, which presented different initial weights at the time of hatching. The paralarvae of the first female weighed an average of 0.24 mg of dry weight, while the paralarvae of the second female had an average of 0.29 mg of initial dry weight (both within the usual margins of weight in this species). . Both groups of paralarvae were placed in separate 100L tanks under the same culture conditions of the previous examples 2 to 5 and one density of 4 larvae per liter. In both cases, amphipods of the genus Jassa spp. (20-30%) together with Artemia sp. (80-70%) from the first day of life to the density of 3-5 prey / paralarva.
    [0157]
    [0158] In both cases it was observed that all amphipods were captured. Those paralarvae that had captured any prey and kept it for 2-3 minutes were removed from the tank (with prey included) and observed at the magnifying glass (40 times) to confirm that they had food in their digestive system. The technical details for this observation are found in Nande, M. et al (2017). Prey Capture, Ingestion, and Digestion Dynamics of Octopus vulgaris Paralarvae Fed Live Zooplankton. Frontiers in Physiology, 8, 573. In the present example, it was observed that freshly hatched paralarvae of 0.24 mg of dry weight attacked the prey from the first day of life, but the percentage of ingestion was only 30%, so it could be considered too early to supply such a diet. On the other hand, the paralarvae with 0.29 mg of dry weight showed an 80% ingestion range from the first day of life, which is considered adequate to start supplying the amphipods of the genus Jassa spp.
    [0159]
    [0160] This test shows that the start of feeding with these amphipods can vary from one set to another and it would be advisable, although not necessary, a parallel capture and ingestion test to optimize the start of feeding with this prey. However, these dams (amphipods) could begin to be administered from the first day.
    [0161]
    [0162] ^ Comparison of results between control experiments and those of the present invention.
    [0163]
    [0164] When comparing the results of the experiments based on the present invention with respect to the one known in the state of the art, the improvements are clearly significant for all the presented examples (2, 3, 4 and 5) and that it includes tests both in 100 and in 1000L with the process of the present invention. The survival of the paralarvae in the procedure of the state of the art at 30 days of age (Garrido et al., 2017) is between 0.14 and 3.77% (the trial consisted of a total of 18 tanks), giving closed the experiment at that age. In contrast, the test conditions in 1000L tanks of the present invention with amphipods obtained a survival of more than 90% at 40 days and 65% at 60 days of age. In turn, the culture conditions in tanks of 100L with amphipods obtained a survival of more than 70% at 25 days of age. Also, it is noteworthy that in the 1000L trial, the treatment with Artemia sp., Despite having a suboptimal diet showed a 40-day survival of more than 65%, which indicates that it is not only the use of amphipods what makes the difference, but that the culture protocol has also improved survival. Regarding the weight, in the procedure of the state of the art (Garrido et al., 2017), a growth index is obtained with an increase between 3.9-6.4% of dry weight per day, but there are only values up to 15 days of age, while the data of the procedure of the present invention for the tanks with amphipods, an increase similar to the maximums of the previous study (between 5 and 6% of dry weight per day) is obtained. 1000L tanks, and an increase of 6.5 and 7.5% dry weight per day for 100L tanks. In the case of 1000L tanks, settled larvae with weights greater than 9 mg of dry weight between 65 and 75 days of life were obtained. Figures 4A and 4B show the weight increase in examples 2, 3, 4 and 5 with the method of the present invention. In this case, you can also observe the differences between treatments with amphipods and with Artemia sp ..
    权利要求:
    Claims (23)
    [1]
    1. Procedure for the culture of common Octopus vulgaris octopus paralarvae emplaced in a culture tank containing water, characterized in that said procedure comprises the addition of selected prey of:
    - gammarids of the genus Jassa spp.,
    - caprélidos of the genera Phtisica spp. and / or Caprella spp. or
    - combinations of them
    to said culture tank.
    [2]
    2. Procedure according to claim 1 where the gammarids of the genus Jassa spp. they are selected from Jassa falcata and Jassa marmorata.
    [3]
    3. Method according to any of the preceding claims wherein the caprolids of the genus Phtisica spp. they are Phthisica marina and the caprélidos of the genus Caprella spp. they are Caprella equilibra.
    [4]
    4. Method according to any of claims 1-3 wherein from day 1 of life to 20-30 days of life of paralarvae gammarids are added between 1-4 mm, from this time and up to 50-70 days of life are added gammarids between 2 and 8 mm in length and caprelids between 4 and 30 mm in length, from 50-70 days and until the settlement of the paralarvae are administered only gammarids with a size between 1 and 4 mm in length ..
    [5]
    5. Method according to any of claims 1-3 wherein the gammarids are added from a day comprised between 1-10 days of life of the paralarva until the settlement thereof, while the caprolids are added from 20-30 days after life and until the settlement of them.
    [6]
    6. Process according to any of claims 1-3 where gammarids are added from the first day of life to their settlement and paralarvae caprellids s and added from day 10 of life until its settlement.
    [7]
    7. Method according to any of claims 1-3 wherein both gammarids and caprolids are added from the 30th day of life of the paralarvae to their settlement.
    [8]
    The method according to any of the preceding claims, wherein, when combinations of gammarids and caprolids are added, between 1 and 10% of caprolids are administered with respect to the total number of prey and between 90 and 99% of gammarids with respect to to the total number of prisoners.
    [9]
    9. Procedure according to claim 8, where 5% of caprolids and 95% of gammarids are administered with respect to the total number of prey.
    [10]
    10. Method according to any of the preceding claims wherein the density of paralarvae in the culture tank is between 3-7 paralarvae / liter.
    [11]
    11. Method according to any of the preceding claims wherein a total of between 3 and 5 prey per parava and day are supplied to the culture tank.
    [12]
    Method according to any of the previous claims where the culture tanks used between the first day of life and until 50-70 days of life are frustroconical and black and from 50-70 days, the paralarvae are transferred to another flat-bottom crop tank and gray color.
    [13]
    13. Method according to claim 12 wherein the density of paralarvae in the flat bottomed culture tank and a gray color will be 0.1 and 0.5 larvae per liter.
    [14]
    14. Method according to any of the preceding claims wherein the culture tank is artificially illuminated by a light placed on the edge of the tank.
    [15]
    15. Method according to claim 14 where the intensity levels of the supplied light vary between 1077 and 1436 W / m2 for the first 10-15 days of life of the paralarvae; between 517 and 1077 W / m2 from 10-15 days of life and up to 50 70 days of life, from this moment and until the settlement the light is reduced to a range between 69 and 517 W / m2.
    [16]
    16. Method according to any of the preceding claims wherein the water of the culture tank is renewed in the following manner:
    during the first two days no type of water renewal is carried out in the tanks, afterwards, it begins to renew with a flow that renews between 4 and 10% of the total volume of the tank every hour, the renewal time is increases gradually, so that it is renewed 20% daily in the first 5 days and from there it increases until it reaches 100% daily at 10-15 days, this renewal is maintained at 100% until 30 days and From there, the renovation is left open permanently for 24 hours a day, which represents a daily renewal of approximately 200%, this renewal percentage will be maintained until the paralarvae settle.
    [17]
    17. Method according to any of the preceding claims wherein, in addition, a mixture of microalgae Isochrysis spp. Is added to the tank. and Nannochioropsis spp. until obtaining a concentration of them between 0.7 and 1.5 x 106cls / mL at the moment of said addition.
    [18]
    18. Process according to claim 17, wherein the microalgae mixture is added once a day.
    [19]
    Method according to any of the preceding claims, characterized in that the concentration of oxygen in the water of the culture tank is greater than 5.5 mg / L.
    [20]
    Method according to any of the preceding claims, characterized in that the water salinity of the culture tank is 35-36 g / L
    [21]
    21. Method according to any of the preceding claims characterized in that the water temperature of the culture tank is between 18 and 22 ° C.
    [22]
    22. Method according to any of the preceding claims wherein the bottom of the culture tank is maintained without siphoning.
    [23]
    23. Method according to any of the preceding claims wherein the dams are added to the culture tank from a storage tank thereof by siphoning the walls and bottom of the storage tank of the dams or by a concentrator using the tube itself water outlet from the culture tank, so that in said tube a mesh is placed that allows to enter the dams and in the bottom of the tube a smaller mesh is placed (which retains them inside the tube) and once the prey is inside the tube, it will be removed and the prey will be transferred to a container to be added to the paralarvae tank.
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    同族专利:
    公开号 | 公开日
    CN111432633A|2020-07-17|
    WO2019106212A1|2019-06-06|
    ES2714930B2|2020-01-15|
    EP3718399A1|2020-10-07|
    JP2021503966A|2021-02-15|
    PT3718399T|2021-08-11|
    MA51220B1|2021-10-29|
    CN111432633B|2022-03-01|
    EP3718399B1|2021-05-12|
    US20200367476A1|2020-11-26|
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    CN104129856A|2014-05-19|2014-11-05|中山大学|Combined ecological floating bed and culture method thereof|
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    US16/766,581| US20200367476A1|2017-11-29|2018-11-21|Method for cultivating paralarvae of the common octopus, octopus vulgaris|
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