JP7096548B2 - How to make detritus feed for aquatic animals - Google Patents

Description

The present invention relates to a method for producing a feed for detritus-eating aquatic animals.

Japanese eel (Anguilla japonica) is a traditional foodstuff and one of the important foodstuffs in the food service industry. Most of the seedlings for aquaculture, glass eels, are collected from natural waters. In recent years, the amount of glass eels collected has decreased, which limits the production of Japanese eels in aquaculture.

The complete aquaculture technique for Japanese eels has already been established, but it has not yet become widespread. One of the reasons why the complete aquaculture technique for Japanese eels has not become widespread is that an effective diet for larvae to grow into fry has not been developed. When the complete aquaculture technique for Japanese eels was established, a diet made by processing the eggs of the Pacific spiny dogfish was used. However, since the spiny dogfish is a fish with a small amount of resources, it is difficult to secure enough female eggs of the spiny dogfish on a continuous basis. Therefore, it is not appropriate to use the eggs of the Pacific spiny dogfish as a food material for the larvae of the Japanese eel.

The food for Japanese eel larvae in the natural area is marine snow. Marine snow is a high molecular weight organism derived mainly from detritus, that is, animal and plant plankton. Not only Japanese eels, but also crustaceans and shellfish ingest detritus. For crustaceans such as Lajonkairia lajonii and whiteleg shrimp to be cultivated, and shellfish such as Lajonkairia lajonii, clams and whiteleg shrimp, detritus as well as microalgae are used as food when they show filtered feeding habits even after growing into larvae or adults. be able to.

Microalgaes are used as feed in the crustacean and shellfish aquaculture techniques that are being established. However, these crustaceans or shellfish often have a sharp decrease in survival during the growth stage. At present, there is no substitute for microalgae when the cause of the decrease in the number of survivors is feed.

Patent Document 1 discloses a method for aquaculture of eels in which eel larvae are bred in a water tank in which marine rotifers (hereinafter, also referred to as “rotifers”) are cultivated using chlorella as a feed. In the aquaculture method of Patent Document 1, eels are bred by feeding on marine snow containing rotifer droppings, rotifer eggs, rotifers or rotifer carcasses in a water tank.

Japanese Unexamined Patent Publication No. 2015-107805

In the eel aquaculture method disclosed in Patent Document 1, since the supply amount of marine snow depends on the state of rotifer, it may not be possible to stably supply marine snow. Also, rotifer droppings are the product of digestion and the nutritional value cannot be strictly controlled.

The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for producing a feed for detritus- eating aquatic animals, which is suitable for breeding detritus -eating aquatic animals.

The method for producing a feed for detritus-eating aquatic animals according to the present invention is as follows.
A freezing step to freeze marine brachionus plicatilis,
A thawing step in which the marine worms are thawed and the thawed marine worms are placed at room temperature for 15 to 30 hours to promote the decomposition of the marine worms by the activity of the enzyme possessed by the marine worms .
including.

In addition, the method for producing a feed for detritus-eating aquatic animals according to the present invention is as follows.
Following the thawing step further comprises a refreezing step of freezing the marine brachionus plicatilis.
It may be that.

In addition, the marine worms are
The first marine worms fed and cultivated with the first microalgae and the second marine worms fed and cultivated with the second microalgae different from the first microalgae.
It may be that.

Further, the first microalgae and the second microalgae are
Selected from the group consisting of cryptophytes, haptophytes and diatoms, respectively.
It may be that.

In addition, the first microalgae is
Isochrysis sp. And
The second microalgae is
Chaetoceros calcitrans,
It may be that.

According to the present invention, a feed suitable for breeding detritus-eating aquatic animals can be obtained.

It is a figure which shows the result of Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It is a figure which shows the OD integral value of the band obtained by SDS-PAGE shown in FIG. It is a figure which shows the relative value of the absorbance of the sample liquid prepared from the thawed rotifer. It is a figure which shows the content of fatty acid per 1 g of the dry weight of a thawed rotifer. It is a figure which shows the content for each kind of fatty acid per 1 g of a dry weight of a thawed rotifer. It is a figure which shows the content of the saturated fatty acid, the monounsaturated fatty acid and the polyunsaturated fatty acid per 1 g of the dry weight of the thawed worm. It is a figure which shows the survival rate of each test group of the breeding test 1. (A) and (B) are diagrams showing images of individuals in a test plot that feed on rotifers that have been re-frozen immediately after thawing. (C) and (D) are diagrams showing images of individuals in a test group fed with rotifers whose time from thawing to refreezing was 12 hours. (E) and (F) are diagrams showing images of individuals in a test plot using rotifers as feed, with a time from thawing to refreezing of 24 hours. It is a figure which shows the survival rate of each test group of the breeding test 1 and the breeding experiment 2. (A) is a figure which shows the image of the individual of the test group which fed diatoms, and the time from thawing to refreezing was 24 hours, and fed rotifer. In (B), a rotifer fed with haptoalgae and having a time from thawing to refreezing for 24 hours and a rotifer fed with diatoms and having a time from thawing to refreezing for 24 hours were mixed. It is a figure which shows the image of the individual of the feeding test group.

An embodiment of the present invention will be described with reference to the drawings. The present invention is not limited to the following embodiments.

The method for producing aquatic animal feed according to the present embodiment includes a freezing step and a thawing step. In the freezing step, rotifers, which are components of aquatic animal feed, are frozen. The rotifer is not particularly limited, and for example, L-type (Brachionus plicatilis OF Mullre), S-type (Brachionus rotundiformis Tschugunoff), SS-type, L-type Obama strain, S-type Yaeyama strain and the like are used.

The rotifer may be a commercially available one or one cultured by a known method may be used. Examples of the rotifer culture method include a subculture method, an outdoor culture method, a thinning type culture method, and a high-density culture method. In rotifer culture, it is preferable to feed microalgae. The microalgae is not particularly limited as long as it feeds on rotifers, but is preferably at least one of cryptoalgae, haptophytes and diatoms.

Examples of the cryptoalgae include Cryptomonas, Rhodomonas, Chroomonas and the like.

Examples of haptophytes include Isochrysis sp., Isochrysis galbana, Pavlova luseri and the like. Specifically, examples of Isocrisis SP include CCAP927 / 12, CCAP927 / 14, UTEX LB1292, and UTEX2307. Examples of isocrisis galvana include CCAP927 / 1 and UTEX LB987. Preferably, the haptophyte is an Isocrisis sp. Tahitian strain.

Examples of diatoms include Chaetoceros, Cyclotella, Fragilaria, Navicula and Amphora. Preferably, the diatom is Chaetoceros calcitrans.

As the microalgae, microalgae that can be cultivated by a known method may be used. When culturing microalgae, a well-established culture strain of microalgae may be used. The microalgae are cultivated in a culture medium suitable for the microalgae to be cultivated, such as Girard F medium, KW21 medium and MNK medium. As the culture solution, a commercially available Girard (F / 2) seawater nutrient solution or a culture solution for microalgae containing various vitamins and amino acids may be used.

Preferably, Girard F medium or Kw21 medium is used as the culture medium for microalgae. As the Kw21 medium, for example, 1 mL of Kw21 medium (manufactured by Daiichi Net Co., Ltd.) may be added to 1 L of diluted seawater. Sodium silicate may be further added to Girard F medium and Kw21 medium, especially when used for diatom culture. The amount of sodium silicate added is adjusted as appropriate. For example, in the case of Girard F medium, sodium silicate nine hydrate may be added at a concentration of 10 to 50 mg / L, 20 to 40 mg / L, preferably 30 mg / L. On the other hand, in the case of Kw21 medium, sodium silicate nine hydrate may be added at a concentration of 70 to 110 mg / L, 80 to 100 mg / L, preferably 90 mg / L.

The culture may be carried out by maintaining the temperature of the culture solution at about 18 to 25 ° C, preferably 20 ° C, and culturing under aeration. Preferably, the aerated air is aseptically treated with a filtration filter or the like.

From the viewpoint of nutritional enhancement, the above-mentioned rotifer is a first rotifer fed and cultivated by feeding the first microalgae and a second rotifer fed and cultivated by a second microalgae different from the first microalgae. It may be a rotifer. In this case, preferably, the first microalgae is Isocrisis SP and the second microalgae is Quitocellos calcitrans. The mixing ratio of the first rotifer and the second rotifer is not particularly limited, and may be adjusted according to the nutrition such as fatty acid possessed by each rotifer.

The method of freezing the rotifer is not particularly limited. To freeze the rotifer, it is convenient to keep the rotifer in the freezer. The temperature of the freezer is not particularly limited as long as the temperature of the rotifer freezes. The freezing temperature of rotifers is, for example, −20 to −80 ° C. When freezing the cultured rotifer, it is preferable to drain the harvested rotifer to make the rotifer into a paste and then freeze it.

Next, the decompression step will be described. In the thawing step, the frozen rotifer is thawed. In the thawing step, for example, the frozen rotifer may be allowed to stand at room temperature. The normal temperature is 20 ° C. ± 15 ° C. (5 to 35 ° C.). Preferably, in the thawing step, the frozen rotifer is placed at a temperature of 15-35 ° C, 18-30 ° C, 20-28 ° C, or 22-26 ° C.

In the thawing step, the frozen rotifer may be placed in water at room temperature to thaw the rotifer. In the thawing step, the frozen rotifer may be thawed while being gently heated in a constant temperature bath or the like. However, when heating, it is preferable to maintain the temperature of the rotifer at a temperature at which the rotifer enzyme is not inactivated.

Preferably, in the thawing step, the thawed rotifer is placed at room temperature for 15-30 hours. The time of leaving at room temperature is, for example, 18 to 28 hours, 20 to 26 hours, preferably 24 hours. When placed at room temperature, the activity of the enzyme contained in the rotifer promotes the decomposition of the rotifer and the gelation of the rotifer. In order to further promote the decomposition of the rotifer, a predetermined degrading enzyme, particularly a degrading enzyme possessed by the rotifer, may be allowed to act on the rotifer.

The production method may further include a refreezing step of freezing the rotifer after the thawing step. The method of refreezing is the same as the above-mentioned freezing step. By refreezing, the enzyme activity of rotifers can be reduced. This can prevent excessive decomposition of the rotifer.

The rotifer obtained in the thawing step or the rotifer thawed after the refreezing step is in the form of a gel (for example, the particle size when dispersed in the breeding water is about 0.1 mm or less), and is a detritus-eating aquatic animal. Can be used as feed. In addition to the above-mentioned rotifer, the feed may contain various additives and the like, if necessary.

The detritus-eating aquatic animal is, for example, Japanese eel, conger eel, crustacean, shellfish and the like. Examples of crustaceans include gazami crab and whiteleg shrimp. Examples of shellfish include lajonkairia lajonii, clams and pen shells. The feed is suitable not only for larvae but also for aquatic animals that show filter feeding habits even after they have grown into adults. When used as a feed for Japanese eels, the feed is preferably given at the time of leaf-shaped larvae and fry.

As described in detail above, according to the method for producing feed for aquatic animals according to the present embodiment, rotifers are decomposed in the thawing step, so that they are dispersed in the breeding water, easily enter the mouth of aquatic animals, and are digested. It is possible to produce feed that is easy to get rid of. The feed is suitable for breeding detritus-eating aquatic animals.

Further, in the thawing step according to the present embodiment, the thawed rotifer may be placed at room temperature. By doing so, the decomposition of the rotifer is further promoted by the activity of the enzyme possessed by the rotifer, and it is easily taken up into the narrow pharynx of leaf-shaped larvae such as eels, and a gel-like food that is easily digested can be obtained.

Further, in the thawing step according to the present embodiment, after the thawing step, a refreezing step of freezing the rotifer may be further included. This can prevent the decomposition of nutrients required for aquatic animals.

Further, the rotifer according to the present embodiment may be a first rotifer and a second rotifer fed and cultured with different microalgae. By combining rotifers fed with abundant microalgae with different nutrition, it is possible to flexibly control the nutrition of the feed. For example, by using a rotifer fed with docosahexaenoic acid (DHA) -rich Isocrisis SP and a rotifer fed with eicosapentaenoic acid (EPA) -rich Quitoseros carcitrans, the rotifer can be used as a feed for the aquatic animals. It can fortify the contained fatty acids that are important for aquatic animals such as Japanese eels.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the examples.

(Preparation of thawed rotifer)
As a rotifer, a marine brachionus L-type Obama strain was used. Rotifers were cultivated by a subculture method. The culture water was seawater (22 psu) diluted to 75%. The rotifer culture was started from a 5 L aquarium and a 30 L circular transparent polycarbonate aquarium was used depending on the growth of the rotifer. The rotifer was cultivated while sufficiently aerating the culture water through the air stone. The temperature of the culture water was maintained at 26 ° C. using a heater. The culture water was exchanged every 3 days using a plankton net (25XX, manufactured by Tanaka Sanjiro Shoten) with a mesh size of 63 μm.

The rotifers were fed with Quitocelos calcitrans (hereinafter referred to as "Quitocellos") or Isocrisis SP Tahitian strain (hereinafter referred to as "Isocrysis"). The rotifers were fed once a day. The feeding amount per rotifer was 20000 cells of Quitoceros or Isocrisis per rotifer.

Here, a method for culturing microalgae will be described. In the preparation of the culture solution of Quitocelos, seawater (about 33 psu) was filtered using cartridge filters having a mesh size of 100 μm, 25 μm, and 10 μm in descending order of mesh size. Seawater was sterilized by ultraviolet rays with a forced circulation germicidal lamp. Subsequently, 10 L of the above seawater and 5 L of distilled water were mixed in a 30 L circular transparent polycarbonate water tank to prepare diluted seawater (about 22 psu).

Sodium silicate nine hydrate (Na ₂ SiO _{3.9H 2} O) was added to diluted seawater (30 mg / L). Using the obtained diluted seawater, Gillard F medium as a culture medium was prepared according to the method of Guillard and Ryther (Canadian Journal of Microbiology, 8, 229-239, 1962). The components added to the diluted seawater in the preparation of Girard F medium are thiamine, biotin, vitamin B ₁₂ , copper sulfate, zinc sulfate, cobalt chloride, sodium molybdate, sodium metasilicate, sodium nitrate, sodium dihydrogen phosphate and iron ( Valence III) -EDTA. Quitoceros was cultured in the culture medium under strong aeration under the conditions of room temperature of 20 ° C. and lighting for 24 hours.

As the medium for isocrisis, a medium prepared in the same manner as the Girard F medium used for culturing the above-mentioned keetoseros was used except that sodium silicate nine hydrate was added. Isocrisis was cultured by batch culture in a 1 L flask under continuous irradiation of light at a water temperature of 20 ° C. and 150 μmol photons / m ² / s for 24 hours.

The cultured rotifer was filtered through a plankton net having a mesh size of 63 μm and washed with distilled water. Moisture was wiped from under the plankton net, and the rotifer was placed in a 1.5 mL microtube. Then, the rotifer was frozen at −80 ° C. and thawed at 25 ° C. using a water bath (SB-1200, manufactured by Tokyo Rika Kikai Co., Ltd.). Rotifers immediately after thawing and rotifers that had been allowed to stand at 25 ° C for 12 or 24 hours after thawing were frozen again at −80 ° C.

The rotifer that has been cultivated by feeding the above-mentioned keet cellos and that has been re-frozen immediately after thawing is referred to as "C0". A rotifer fed with Quitocelos and cultivated with a time from thawing to refreezing of 12 hours is referred to as "C12". "C24" is a rotifer that has been cultivated by feeding Quitocelos and has a time from thawing to refreezing of 24 hours. The following analysis and breeding experiments were performed using C0, C12 and C24 as samples.

(Analysis of components of rotifer thawed by SDS-PAGE)
The degree of protein degradation at C0, C12 and C24 was evaluated by SDS-PAGE as follows. A small amount of the sample was added to a 1.5 mL microtube, and 500 μL of 1 × PBS buffer (sodium chloride, potassium chloride, disodium hydrogen phosphate / 12hydrate, potassium dihydrogen phosphate) was added. Next, the mixture was stirred with a stirrer (Vortex-Genie 2, manufactured by Scientific Industries) and centrifuged (3740, manufactured by Kubota Shoji Co., Ltd.) (12,000 rpm, 10 minutes). After centrifugation, the supernatant was discarded.

In a new centrifuge tube, 4500 μL of 1 × PBS buffer, 500 μL of 10% TritionX-100 solution, 25 μL of 2 mg / mL leupeptin solution, 10 μL of 0.5M EDTA solution, and 5 μL of 0.2M PMSF solution were mixed (hereinafter, with Reagent A). do). Nine times the amount of sample A was added to a 1.5 mL microtube containing the sample. The microtube was allowed to stand on ice for 15 minutes. The sample was stirred with a stirrer and centrifuged (12,000 rpm, 4 ° C., 5 minutes). After centrifugation, the supernatant was collected and diluted 10-fold to obtain a sample solution.

A BSA solution containing bovine serum albumin (FV, pH 5.2) (hereinafter referred to as “BSA”), which is a reference for the amount of protein, at concentrations of 1 mg / L, 0.5 mg / L and 0.25 mg / L. 20 μL of each was prepared. 500 μL of a 5-fold diluted CBB solution (Coomassie Brilliant Blue, G-250, ethanol and phosphoric acid) was added to each of the sample solution and the BSA solution. The sample solution was stirred with a stirrer, and the absorbance of the sample solution was measured using a spectrophotometer (U-5100, manufactured by Hitachi High-Tech Science Co., Ltd.). A standard curve as an index of protein mass was created from the concentration of BSA in the BSA solution and its absorbance. The protein concentration of the sample solution was estimated from the regression equation of the standard curve.

The sample solution, pure water and 3 × Sample Buffer were placed in a 1.5 mL microtube so that the protein concentration was constant (mg / mL) and the volume was 24 mL. The sample solution was stirred with a stirrer and centrifuged for 5 seconds. Subsequently, it was incubated at 100 ° C. for 3 minutes in an incubator (Mini T-100, manufactured by Chiyoda Science Co., Ltd.).

To prepare a running gel, 3.4 mL of acrylamide aqueous solution, 4.5 mL of pure water, 2.0 mL of Tris-HCl 2.0 mL, 100 μL of sodium dodecyl sulfate (SDS), 100 μL of 5% ammonium peroxodium sulfate solution (APS), and N, N. , N', N', -Tetramethylethylenediamine (TEMED), 10 μL of a 1,4-butanediamine aqueous solution was mixed. A gasket (RMS-01, manufactured by Atto) was sandwiched between glass plates (MAB-10 and MB-00, manufactured by Atto) and fixed with a clip (Rapidas Magne Clip Mini, manufactured by Atto). After pouring the running gel between the glass plates, pure water was layered on the running gel and allowed to stand for 30 minutes.

To prepare a stacking gel, 500 μL of acrylamide aqueous solution, 3.9 mL of pure water, 500 μL of Tris-HCl 500 μL, SDS 50 μL, 5% APS 50 μL, and 5 μL of 1,4-butanediamine aqueous solution containing TEMED were mixed. The pure water on the running gel was removed, the stacking gel was poured in, a comb (RM10-12, manufactured by Atto) was attached, and the mixture was allowed to stand for 30 minutes.

Approximately 400 ml of an electrophoresis buffer (Tris-HCL, glycine and SDS) was placed in an electrophoresis tank (AE-6530, manufactured by Atto). The gasket and comb were removed from the gel plate and installed in the electrophoresis tank. The migration buffer was added until the migration tank was filled. Unpolymerized acrylamide remaining in the wells was removed using a 5 mL syringe. The sample solution was placed in the well with a syringe.

The electrophoresis tank was connected to a power supply device (Powerpack (trademark) HC, manufactured by BIO-RAD), and the electrophoresis was performed at 0.02A. After the electrophoresis, the running gel was cut out and immersed in a fixative (methanol, acetic acid and water) for 10 minutes. On a rotary stirrer (DSR-2100P, manufactured by Primaco Group), the cells were stained with a CBB stain (Coomassie Brilliant Blue, ethanol and acetic acid) for one day.

The gel was photographed with a gel-dedicated imaging system (ChemiDoc Touch MP Imaging System, manufactured by BIO-RAD). A specific band was quantified by image analysis using image analysis software imagej (National Institute of Health).

(Results of SDS-PAGE)
FIG. 1 shows an image of SDS-PAGE gel. FIG. 2 shows the OD integral value of the band around 37 kDa (the part surrounded by the solid line) for each sample. The OD integral value decreased as the standing time from thawing to refreezing became longer.

(Analysis of components of rotifer thawed by OPA method)
For C0, C12 and C24, the amount of free amino acids separated by decomposition was measured by the OPA (o-Phythalaldehyde) method. Mix 50 mL of 100 M buffer borate (disodium tetraborate decahydrate and distilled water, pH 9.5), a solution of 80 mg of orthophthalaldehyde in 2 mL of methanol, and 200 μL of β-mercaptoethanol, and 100 mL with pure water. (Hereinafter referred to as "solution B").

Each sample and solution B were mixed and stirred with a stirrer for 5 seconds to obtain a sample solution. After allowing to stand for 2 minutes, the absorbance of the sample solution at 340 nm was measured using a spectrophotometer (U-5100, manufactured by Hitachi High-Tech Science Co., Ltd.). Statistical analysis software (Statview 5.0, manufactured by SAS Institute) was used for statistical analysis of the results.

(Result of OPA method)
FIG. 3 shows the relative values of the absorbances of C12 and C24 with respect to the absorbance of C0. The values of C12 and C24 were more than four times higher than those of the rotifer sample refrozen immediately after thawing. Multiple comparison tests between C0 and C12 and between C0 and C24 showed a significant difference (p <0.05, ANOVA, n = 3, Fisher PLSD method).

As described above, from the results of the SDS-PAGE and OPA methods, it is considered that the thawed rotifer passes through the pharynx and the digestive tract of larvae such as eels because the protein is decomposed in the thawed rotifer.

(Analysis of rotifer fatty acids thawed by gas chromatography)
It was investigated whether the fatty acid composition and the amount of lipid in the thawed rotifer changed depending on the length of the standing time from thawing to refreezing. Each 1 g of each sample was freeze-dried in a freeze-dryer (FDU-1200 type, manufactured by Tokyo University of Science) for 24 hours to obtain a dry sample having a dry weight of about 0.1 g. Lipid extraction was performed by the method of Folch et al. As follows. 20 mL of a chloroform-methanol mixed solution (2: 1) was added to a dry sample, allowed to stand for 10 minutes or more, and crushed with a homogenizer (VH10, manufactured by AS ONE Corporation) for 1 minute. The lipid adhering to the homogenizer was recovered with 10 mL of a chloroform-methanol mixed solution (2: 1), filtered through a glass fiber filter paper (Whatman No. 1, manufactured by GE Healthcare), and the solution was transferred to a 100 mL volumetric flask. The eggplant flask was immersed in a constant temperature water tank (SB-1200, manufactured by Tokyo University of Science) set at 35 ° C., and distilled with a rotary evaporator (N-1110, manufactured by Tokyo University of Science).

Subsequently, the sample was vacuum dried with a digitalizer (MDA-006, manufactured by ULVAC Kiko Co., Ltd.), 30 mL of chloroform was added, the sample was filtered again with a glass fiber filter paper, and the sample was transferred to a 100 mL eggplant flask. This was distilled, vacuum dried with a digitalizer, and then the total lipid (TL) content was measured with an electronic balance (AX224, manufactured by Sartorius). After the measurement, the total lipid was collected in a vial using 3 mL of a chloroform-methanol mixture (1: 1), and then stored in a freezer set at −80 ° C.

The lipid extracted from the sample was dissolved in a chloroform-methanol mixture (1: 1) and transferred to a glass test tube. After removing the solvent with nitrogen gas, the sample was resuspended in 0.5 mL of chloroform containing 1 mg / mL of C19: 0, which is the reference for each lipid. The suspension was transferred to a test tube, dissolved in 1 mL of a 5% hydrogen chloride methanol solution, and heated at 80 ° C. for 3 hours for methyl esterification. After heating, the solution was allowed to cool, 5.5 mL of ion-exchanged water and 1 mL of hexane were added, and the mixture was centrifuged (2000 rpm, 5 minutes). After recovering the hexane layer with a Pasteur pipette, fatty acids were analyzed with a gas chromatograph analyzer (GC-2010, manufactured by Shimadzu Corporation). Analysis was performed using workstation software for GC (GC-solution, manufactured by Shimadzu Corporation). Statistical analysis software (Statview 5.0, manufactured by SAS Institute) was used for statistical analysis of the results.

(Results of fatty acid analysis)
FIG. 4 shows the content of fatty acid per 1 g of dry weight of rotifer. According to the multiple comparison test, there was no significant difference in the standing time from thawing to refreezing.

The content of each type of fatty acid per 1 g of dry weight of rotifer is shown in FIG. Due to the difference in the standing time from thawing to refreezing, some fatty acids had different contents and some had almost no change in content.

FIG. 6 shows the content per 1 g of dry weight of worms when the fatty acids are divided into three groups of saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids. According to the multiple comparison test, there was no significant difference in the standing time from thawing to refreezing. Therefore, it was shown that the nutritional value did not deteriorate even if the standing time from thawing to refreezing was lengthened.

(Breeding experiment 1)
As the test group, a C0 test group for feeding C0 as a feed, a C12 test group for feeding C12 as a feed, and a C24 test group for feeding C24 as a feed were set. A 4L aquarium (579975, manufactured by Kamihata Fish Farm) was used in each test plot. As the breeding water, seawater (about 33 psu) filtered with a cartridge filter having a mesh size of 100 μm, 25 μm, and 10 μm in descending order of mesh size was used. The breeding water was sterilized by ultraviolet rays with a forced circulation germicidal lamp (UVF-1000, manufactured by Iwaki), and then suction filtered using a glass fiber filter paper (GF / F, manufactured by GE Healthcare).

Fertilized eggs of the spotted garden eel (Heteroconger hassi) were housed in 42 in the C0 test group, 32 in the C12 test group, and 30 in the C24 test group. The water tank was installed in a water bath in which the water temperature was adjusted to 26 ° C. with a heater. The breeding water was aerated with ceramic air stones (13 ml / min). The breeding was carried out in a blackout curtain (shading rate 95%, wide screen BK2012, manufactured by Japan Wide Cloth Co., Ltd.) provided in a room illuminated by fluorescent lamps. During the breeding period, 0.02 g of C0, C12 or C24 was fed every two hours between 9:00 and 17:00. Water was changed every day to prevent deterioration of water quality.

The number of surviving individuals in each test plot was counted daily to calculate the survival rate. Statistical analysis software JMP (manufactured by SAS Institute) was used for statistical analysis of the breeding test.

(Result of breeding test 1)
The survival rate of each test plot is shown in FIG. In the C0 test group and the C12 test group, the survival rate was 0% at 4 days of age. On the other hand, in the C24 test group, the survival rate after the opening day was stable as compared with the other test groups, and survival up to 5 days of age could be confirmed. As a result of survival time analysis, there was a significant difference between the C0 test group and the C12 test group, and between the C0 test group and the C24 test group, but there was no significant difference between the C12 test group and the C24 test group. (Kaplan-Meier method, p <0.05).

An image of the longest surviving individual in each test group taken with a stereomicroscope (C-BD115, manufactured by Nikon Corporation) is shown in FIG. As shown in FIGS. 8A and 8B, the contents could not be confirmed in the gastrointestinal tract in the 3-day-old individuals in the C0 test group. On the other hand, in the 3-day-old individuals in the C12 test group shown in FIGS. 8 (C) and 8 (D) and the 5-day-old individuals in the C24 test group shown in FIGS. 8 (E) and 8 (F), the contents were contained in the digestive tract. I was able to confirm.

(Breeding experiment 2)
A CI24 test plot was set up to feed a rotifer (CI24) in which rotifers fed with isocrisis and the time from thawing to refreezing was 24 hours and C24 were mixed at a weight ratio of 1: 1. Forty-four fertilized spotted garden eel eggs were housed in the CI24 test plot. Except for the feed, the other conditions of the CI24 test plot are the same as those of the C0 test plot. The number of surviving individuals in the CI24 test plot was counted daily, and the survival rate was calculated in the same manner as in breeding experiment 1.

After fixing the spotted garden eel larvae bred in the CI24 test group with 100% formalin, a micro ruler (MR-2, manufactured by KENIS) was placed on the larva, and the images were taken with a stereomicroscope and a single-lens reflex camera (D5200, manufactured by Nikon). The head length of the spotted garden eel was measured by image analysis using the image analysis software Image J.

(Result of breeding test 2)
FIG. 9 shows the survival rate of the CI24 test group together with the results of the breeding experiment 1. The survival rate of the CI24 test group was higher than that of the C24 test group, and survival up to 11 days of age could be confirmed. There were significant differences between the C0 and CI24 test plots, between the C12 and CI24 test plots, and between the C24 and CI24 test plots. (Kaplan-Meier method, p <0.05). A diet in which rotifers fed with EPA-rich quilt cellos and rotifers fed with DHA-rich isocrisis are thawed and mixed to supplement the nutrients required for spotted garden eel larvae, improve survival rates, and increase survival days. It is thought that it has grown.

Images of the longest surviving individuals in the C24 test group and the CI24 test group taken with a stereomicroscope are shown in FIGS. 10 (A) and 10 (B), respectively. The 11-day-old individual in the CI24 test group shown in FIG. 10 (B) had a longer body length than the 5-day-old individual in the C24 test group shown in FIG. 10 (A), and the development of the head was confirmed.

The average head length of 3-day-old individuals in the CI24 test group was 8.7 ± 1.1 mm. On the other hand, it was 12.3 ± 0.9 mm at the age of 11 days in the CI24 test group. There was a significant difference between 3 days and 11 days (n = 3, t-test, p <0.05). Since the head length of 11 days was longer than that of 3 days, it was confirmed that the rotifers given Quitocellos and the rotifers given Isocrisis were thawed and mixed with each other to grow. Was done.

The embodiments described above are for the purpose of explaining the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is shown not by the embodiment but by the scope of claims. And, various modifications made within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.

The present invention is suitable for aquaculture of detritus-eating aquatic animals.

Claims (5)

A freezing step to freeze marine brachionus plicatilis,
A thawing step in which the marine worms are thawed and the thawed marine worms are placed at room temperature for 15 to 30 hours to promote the decomposition of the marine worms by the activity of the enzyme possessed by the marine worms .
A method for producing a feed for detritus-eating aquatic animals, including. Following the thawing step further comprises a refreezing step of freezing the marine brachionus plicatilis.
The method for producing a feed for a detritus-eating aquatic animal according to claim 1 . The marine worms are
The first marine worms fed and cultivated with the first microalgae and the second marine worms fed and cultivated with the second microalgae different from the first microalgae.
The method for producing a feed for a detritus-eating aquatic animal according to claim 1 or 2 . The first microalgae and the second microalgae are
Selected from the group consisting of cryptophytes, haptophytes and diatoms, respectively.
The method for producing a feed for detritus-eating aquatic animals according to claim 3 . The first microalgae is
Isochrysis sp. And
The second microalgae is
Chaetoceros calcitrans,
The method for producing a feed for detritus-eating aquatic animals according to claim 3 .

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