Aquatic feed composition

Abstract

This invention provides an aquatic feed composition suitable for farmed fish or farmed crustaceans, the composition comprising hemp seed product, wherein the hemp seed product contains 25% to 100% by weight of protein and is present in the composition in an amount of 5% to 100% by weight. A method for producing and using the aquatic feed composition is also provided.

Description

[0001] Invention Field

[0002] This invention relates to aquatic feed compositions suitable for commercially farmed fish or crustaceans, and methods for their manufacture and use. The compositions comprise proteins derived from hemp seed products and / or contain hemp seed oil.

[0003] background

[0004] Aquaculture (also known as hydroponics) is a significant and continuously growing industry in the UK and globally. This industry involves the cultivation of aquatic organisms such as fish, crustaceans, mollusks, and plants in freshwater or seawater. The industry is poised for further growth as global demand for edible protein continues to increase while wild populations of aquatic life decline.

[0005] However, this growing industry itself places demands on the planet, as fish and crustacean farming, in particular, requires suitable feed. For example, over 90% of the environmental impact of salmon production stems from feed supply and use, including carbon, land, and freshwater footprints (Newton RW, Little DC (2018) Mapping the impacts offarmed Scottish salmon from a life cycle perspective. The International Journal of Life Cycle Assessment 23, 1018-1029). Therefore, reducing the impact of feed production while maintaining good feed conversion ratios is crucial for the sustainable development of this industry. Major alternatives to marine feedstocks have been shown to be environmentally damaging, with high processing energy consumption and / or significant impacts through land-use changes such as soybean cultivation.

[0006] Currently, several alternative protein sources exist to land-based or fish-based proteins, each exhibiting different advantages and disadvantages. In recent years, the production of microbial or single-cell protein (SCP) has flourished, offering protein yields of up to 80% and meeting sustainability requirements due to the availability of byproducts from other industries as substrates. However, dietary processing incurs high energy costs, which in turn affect price and CO2 footprint. This, coupled with palatability and nucleic acid content, may limit its addition levels in aquatic feeds (Pilmer et al., (2022) Using dietary additives to improve palatability of diets containing single-cell protein from methanotrophic bacteria in yellowtail kingfish). Seriola Ialandi ) diets. Aquaculture Research 53(14):5006-5017).

[0007] In recent years, insect meal has gained attention due to its favorable amino acid profile and palatability, similar to fishmeal. However, due to regulatory restrictions, insects cannot feed on waste and must instead feed on feed-grade substrates. This means that ingredients that can be used directly in aquaculture feed are now being used to feed insects. Similar to SCPs, scalability may be an issue, and the potential transmission of pathogens to farmed animals (Baiano, (2020) Edible insects; An overview on nutritional characteristics, safety, farming, production technologies, regulatory framework, and socio-economic and ethical implications. Trends Food Sci. Technol. 100: 35–50).

[0008] Another alternative approach is to use microalgal biomass produced in bioreactors. This source has added value due to its naturally occurring high levels of health-beneficial long-chain polyunsaturated fatty acids. Nevertheless, processing biomass requires high energy costs and has been found to have digestibility issues in various fish species (Gong et al., (2017) Digestibility of the defatted microalgae Nannochloropsis sp. and Desmodesmus sp. when fed to Atlantic salmon, Salmo salar. Aquaculture Nutrition 24, 56-64).

[0009] Therefore, in order to support the growing aquaculture industry, there is ongoing interest in alternatives and improved raw materials for producing aquatic feed compositions suitable for farmed fish or crustaceans.

[0010] Brief Description of the Invention

[0011] Therefore, the present invention provides an aquatic feed composition suitable for farmed fish or farmed crustaceans, the composition comprising hemp seed product, wherein the hemp seed product comprises 25% to 100% by weight of protein (based on the weight of the hemp seed product) and is present in the composition in an amount of 5% to 100% by weight (based on the total weight of the composition).

[0012] The present invention also provides a method for producing the aquatic feed composition described herein, the method comprising mixing a hemp seed product containing 25% to 100% protein with one or more additional aquatic feed ingredients to form a mixture, wherein the hemp seed product is present in the mixture in an amount of 5% to 95% by weight.

[0013] On the other hand, the present invention provides a method for raising fish or crustaceans, comprising feeding said fish or crustaceans the aquatic feed composition described herein.

[0014] Furthermore, as described below, the present invention also provides the use of hemp seed products in the preparation of the aquatic feed compositions described herein.

[0015] The aquaculture feed compositions described herein contain protein derived from hemp seed products, such as hemp seed meal or hemp seed protein concentrate. The use of this unconventional protein source in aquaculture feed has significant advantages; hemp seed products are easily digestible and supportive of growth. Furthermore, hemp seed products offer significant advantages over soybeans (currently one of the main protein sources used in aquaculture feed), whose cultivation and application in aquaculture feed contribute to deforestation and a high CO2 footprint (both from the cultivation process and international transport).

[0016] Furthermore, this study shows that in trials comparing aquatic diets containing hemp seed protein with those containing soy protein concentrate, the hemp seed protein-containing diet was associated with reduced intestinal inflammation and a healthier overall gut in fish. This surprisingly suggests that aquatic diets containing hemp seed protein may also be beneficial to the health of farmed fish or crustaceans.

[0017] In a further aspect, this document describes aquatic feed compositions containing hemp seed oil suitable for farmed fish or farmed crustaceans. Specifically, the aquatic feed compositions may contain 5% to 50% by weight of hemp seed oil. Similar to the aquatic feed compositions described above, hemp seed oil can be advantageously used in feed to avoid or reduce the use of fish oil or soybean oil.

[0018] Detailed description

[0019] As described above, the present invention provides an aquatic feed composition suitable for farmed fish or farmed crustaceans, the composition comprising hemp seed product, wherein the hemp seed product comprises 25% to 100% by weight of protein and is present in the composition in an amount of 5% to 100% by weight. Specifically, the protein referred to is derived from hemp seeds, i.e., hemp seed protein.

[0020] The hemp seed product is incorporated into the composition in an amount of 5% to 100% by weight, 10% to 100% by weight, 10% to 80% by weight, or 10% to 50% by weight, preferably 20% to 40% by weight, more preferably 25% to 35% by weight. When the hemp seed product constitutes less than 100% of the aquatic feed composition, the remainder of the composition consists of one or more other ingredients suitable for the farmed fish / crustaceans targeted by the composition.

[0021] Specifically, the hemp seed product included in the aquaculture feed composition provides at least a portion of the protein component of the composition. (As discussed further below, additional protein in the composition may be derived from other ingredients, such as fishmeal, vegetable meal, or animal meal). The hemp seed product itself contains 25% to 100% by weight of protein, or 25% to 99% by weight, 25% to 95% by weight, 25% to 80% by weight, 40% to 80% by weight, 50% to 80% by weight, 55% to 80% by weight, or 60% to 80% by weight of protein. In one example of an aquaculture feed that can be used for farmed salmon, the hemp seed product is hemp seed meal and contains 55% to 80% by weight of protein.

[0022] Protein content assessment was performed according to the Official Analytical Chemists Association (AOAC) Methods of Analysis (2000) standard procedure. Specifically, protein content was measured using the Kjeldahl method based on analytical nitrogen content (N × 6.25) after sulfuric acid digestion, using an Opsis AB LiquidLINE KjelROC analyzer.

[0023] The percentage of protein content mentioned above can also be based on the weight percentage of dry matter.

[0024] The hemp seed product is a product derived from hemp seeds and containing hemp seed protein. Specifically, the hemp seed product may be hemp seed meal or hemp seed protein concentrate, which will be described further below.

[0025] Industrial hemp ( Cannabis sativa Industrial hemp is cultivated worldwide for a variety of industrial uses. The industrial hemp plant has low concentrations of tetrahydrocannabinol (THC), for example, less than 1% THC. Many different industrial hemp cultivars (plant genetic variations) are known, and these can be used to provide hemp seeds from which hemp seed products are derived. In particular, cultivars that are particularly well-suited for producing hemp seeds (rather than hemp fiber) exist, and these varieties are especially useful for preparing the compositions of the present invention.

[0026] Hemp seeds consist of a high-fiber outer shell and a high-protein and high-fat inner kernel. The hemp seed products used in the compositions described herein can be produced by dehulling the seeds to separate the outer shell, or they can be prepared from hemp seeds with their outer shells retained, such that products derived from the outer shell, such as fiber, are retained in the hemp seed products used in aquaculture feed compositions.

[0027] Hulled or unhulled hemp seeds can be used to produce hemp seed meal or hemp seed protein concentrate of the aquatic feed composition of the present invention. Hemp seed meal (also known as hemp seed cake) can be produced by cold pressing hemp seeds and typically contains up to 35% by weight of protein. Hemp seed protein concentrate is produced from hemp seed meal (hemp seed cake) through protein concentration steps such as milling (including screening / sieving) or solvent / enzymatic extraction. These steps can be used to produce hemp seed products with the desired percentage of protein. Specifically, these steps can be used to produce hemp seed protein concentrate containing 50% by weight or more of protein. Hemp seed protein concentrate may be in the form of flour.

[0028] Preferably, the hemp seed product is a hemp seed protein concentrate.

[0029] In some instances, hemp seed products contain 20% to 35% by weight of protein. In these instances, the hemp seed products are obtained from hemp seeds through cold pressing, without requiring further steps to increase the weight percentage of protein in the product.

[0030] The hemp seed products produced as described above have an amino acid profile similar to that of soybean protein concentrate, and are known to contain amino acids essential for the growth of fish / crustaceans.

[0031] Other components of the aquatic feed composition may be selected based on the farmed fish / crustaceans to which the aquatic feed composition will be fed. Commercial feeds for farmed fish / crustaceans are typically formulated based on digestible nutrients and energy. In particular, the composition may also contain one or more of the following: fishmeal, soybean meal, soybean protein concentrate, rapeseed meal, marine animal meal, crustacean meal, insect meal, single-cell protein, processed animal protein, bacteria, algae, fish protein concentrate, fish oil, hemp seed oil, vegetable oil, wheat, vitamins, and minerals. Recommended diets for farmed fish and crustaceans based on their species are known in the art (e.g., on the FAO website - https: / / www.fao.org / fishery / affris / species-profiles / en / ).

[0032] For example, for salmonid fish such as salmon and trout, dietary requirements typically include 10 essential amino acids, omega-3 fatty acids, energy (primarily provided by lipids and proteins), 15 essential vitamins, and astaxanthin. Typical dietary formulations known in the art may include fishmeal, soybean meal, wheat / wheat by-products, vitamins / minerals, and fish oil or soybean oil, wherein approximately 40-45% of the feed is protein and approximately 30-40% is lipid. Hemp seed oil, which can be prepared from hemp seeds, can be used in the aquatic feed formulations described herein to provide at least a portion of the lipid component. Furthermore, one aspect of the invention described herein is an aquatic feed composition comprising hemp seed oil suitable for farmed fish or farmed crustaceans. Specifically, the aquatic feed composition may contain 5% to 50% by weight of hemp seed oil, preferably 10% to 40% by weight, and more preferably 25% to 35% by weight.

[0033] The total protein content of the aquatic feed composition may be from 40% to 80% by weight, preferably from 40% to 70% by weight, and more preferably from 50% to 60% by weight. These values ​​may be based on dry matter. It may be necessary to incorporate fishmeal into the aquatic feed composition to ensure the presence of the necessary nutrients; therefore, a portion of the protein in the aquatic feed composition will come from the fishmeal.

[0034] The aquatic feed composition of the present invention can be prepared by mixing hemp seed products with one or more additional ingredients to form a mixture or paste. The aquatic feed may be in granular or powder form, or it may be in flake form. Preferably, the aquatic feed composition is in granular form.

[0035] Aquatic feed compositions can be formed by hot or cold extrusion. In particular, extrusion processing technology can be used to provide products with good water stability. Typically, during extrusion, the mixture is forced through a barrel by one or more screws and then through a die located at the end of the barrel. Hot extrusion may be preferred because it allows for obtaining higher fat content.

[0036] The farmed fish / crustaceans are preferably commercially farmed. There are no particular limitations on the fish or crustaceans suitable for the aquatic feed composition; they can be freshwater or marine farmed fish / crustaceans. Specifically, the farmed fish or crustaceans are selected from salmon, Arctic char, carp, trout, sea bass, sea bream, sturgeon, turbot, halibut, kingfish, barramundi, grouper, catfish (including shark catfish), marine finned fish, eel, halibut, marine fish, tilapia, herring, luscat, flounder, cod, tuna (especially yellowfin tuna), sardines, mackerel, lobster (freshwater, brackish water, or marine), prawns, shrimp, crabs (freshwater, brackish water, or marine) or freshwater crayfish (e.g., American or Australian crayfish). Alternatively, the farmed fish or crustaceans are selected from salmon, trout, sturgeon, shrimp, tilapia, and carp.

[0037] Preferably, the farmed fish or farmed crustacean is a salmonid fish. Specifically, the farmed fish is salmon or trout (e.g., juvenile or post-juvenile salmon or trout), more preferably Atlantic salmon, Pacific salmon, or rainbow trout (e.g., juvenile or post-juvenile Atlantic / Pacific salmon or rainbow trout). Most preferably, the farmed fish is Atlantic salmon (e.g., post-juvenile Atlantic salmon).

[0038] In another embodiment, the farmed fish may be tilapia or basa fish, preferably tilapia.

[0039] In a further embodiment, the farmed crustacean is a warm-water crustacean, preferably shrimp or prawn, more preferably Litopenaeus vannamei (Penaeus vannamei). Litopenaeus vannamei ), tiger prawn ( Penaeus monodon Tiger prawn or giant freshwater prawn ( Macrobrachium rossenbergii (giant freshwater shrimp), more preferably Litopenaeus vannamei.

[0040] Aquatic feed compositions can be fed to farmed fish / crustaceans over a period of time to ensure efficient and healthy growth. Specifically, this period can be at least one week, at least one month, at least six months, at least one year, at least two years, or at least three years. This period can begin when the fish / crustaceans are juveniles or young adults and continue until harvest. The time cycle can be one month to three years, one month to three months, three months to six months, or one to two years. The time period will depend on the species of fish / crustaceans, the farming conditions, and the desired results. For example, to obtain 4 kg of salmon or 2 kg of trout, the rearing period can be approximately two years; to obtain 400 g of trout, the rearing period can be approximately eight months; and for shrimp, the rearing period can be approximately three to six months.

[0041] In the aquaculture methods described herein, fish / crustaceans can be cultured in open-water aquaculture systems or recirculating aquaculture systems. Preferably, open-water aquaculture systems are used. Specifically, when the fish are salmon or trout, they can be cultured in an open-water aquaculture system. Alternatively, when the crustaceans are shrimp, a recirculating aquaculture system can be used.

[0042] The present invention will be further described with reference to the following embodiments. Detailed Implementation

[0043] Example 1 – Analysis of Hemp Seed Protein Concentrate

[0044] Nutritional characterization (moisture, oil, protein, ash, minerals, amino acid composition, and fatty acid profile) can be performed on hemp seed meal / hemp seed protein concentrate, and anti-nutritional factors (phytic acid and glycosylation) can be analyzed. Hemp seed products with the desired protein content can be selected. In this example, hemp seed protein concentrate produced from hulled hemp seeds through cold pressing and milling steps was analyzed.

[0045]

[0046] Table 1. Results of analysis of hemp seed protein concentrate

[0047] Example 2 – Testing of Aquatic Feed Composition

[0048] The suitability of the aquatic feed compositions described herein can be verified in in vivo testing.

[0049] For example, in a suitability test for feeding Atlantic salmon, a reference composition may comprise 70% fishmeal, 10% wheat, 15% fish oil, and 5% vitamin mixture (which may include yttrium oxide as an inert marker). A test aquaculture feed composition may have the same composition as the reference composition, but replace 30% of the dry matter with a hemp seed product described herein (such as the hemp seed product of Example 1). Alternatively, the reference composition may comprise 40% fishmeal, 30% soybean meal, 10% wheat, 15% fish oil, and 5% vitamin mixture (which may include yttrium oxide as an inert marker), and in the test composition, all soybean meal may be replaced with the hemp seed product described herein, or in the test composition, a portion of the soybean meal and a portion of the fishmeal may be replaced with the hemp seed product described herein.

[0050] Palatability testing can be conducted to ensure that both the reference and test feeds are ingested by the test organisms.

[0051] Preliminary trials can be conducted to test digestibility (e.g., Betancor et al., (2016) Nutritional Evaluation of an EPA-DHA oil from transgenic Camelina sativa in feeds forpost-smolt Atlantic salmon ( Salmo salar (L.) PLOS One, e0159934). For example, when testing Atlantic salmon, the reference composition and the test aquaculture feed composition can be fed to post-juvenile Atlantic salmon weighing approximately 500g in different marine aquaculture ponds for two weeks. At the end of the experiment, all fish in each pond can be painlessly killed approximately 6 hours after feeding, and weight, length, and operational welfare indicators can be measured. Fecal samples can be collected by stripping for digestibility analysis according to the method reported by Austeng (Austreng (1978) Digestibility determination in fish using chromic oxide marking and analysis of contents from different segments of the gastrointestinal tract. Aquaculture 13, 265-272). Fish in each culture pond can be dissected, and the foregut, hindgut, and liver can be collected and fixed in 4% buffered formalin for histopathological evaluation using ImageJ (FIJI) software (Schindelin et al. (2012) Fiji: an open-source platform for biological-imageanalysis. Nature Methods, 9, 676–682).

[0052] A comprehensive nutritional trial (e.g., lasting 2.5 months) can also be conducted using the test composition and a reference composition to assess the effects of the test composition on fish growth, fillet quality, welfare, and health. For example, for Atlantic salmon, the trial can be conducted on post-juvenile (seawater) Atlantic salmon weighing approximately 300 g. At the end of the experimental period, the fish can be humanely and painlessly killed, their length and weight measured, and operational welfare indicators (OWI) recorded. Furthermore, multiple samples can be collected: meat samples can be used for fillet quality (moisture, protein, ash, fat content, fatty acid and amino acid profiles, mineral and pigment deposition); liver, as well as foregut and hindgut samples, can be fixed in 4% buffered formalin for histological evaluation; fecal samples can be collected for short-chain fatty acids; and liver and foregut samples can be used for qPCR.

[0053] Example 3 – Testing Aquaculture Feed Compositions in Atlantic Salmon

[0054] In addition to the scheme described in Example 2, this example also provides the testing of the aquatic feed composition described herein in Atlantic salmon, and further demonstrates the suitability of hemp seed as a protein source for Atlantic salmon by showing the digestibility of hemp seed and its effects on fish welfare and performance.

[0055] method

[0056] The experiment was conducted on late-juvenile Atlantic salmon (409 ± 21.4 g). The fish were approximately one year old. Nine fish ponds were used, with 40 fish in each pond. After an initial acclimatization period, the following three feeds were used for two weeks, with each feed given to three ponds, i.e., 120 fish per feed. Feeding was performed twice daily at full capacity using an automatic feeder, and uneaten feed was collected.

[0057] These three diets contain yttrium as an inert marker. A diet containing high levels of fishmeal (FISH diet) was used as a control (in particular, this diet is known to have high digestibility). The second and third diets were based on the first FISH diet. In the second diet, designated "SPC", 30% (by weight) of the FISH diet was removed and replaced with soybean protein concentrate (notably, soybean protein is currently the leading commercial alternative to fishmeal in aquaculture feeds). In the third diet, designated "HM", 30% (by weight) of the FISH diet was removed and replaced with hemp seed meal, which has high levels of protein. The amounts of ingredients (g / kg) in each diet are shown in Table 2 below, and the diet compositions are shown in Table 3. All diets were extruded into 4 mm pellets.

[0058]

[0059] Table 2. Feed formulations for the three feeds used in the examples.

[0060]

[0061] Table 3. Analyzed composition of the three feeds used in the examples.

[0062] The percentages of protein, lipids, dry matter, and ash in the control, hemp seed meal, and SPC powder are shown in Table 4 below.

[0063]

[0064] Table 4. Composition of fishmeal, soybean protein concentrate, and hemp seed meal

[0065] Further details regarding the content of soybean protein concentrate (SPC) and hemp seed meal (HM) are shown in Table 5 below.

[0066]

[0067] Tables 5A and 5B. Further details on the amino acid content (5A) and elemental content (5B) of soybean protein concentrate and hemp seed meal.

[0068] All compositional assessments were performed according to the Official Analytical Chemist Association (AOAC) Methods of Analysis (2000) standard procedures. Specifically, protein was determined using the Kjeldahl method based on analytical nitrogen content (N × 6.25) after sulfuric acid digestion, using an Opis AB LiquidLINE KjelROC analyzer. Crude lipid content was determined by gravimetric analysis following Soxhlet extraction (Tecator Soxtec system 2050 automated extraction system).

[0069] Moisture content was obtained after drying at 110℃ for 24 h in an oven, and ash content was determined after incineration at 600℃ for 16 h.

[0070] As shown in Table 4, the protein content of hemp seed meal exceeds 60%.

[0071] At the end of the experiment, all fish were painlessly euthanized approximately 6 hours after feeding, and their weight, length, and operating welfare index (OWI) were measured. Feces were collected by squeezing according to the method of Austreng E. 1978 (Aquaculture 13:265-272). Specifically, feces were collected from different fish and combined into two samples (A and B) in each rearing tank (i.e., each sample contained feces from 20 fish).

[0072] The protein, lipid, and amino acid contents were determined, and the apparent digestibility coefficient (ADC) of the feed and the apparent component digestibility coefficient (AIDC) of hemp seed and SPC powder were calculated according to the methods of Aas et al. 2015 (NofimaReport 32 / 2015:1–22) and Bureau et al. 1999 (Aquaculture 180(3-4):345-358), respectively.

[0073] ADC (%) = 100 × ((a - b) / a)

[0074] Where 'a' represents the ratio of nutrients to markers in the feed, and 'b' represents the ratio of nutrients to markers in the feces.

[0075] AIDC (%) = ADC REF + ((1 – s) × DR / sDI) × (ADC T – ADC REF )

[0076] ADC T It is the apparent digestibility coefficient of the test diet (i.e., SPC feed or HM feed); ADC REF 1 is the apparent digestibility coefficient of the reference diet (i.e., FISH feed); DR is the % of nutrients (or kJ / g total energy) in the reference diet meal; DI is the % of nutrients (or kJ / g total energy) in the test component; s is the proportion of the test component in the test diet meal; 1–s is the proportion of the reference diet meal in the test diet meal.

[0077] The description of the nutritional biochemical analyses required for these calculations can be found in McMillan et al., 2024 (Aquaculture, Vol. 585, No. 740686), except for the amino acid analysis, which is described in Glencross et al., 2021 (Aquaculture, Vol. 544, No. 737133).

[0078] Condition factor k The specific growth rate (SGR) and other parameters are calculated based on measurements of fish weight (W) and length (L). Specifically, they are based on the formula... k = 100 (W / L) 3 ) Calculate fullness ( k ).

[0079] The specific growth rate (SGR) was calculated according to the method described by Ricker, 1975 (Bulletin of the Fisheries Research Board of Canada 191, 1–382).

[0080] SGR (% BW / day) = 100 × (e g - 1)

[0081] Where g = (ln (BW) f ) - ln (BW i )) / (t2 - t1)

[0082] Or g = (ln (BW) f ) - ln (BW i )) × (t2 - t1) -1

[0083] In addition, samples were collected from six fish per culture tank (n=18 per diet treatment) to assess fecal levels of short-chain fatty acids. Blood was collected from these six fish using a heparinized syringe, centrifuged at 3000 g for 15 minutes, and plasma was collected for analysis of circulating short-chain fatty acid levels. Furthermore, two segments each of the foregut and hindgut, as well as the liver, were collected from the same fish and stored overnight at 4°C in RNA-Later, then maintained at -20°C until RNA extraction for assessing inflammation via molecular biomarkers. Samples from the same fish / tissue were fixed in 4% buffered formalin for histopathological evaluation of the tissues.

[0084] Analysis of short-chain fatty acids in feces and blood plasma

[0085] Fecal samples were freeze-dried for at least 12 hours (Christ Alpha 1-4 LSC, SciQuip Ltd., Shropshire, UK). A Sigma-Aldrich 13C-short-chain fatty acid fecal mixture (Merck KGaA, Darmstadt, Germany), acetonitrile (ACN), 3-nitrophenylhydrazine hydrochloride solution, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution were added to the fecal and plasma samples. The samples were vortexed, incubated on ice at 40°C for 50 minutes, and centrifuged (2000 × 10⁻⁶). g Extract the supernatant, dry it under nitrogen, and redissolve it in methyl tert-butyl ether (MTBE) solution. Vortex and centrifuge the dissolved sample (2000 × g, 5 min) to extract the aqueous MTBE layer. Dry the MTBE layer again under nitrogen and redissolve it in equal volumes of ACN and water. Centrifuge the sample again (2000 × g, 5 min). g Extract the supernatant (after 5 minutes) and transfer it to a liquid chromatography vial. Analyze all samples using a liquid chromatography-mass spectrometry system (Waters Corporation, Massachusetts, USA).

[0086] Quantitative RT-polymerase chain reaction analysis

[0087] Expression of genes of interest in all RNA samples was determined by qPCR, with elongation factor-1α (elf1α), hprt, and rpl2 used as reference genes (see table below). cDNA was diluted 20-fold with milliQ water. Primer efficiency for each gene was pre-evaluated by serial dilutions of cDNA pooled from samples to ensure >85% efficiency for all primers. qPCR was performed in duplicate in 20 μL reaction volumes in 96-well plates using a Biometra TOptical thermal cycler (Analytik Jena, Goettingen, Germany). The reaction volume contained 10 μL of Luminaris Color HiGreen qPCR premix (Thermo Scientific, Hemel Hempstead, UK), 1 μL of primers (10 pmol) corresponding to the analyzed genes, 3 μL of molecular biology grade water, and 5 μL of cDNA (diluted 1 / 20). Amplification was also performed using a systemic negative control (NTC, template-free control) without cDNA. The standard amplification parameters include UDG pretreatment at 50°C for 2 minutes, initial denaturation at 95°C for 10 minutes, followed by 35 cycles: 15 s at 95°C, 30 s at annealing temperature, and 30 s at 72°C.

[0088]

[0089] Table 6. Primers used in the qpCR analysis of this study, including pCR efficiency and function.

[0090] Histological assessment

[0091] Cross-sectional sections of the liver and midgut from six fish per culture pond were fixed in 4% buffered neutral formaldehyde and embedded in paraffin. 4 μm thick sections were cut using a microtome (Leica RM 2035, Leica Biosystems, Wetzlar, Germany) and stained with hematoxylin and eosin (H&E) (Betancor et al., 2015). Furthermore, slides containing intestinal samples were stained with Alsin Blue / Periodic Acid Schiff (AB-PAS) to differentiate between neutral / mixed mucus and acidic mucus in goblet cells (Betancor et al., 2016b). Scanned slides (Axio Scan Z1, ZEISS, Oberkochen, Germany) were visualized using QuPath 0.2.3 (Bankhead et al., 2017). Digital images were analyzed using ImageJ (FIJI) software (Schindelin et al., 2012).

[0092] The presence of intracytoplasmic lipid vacuoles was examined in liver micrographs from each diet group at 20x original magnification. The micrographs were converted to black-and-white 2D images, where white corresponds to the area occupied by lipid droplets, and the percentage of area covered by lipid droplets was measured. In the intestine, 30 measurements were collected for each fish for each assessment parameter, including intestinal epithelial cell height (hE), intestinal epithelial cell width (nNE) (measured as the number of intestinal epithelial cell nuclei along a 100 µm distance), circular muscle layer thickness (CMT), maximum (Lmax) and minimum (Lmin) intestinal length passing through the center of the lumen in cross-section, and goblet cells (GB, calculated per 100,000 µm). 2 (Number of goblet cells in the intestinal mucosa). Length measurements are shown in µm.

[0093] Results and Conclusions

[0094] The digestibility results are shown in Table 7, and the fish performance results are shown in Table 8.

[0095]

[0096] Table 7. Digestibility of Diets and Experimental Components

[0097] The results showed that the digestibility of the HM diet containing hemp seed meal was comparable to that of the control FISH diet and the SPC diet containing soybean protein concentrate. In particular, the protein digestibility of the hemp seed meal component was higher than that of the SPC powder component.

[0098]

[0099] Table 8. Fish performance at the end of the experiment

[0100] At the end of the experiment ， No significant differences were found in growth or condition among the different feed groups. p > 0.05). The assessed operational welfare metrics (data not shown) also did not reveal any welfare issues with any feed given to the fish.

[0101] Short-chain fatty acids (SCFAs) in the gut and feces

[0102] Short-chain fatty acids (SCFAs) are fatty acids with fewer than six carbon atoms, primarily produced by the gut microbiota, and are generally essential for gut health. Therefore, changes in their concentration indicate changes in the gut microbiota.

[0103] The table below shows the SCFAs identified in the obtained fecal and plasma samples.

[0104]

[0105] Table 9. Short-chain fatty acid levels identified in feces and plasma of Atlantic salmon fed three different experimental diets over a two-week period. Values ​​are shown as mean ± SD for 18 fish per diet (n = 3 culture tanks per diet). Differences (p < 0.05) are indicated by different superscript letters.

[0106] Generally, SCFA concentrations were higher in the feces of fish fed with control hemp seeds and lowest in fish fed with hemp seeds. Conversely, hemp seed-fed fish showed the highest circulating SCFA levels in plasma. Therefore, no correlation was found between plasma and fecal SCFA levels, similar to what has been observed in humans (Chen et al., 2022; Mayo-Martinez et al., 2024).

[0107] Nevertheless, when observing differences in SCFA levels between different treatments, the only difference was observed in plasma valerate levels, with fish fed a hemp seed diet showing significantly higher levels. Valerate supplementation in mice has been shown to have anti-inflammatory effects (downregulating IL-17 and upregulating IL-10 expression levels) (Luu et al., 2019), potentially suggesting that hemp seed meal exerts a protective effect against inflammation by promoting the production of this SCFA.

[0108] Histological results

[0109] Quantitative scores of intrahepatic liver vacuolization showed no difference among the three dietary treatments (see table below), and a uniform and regular distribution of lipids was generally observed in tissues (data not shown). These results indicate that all fish exhibited adequate nutritional status with acceptable fat storage and no signs of starvation.

[0110]

[0111] Table 10. Histological parameters assessed in the livers of Atlantic salmon fed with the experimental diet for 2 weeks. Values ​​show mean ± SD for 18 fish per diet (n = 3 culture tanks per diet).

[0112] Histological evaluation of the foregut and hindgut revealed significant differences (see table below). Specifically, changes associated with goblet cells included total goblet cell count (foregut) and acidic / mixed goblet cell count (%) and neutral goblet cell count (%) in the hindgut.

[0113]

[0114] Table 11. Histological parameters of the foregut in Atlantic salmon fed with the experimental diets for two weeks. GB, goblet cells; hE, intestinal epithelial cell height (µm); nNE, intestinal epithelial cell width; CMT, circular muscle layer thickness; Lmax and Lmin, maximum and minimum intestinal lengths in cross-section (µm); CSP, cross-sectional perimeter (µm). Numerical values ​​are shown as mean ± SD for 18 fish per diet (n = 3 culture ponds per diet). Differences (p < 0.05) are indicated by different superscript letters.

[0115]

[0116] Table 12. Histological parameters assessed in the hindgut of Atlantic salmon fed with the experimental diets for two weeks. GB, goblet cells; hE, intestinal epithelial cell height (µm); nNE, intestinal epithelial cell width; CMT, circular muscle layer thickness; Lmax and Lmin, maximum and minimum intestinal lengths in cross section (µm); CSP, cross section perimeter (µm). Numerical values ​​are shown as mean ± SD for 18 fish per diet (n = 3 culture ponds per diet). Differences (p < 0.05) are indicated by different superscript letters.

[0117] It is noteworthy that, compared to soybeans, fish fed a hemp seed diet had neutrophil (GB) counts closer to those on a fishmeal control diet. Without being bound by theory, it is believed that if soybeans stimulate the intestinal lining, we can expect to see a reduction in the production of neutral mucin.

[0118] In particular, goblet cells (GCs) are specialized intestinal epithelial cells (IECs) that play a crucial role in intestinal defense. They are distributed throughout the epithelial lining of the small and large intestines, and are especially abundant in the human colon, where a robust mucus barrier is particularly essential (Tonetti et al., eGastroenterology, 2024; 2:3100098). As Tonetti et al. noted, the apical surface of GCs is characterized by microvilli, which significantly increase the surface area available for mucin secretion into the intestinal lumen. Their cytoplasm is characterized by numerous secretory granules containing mucin precursors, highlighting their role in mucin production and secretion. They continuously secrete and renew the mucus layer, physically repelling pathogens from the intestinal lining. More than 20 mucins have been identified (labeled MUC1 to MUC21), each with slightly different structures and functions. In the intestine, the dominant mucin is MUC2. MUC2 deficiency in mice led to increased inflammation and susceptibility to infection, highlighting its importance in gut health. Mucins also serve as binding sites for bacteria, further hindering their invasion. Some bacterial species in the gut utilize components of the mucus layer as an energy source, influencing mucus production and the overall composition of the gut microbiome.

[0119] Therefore, the measurement of goblet cell count in this experiment can be considered to correspond to the positive functional effect of hemp meal on the production of intestinal mucus beneficial to fish.

[0120] Gene expression

[0121] The expression of 13 target genes (listed in the Methods section above) in the foregut and hindgut of fish fed to the control group, HEMP group and SPC group was analyzed, and no welfare issues were found overall.

[0122] The table below shows the gene expression levels of 13 target genes analyzed in the foregut and hindgut. It appears that gene expression was generally upregulated in the foregut of fish fed hemp seed meal, while upregulation was observed in the hindgut of animals fed SPC.

[0123]

[0124] Figure 13. The table shows the expression levels of different genes assessed in the foregut and hindgut of Atlantic salmon fed with three experimental diets for two weeks. Expression levels were assigned numbers 1 to 7, where 1 represents the least expression and 7 represents the most expression.

[0125] Studies have shown that hemp seed meal diets have an impact on inflammatory processes and tolerance at the intestinal mucosal interface, as several genes indicate positive shifts in fish fed this diet compared to those fed a soybean meal-based diet. In particular, feeding hemp seed meal diets… anxa1 The gene is upregulated in the hindgut. This gene protects cell membranes from the inflammatory cascade triggered by specific cytokines. Hsp70 The upregulation of this gene was also significant. Intracellularly localized Hsp70s are crucial components of cellular protein folding mechanisms, performing molecular chaperone functions and helping to protect cells from the adverse effects of oxidative stress. This gene appeared to be upregulated in the foregut of salmon fed a hemp seed meal diet and downregulated in the hindgut. This suggests that the hemp seed meal diet reduces the need for hindgut secretion of this protein, as less inflammation occurred in this region compared to fish fed a soy protein-based diet. Other significant genes with similar positive effects observed include... muc2 Genes (particularly prominent in the gut, where they are secreted into the lumen of the large intestine (hindgut) by goblet cells in the epithelial cell layer), Mastitis protein and close Synthesis Protein 15 .

[0126] Overall, no welfare issues were identified through histological, gene expression, or SCFA results.

[0127] Therefore, these results further demonstrate that hemp seed meal containing a high percentage of protein has no effect on the growth and health of Atlantic salmon.

[0128] Example 4 – Testing Aquaculture Feed Compositions in Atlantic Salmon

[0129] In addition to the results in Example 3, this example also provides a longer-term test of the aquatic feed composition described herein in Atlantic salmon in a growth trial.

[0130] method

[0131] This experiment was conducted using juvenile Atlantic salmon. After acclimatization, the fish were fed one of two experimental diets for 2.5 months. A total of 12 cylindrical cone-bottom culture tanks were used in the experimental experiment, as the treatment was carried out in six replicates using two different diets.

[0132] A total of 240 fish with an average weight of 619.3 ± 1.6 g and a length of 37.0 ± 0.7 cm were weighed, measured, and evaluated for their operating welfare index (OWI), and then distributed to experimental rearing ponds (n = 40 fish / pond; 12 ponds in total). Throughout the experiment, the fish were fed daily by an automatic feeder at an amount exceeding their expected feeding needs.

[0133] At the end of the experiment, all fish in each rearing tank were subjected to painless lethality approximately 6–12 h after feeding, and their weight, length, and OWI were measured. Next, fecal samples were collected by squeezing, following the method reported by Glencross et al. (2005). Suitable Norwegian Quality Catch (NQC) fillets were collected from two fish per rearing tank (n = 12 per diet) and placed on ice for color assessment using a digital SalmoFan and colorimeter.

[0134] Two isonitrogenous and isolipid diets were formulated to meet the known requirements of Atlantic salmon and prepared with the same inclusion ratios of 15% soybean protein concentrate (control) or 15% hemp seed meal (HEMP), as shown in the table below. The composition of the diets and the composition of the hemp seed meal used in the HEMP diets are shown in the following tables.

[0135]

[0136] Table 14. Feed formulations for the two feeds used in the examples

[0137]

[0138] Table 15. Analyzed chemical composition of the three feeds used in the examples

[0139]

[0140] Table 16. Composition of hemp seed meal used in hemp feed.

[0141]

[0142] Tables 17A and 17B – Further details on the amino acid content (17A) and elemental content (17B) of soybean protein concentrate and hemp seed meal.

[0143] All compositional evaluations were performed as described in Example 3.

[0144] Calculate biometric parameters using the following formula:

[0145] Feed conversion ratio (FCR) = Consumed feed (dry weight) / Weight gain (wet weight).

[0146] Fulton fullness (k) = 100 (W / L3), where W is the weight of the fish (g) and L is the total length (cm).

[0147] Liver body index (HSI) = (LW / W) 100, where LW is liver weight and W is body weight.

[0148] Specific growth rate (SGR) = 100 (lnWo – lnWt) D-1, where Wo and Wt are the starting and ending weights of the fish at a specific period (average weight of the culture pond, n = 3), and D represents the number of feeding days.

[0149] Weight gain (WG, g) = Wt –Wo, where Wo and Wt are the initial and final weights of the fish at a specific time (average of the culture pond, n = 3).

[0150] The visual assessment of flesh color is performed using a colorimeter that measures the light reflectance from the flesh and compares it to a standard calibration plate. Following the recommendations of the International Commission on Illumination (CIE, 1978), the measured color parameter is luminance (L). ), red-green hue (a ) and yellow-blue saturation (b Color measurements were taken on the left side of each fish fillet. The instrument was placed on the meat, and three measurements were taken on the cut surface of the fillet. The value represents the redness of the meat, b The value represents the yellowness of the meat. Use a value and b Calculate the colorimetric value (Cab) Chromaticity is a measure of the intensity and sharpness of a color, expressed by the following formula (Hunt, 1977); Cab. =(a 2 +b 2)½ ...Eq. 1 The hue angle is used to define the relationship between the redness and yellowness of fish fillets and is calculated using the following formula; if a If the angle is greater than 0°, then Hab° = tan-1(b / a …Eq. 2 If a 2) Digital SalmoFan™ is also used to measure the color of salmon flesh. To do this, the digital sensor device is placed on the NQC and four measurements are taken in different fillet areas to obtain the SalmoFan™ value.

[0151] result

[0152] At the end of the experimental trials, no differences were observed in any feed performance parameters (see table below). However, differences were observed in the FCR, with fish fed hemp seed meal showing a reduced FCR (p = 0.026).

[0153]

[0154] Table 18. Fish performance and feed utilization of Atlantic salmon fed a commercial control or a diet containing hemp seed meal for 13 weeks. K, Fulton condition rating; SGR, specific growth rate; FCR, feed conversion ratio; HSI, hepatostomy index; FI, feed intake. Different superscript letters indicate significant differences between experimental diets (p<0.05).

[0155] The effects of the two experimental diets on overall fish welfare were assessed using scores on operational welfare indices, including fin integrity, scale loss, and the presence of cataracts or operculum abnormalities. Overall, the fish exhibited very low OWI scores, indicating good welfare (data not shown). Nevertheless, fish fed the control diet appeared to be more prone to cataracts, although this was not significant (p = 0.168).

[0156] Regarding the color of the Norwegian Quality Crust (NQC), no differences were observed in any parameters evaluated using either the digital SalmoFan or a colorimeter. The digital SalmoFan provides a visual evaluation of color on a scale of 20-34; 20 represents a low level of color. In our case, the SalmoFan score was approximately 24, which is an acceptable value for fish of this size.

[0157]

[0158] Table 19. Instrumental color assessment of Norwegian Quality Crust Atlantic salmon (NQC) fed for 13 weeks with a commercial control or a diet containing hemp seed meal. Measurements were performed using a colorimeter and a digital SalmoFan on top of the NQC. Values ​​are presented as mean ± standard deviation (n=3). brightness, Red / green hue; Blue / yellow hue; Color quality (Ca) ); Hue (H°ab).

[0159] in conclusion

[0160] The results confirmed that hemp seed meal is a highly digestible protein source, and that replacing soybean protein concentrate with hemp seed meal did not affect fish performance. Fish fed diets containing hemp seed meal showed a lower free flow rate (FCR), which we believe reflects the superior digestibility of this component compared to the soybean protein concentrate found in Example 3. Visual color was not affected by the presence of hemp seed meal. No welfare issues were identified by OWI.

[0161] Atlantic salmon, as a relatively sensitive carnivore, serve as a good model for other salmonid species such as trout (rainbow trout, brown trout, and sea trout), which share very similar digestibility, gut-related physiology, and metabolic outcomes.

[0162] Example 5 – Nile tilapia ( Oreochromis niloticus Nutritional trials

[0163] A nutritional trial was conducted to study the effects of replacing 30% of soybean protein concentrate with hemp seed meal on Nile tilapia ( Oreochromis niloticus The effect on growth performance.

[0164] method

[0165] The experiment was conducted in a research facility using eight 60L growth units, each initially stocked with 50 juvenile Nile tilapia (a total of 400 tilapia). A recirculating aquaculture system (RAS) was used, equipped with a drum filter, protein skimmer, UV sterilizer, and biofilter. Water parameters (temperature, oxygen, pH, ammonia, nitrite, nitrate, salinity, and alkalinity) were recorded daily during the experiment. The average water temperature throughout the experiment was 28.6°C, and the oxygen content was >5 ppm.

[0166] The fish used had undergone prior health testing. Their average weight at the start of the experiment was 2.55 g, and their final average total weight reached approximately 10.73 g. Bioassays were performed on days 0, 10, and 21 by collecting samples from all fish in the rearing tanks. After bioassays, the fish were returned to their respective aquaculture tanks.

[0167] The experiment lasted for 3 weeks, with fish fed four times a day until satiated; the feed was measured daily. Two different extruded feeds were used: ONCt (control feed containing soybean protein concentrate - calculated feed protein / oil content: 48.8 wt% / 12.2 wt%) and ONHp (test feed containing hemp seed meal - calculated feed protein / oil content: 47.5 wt% / 12.3 wt%) (the composition of the feed, soybean protein concentrate, and hemp seed meal is shown in the table below). Each feed group contained 200 fish.

[0168] The soybean protein concentrate used contained over 60% protein and underwent extensive processing to remove anti-nutritional factors (ANFs) and non-starch polysaccharides (stachyose, raffinose) that interfere with the digestion of monogastric animals such as fish. As a high-quality product, it is more commonly used in more carnivorous species (salmon, sea bass, snapper, turbot, eel, etc.), while commercial tilapia production typically uses less processed standard-grade soybean meal (47-50% protein). However, in this experiment, soybean protein concentrate was used to provide a high-standard feed, which was then used as a control to measure the performance of the experimental diet.

[0169]

[0170] Table 20. Specifications of hemp seed meal used in the experiment

[0171]

[0172] Tables 21A and 21B. Details of the amino acid content (21A) and elemental content (21B) of the hemp seed meal used in the experiment.

[0173]

[0174] Table 22. Composition of the soybean protein concentrate used in the experiment.

[0175]

[0176] Table 23. Experimental feeds used in the experiment. Due to the difference in oil content between soybean protein concentrate (0.7 wt%) and hemp seed meal (13.3 wt%), soybean oil was increased and wheat flour was reduced in the control diet to create space for this.

[0177] Statistical analysis

[0178] The following formula was used to determine various parameters based on the data collected during the experiment:

[0179] Specific growth rate, SGR (% / day) = (lnWt) end – lnWt0 / number of days) x 100

[0180] Among them Wt end = The weight at the end of the test (in grams), Wt0 is the weight at the beginning of the test (in grams).

[0181] Feed conversion ratio (FCR) = Total feed intake (g) / Total body weight gain (g)

[0182] Survival rate (%) = (last N° fish / first N° fish) x 100.

[0183] Body condition, K = 100 W / L 3

[0184] The results of the performance data were analyzed using t-tests and Mann-Whitney U tests, with a significance level of p<0.05.

[0185] result

[0186] The final average weight, SGR, FCR, survival rate, and condition factor of the fish are shown in the table below. (Phase 1 – up to day 10; Phase 2 – from day 10 to day 21.)

[0187]

[0188] Table 24. Weight results throughout the experiment. Data are presented as mean ± one standard deviation.

[0189]

[0190] Table 25. Specific growth rate (SGR) results throughout the experiment. Results are expressed as mean ± one standard deviation.

[0191]

[0192] Table 26. Feed conversion ratio (FCR) results throughout the experiment. Results are expressed as mean ± one standard deviation.

[0193] 1 Mann-Whitney U test

[0194]

[0195] Table 27. Food intake (FI) results throughout the experiment. Results are expressed as mean ± one standard deviation.

[0196]

[0197] Table 28. Survival results throughout the experiment. Results are expressed as mean ± one standard deviation.

[0198] 1 Mann-Whitney U test

[0199]

[0200] Table 29. Results of body condition (K) throughout the experiment. Results are expressed as mean ± one standard deviation.

[0201] Discussion and Conclusion

[0202] During the experiment, the average weight of the fish reached almost four times their initial weight (five times for fish fed ONCt). Survival rates did not show significant differences between treatments.

[0203] Significant differences were obtained when considering average body weight, SGR, FCR, feed intake, and K. Body weight, SGR, FCR, and feed intake showed significant differences after 10 days (except for FCR), in the second phase, and at the end of the experiment. Condition analysis also showed significant differences at the end of the experiment. However, as mentioned above, the control diet was a high-quality diet, and the SGR of 5.83 achieved with the test diet was very acceptable for this species. Furthermore, palatability issues with the test diet are thought to have contributed to the reduced feed intake. These issues can be easily addressed while maintaining the level of hemp seed meal in the diet, for example, by improving the sensory and olfactory quality of the feed through supplements such as krill meal, mussel meal, and feed stimulants. Additionally, it should be noted that the importance of issues such as dietary palatability and texture decreases significantly as fish reach greater weight and their feeding preferences decrease.

[0204] Therefore, the trial is considered to provide strong evidence that hemp seed meal can support growth comparable to soybean meal, and that feed containing protein from hemp seed meal can be successfully used as commercial feed for tilapia and related species such as basa.

[0205] Example 6 - Nutritional Experiment of White-legged Shrimp (Litopenaeus vannamei)

[0206] A nutritional study investigating the effect of replacing 30% of soybean protein concentrate with hemp seed meal on the growth performance of white-legged shrimp (Litopenaeus vannamei).

[0207] method

[0208] The experiment was conducted in a research facility using 12 500L growth units, each initially stocked with 50 juvenile Litopenaeus vannamei (a total of 600 shrimp). A recirculating aquaculture system (RAS) was used, equipped with a drum filter, protein skimmer, UV sterilizer, and biofilter. Water parameters (temperature, oxygen, pH, ammonia, nitrite, nitrate, salinity, and alkalinity) were recorded daily during the experiment. The average water temperature throughout the experiment was 29°C ± 1°C, and the oxygen content was >5 ppm.

[0209] The shrimp used were obtained from the White Panther hatchery and had previously been tested for health by Genics Ltd. At the start of the trial, their average weight was approximately 16.83 g. Bioassays were performed on days 0, 15, and 30, with all animals collected from each rearing tank. After the bioassays, the shrimp were returned to their respective rearing tanks.

[0210] The experiment lasted for 4 weeks. In the first phase (up to day 14), shrimp were fed four times daily at a rate of 2.5% of body weight, and in the second phase (days 15 to 30), they were fed at a rate of 1.8% of body weight per day. Two different dry diets were used: LVCt (control diet containing soybean protein concentrate - calculated feed protein / oil content: 42.6 wt% / 8.4 wt%) and LVHp (test diet containing hemp seed meal - calculated feed protein / oil content: 41.3 wt% / 8.5 wt%) (see table below). Each diet group contained 300 shrimp. As in Example 5, the soybean protein concentrate represents a specially processed soybean meal that creates a high standard of feed, which was used as a control to measure the performance of the experimental diets.

[0211] The details of the content of hemp seed meal and soybean protein concentrate used in the feed are the same as those given in Example 5 above.

[0212]

[0213] Table 30. Experimental feeds used in the experiment Because of the difference in oil content between soybean protein concentrate (0.7 wt%) and hemp seed meal (13.3 wt%), soybean oil was increased and wheat flour was reduced in the control diet to make room for this.

[0214] Statistical analysis

[0215] The following formula was used to determine various parameters based on the data collected during the experiment:

[0216] Specific growth rate, SGR (% / day) = (lnWt) end – lnWt0 / number of days) x 100

[0217] Among them Wt end = The weight at the end of the test (in grams), Wt0 is the weight at the beginning of the test (in grams).

[0218] Feed conversion ratio (FCR) = Total feed intake (g) / Total body weight gain (g)

[0219] Survival rate (%) = (last N° cells / first N° cells) x 100.

[0220] The results of the performance data were analyzed using t-tests and Mann-Whitney U tests, with a significance level of p<0.05.

[0221] result

[0222] The results for shrimp body weight, SGR, FCR, and survival rate are shown in the table below. (Phase 1 – up to day 15; Phase 2 – from day 15 to day 30.)

[0223]

[0224] Table 31. Body weight and specific growth rate (SGR) results throughout the trial. Results are expressed as mean ± one standard deviation. 1 Mann-Whitney U test

[0225]

[0226] Table 32. Results of feed conversion ratio (FCR) and survival rate throughout the trial. Results are expressed as mean ± one standard deviation. 1 Mann-Whitney U test

[0227] Discussion and Conclusion

[0228] During the trial, no significant differences were observed in FCR, SGR, and survival rate between the two treatments, although nominally better SGR and FCR values ​​were observed overall. However, significant differences were found when considering shrimp weight, with significant differences between the two test groups from the start of phase 2 and when considering overall weight gain throughout the trial.

[0229] Generally, replacing 30% of soybean protein concentrate with hemp seed meal increased shrimp body weight at the same feed intake, without significantly affecting FCR, SGR, and survival parameters. The results indicate that hemp meal promoted shrimp growth compared to soybean protein concentrate. This supports the conclusion that hemp seed meal can successfully replace the soybean component in the diet and have a beneficial effect on the growth rate of shrimp and other cold- and warm-water crustaceans.

Claims

1. An aquatic feed composition suitable for farmed fish or farmed crustaceans, the composition comprising hemp seed product, wherein the hemp seed product comprises 25% to 100% by weight of protein and is present in the composition in an amount of 5% to 100% by weight.

2. The aquatic feed composition according to claim 1, wherein the hemp seed product is present in the composition in an amount of 10% to 100% by weight.

3. The aquatic feed composition according to claim 1 or claim 2, wherein the hemp seed product contains at least 40% by weight of protein.

4. The aquatic feed composition according to claim 3, wherein the hemp seed product contains at least 50% by weight of protein, preferably at least 60% by weight of protein.

5. The aquatic feed composition according to claim 4, wherein the hemp seed product comprises 60% to 85% by weight of protein.

6. The aquatic feed composition according to claim 1 or claim 2, wherein the hemp seed product comprises 20% to 35% by weight of protein.

7. The aquatic feed composition according to any one of claims 1 to 6, wherein the hemp seed product is present in the composition in an amount of 10% to 80% by weight.

8. The aquatic feed composition according to claim 7, wherein the hemp seed product is present in the composition in an amount of 10% to 50% by weight.

9. The aquatic feed composition according to claim 8, wherein the hemp seed product is present in the composition in an amount of 20% to 40% by weight, preferably 25% to 35% by weight.

10. The aquatic feed composition according to any one of the preceding claims, wherein the hemp seed product is hemp seed meal or hemp seed protein concentrate, preferably wherein the hemp seed product is hemp seed protein concentrate.

11. The aquatic feed composition according to any one of the preceding claims, wherein the hemp seed product is prepared from dehulled hemp seeds or hemp seeds with their outer shells retained, preferably from dehulled hemp seeds.

12. The aquatic feed composition according to any one of the preceding claims, wherein the composition further comprises one or more of fish meal, soybean meal, soybean protein concentrate, rapeseed meal, marine animal meal, crustacean meal, insect meal, single-cell protein, processed animal protein, bacteria, algae, fish protein concentrate, fish oil, hemp seed oil, vegetable oil, wheat, vitamins, and minerals.

13. The aquatic feed composition according to any one of the preceding claims, wherein the farmed fish or farmed crustaceans are selected from salmon, Arctic char, carp, trout, sea bass, sea bream, sturgeon, turbot, halibut, kingfish, barramundi, grouper, catfish including shark catfish, marine finned fish, eel, halibut, marine fish, tilapia, herring, lusta mullet, flounder, cod, tuna, sardines, mackerel, lobster, prawn, shrimp, crab, or freshwater crayfish.

14. The aquatic feed composition according to claim 13, wherein the farmed fish is salmon or trout, preferably salmon, more preferably Atlantic salmon.

15. The aquatic feed composition according to claim 13, wherein the farmed fish is tilapia or basa, preferably tilapia.

16. The aquatic feed composition according to claim 13, wherein the cultured crustacean is a warm-water crustacean.

17. The aquatic feed composition according to claim 14 or 16, wherein the cultured crustacean is shrimp or prawn, preferably Litopenaeus vannamei, Penaeus monodon (tiger shrimp) or Macrobrachium rossenbergii (giant freshwater prawn), more preferably Litopenaeus vannamei.

18. The aquatic feed composition according to any one of the preceding claims, wherein the composition is in the form of powder or pellets, or wherein the composition is in the form of flakes.

19. The aquatic feed composition according to claim 18, wherein the composition is in pellet form.

20. The aquatic feed composition according to any one of claims 1 to 19, wherein the aquatic feed composition is produced by extrusion, preferably wherein the aquatic feed composition is produced by thermal extrusion.

21. A method for producing an aquatic feed composition according to any one of claims 1 to 20, the method comprising mixing the hemp seed product comprising 25% to 100% protein with one or more additional aquatic feed ingredients to form a mixture, wherein the hemp seed product is present in the mixture in an amount of 5% to 95% by weight, preferably 10% to 95% by weight.

22. The method of claim 21, wherein the cannabis seed product comprises a protein in weight percentages specified in any one of claims 2 to 6, and / or the cannabis seed product is present in the mixture in an amount specified in any one of claims 7 to 9.

23. The method according to claim 21 or 22, the method comprising extruding the mixture by an extruder to form the aquatic feed composition, preferably wherein the aquatic feed composition is in pellet form.

24. The method according to any one of claims 21 to 23, wherein the method comprises producing the cannabis seed product from cannabis seeds.

25. The method of claim 24, wherein the hemp seed product is hemp seed meal, and the method includes producing hemp seed meal from the hemp seeds; or wherein the hemp seed product is hemp seed protein concentrate, and the method includes producing hemp seed protein concentrate from the hemp seeds.

26. A method for raising fish or crustaceans, the method comprising feeding the fish or crustaceans with an aquatic feed composition according to any one of claims 1 to 20.

27. The method of claim 26, wherein the method comprises feeding the fish or crustacean with the aquatic feed composition for a period of at least one month, and preferably wherein the period is from one month to three years.

28. The method according to claim 26 or claim 27, wherein the fish or crustacean is selected from salmon, Arctic char, carp, trout, sea bass, sea bream, sturgeon, turbot, halibut, kingfish, barramundi, grouper, catfish including shark catfish, marine finned fish, eel, halibut, marine fish, tilapia, herring, lusta mullet, flounder, cod, tuna, sardines, mackerel, lobster, prawn, shrimp, crab, or freshwater crayfish.

29. The method of claim 28, wherein the farmed fish is salmon or trout, more preferably salmon, and most preferably Atlantic salmon.

30. The method of claim 29, wherein the salmon or trout is a juvenile or post-juvenile salmon or trout, preferably a post-juvenile salmon or trout.

31. The method according to claim 28, wherein: (i) The farmed fish is tilapia or basa, preferably tilapia; or (ii) The farmed crustacean is a warm-water crustacean.

32. The method according to claim 28 or 31, wherein the farmed crustacean is shrimp or prawn, preferably Litopenaeus vannamei, tiger prawn or giant freshwater prawn, more preferably Litopenaeus vannamei.

33. The method according to any one of claims 26 to 32, wherein the fish or crustaceans are cultured in an open aquatic system.

34. The method according to any one of claims 26 to 32, wherein the fish or crustacean is cultured in a recirculating aquaculture system, preferably wherein the fish or crustacean is shrimp.

35. Use of a hemp seed product containing 25% to 100% by weight of protein in the preparation of an aquatic feed composition, wherein the aquatic feed composition is as defined in any one of claims 1 to 20.

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