Aquafeed compositions

Abstract

The present invention provides an aquafeed composition suitable for a farmed fish or a farmed crustacean, the composition comprising a hemp seed product, wherein the hemp seed product comprises 25% to 100% by weight protein and is present in the composition in an amount of 5 to 100% by weight. Methods of producing and using the aquafeed composition are also provided.

Description

[0001] AQUAFEED COMPOSITIONS

[0002] Field of Invention

[0003] The present invention relates to aquafeed compositions suitable for commercially farmed fish or crustaceans and their methods of manufacture and use. The compositions comprise protein from a hemp seed product and / or comprise hemp seed oil.

[0004] Background

[0005] Aquaculture (also known as aqua farming) is a significant and growing industry in the UK and around the world. This industry involves the farming of aquatic organisms such as fish, crustaceans, molluscs and plants in fresh water or sea water. The industry is set to grow further as the worldwide demand for food protein increases while at the same time wild populations of aquatic organisms are decreasing.

[0006] However, this growing industry makes its own demands on the planet, since suitable feeds are required in particular for the culture of fish and crustaceans. For example, over 90% of embodied environmental impacts of salmon production are from the provision and use of feed, including carbon, land and freshwater footprints (Newton RW, Little DC (2018) Mapping the impacts of farmed Scottish salmon from a life cycle perspective. The International Journal of Life Cycle Assessment 23, 1018-1029). Reducing the impact of feed production whilst maintaining good feed conversion is therefore critical to the sustainability of the industry going forward. The main substitutes for marine ingredients have shown to be environmentally damaging with high processing energy requirements and / or high impacts through Land Use Change such as soy.

[0007] Presently there are several sources of protein alternative to terrestrial or fish-based ones, all presenting different strengths and drawbacks. The production of microbial-origin or single cell protein (SCP) has thrived in recent years offering protein yields of up to 80% and ticking the box of sustainability as by-products from other industries that can be used as substrates. However, the processing of the meal requires high energy costs which in turn impacts on the price and the CO2 footprint. This together with the palatability and nucleic acid content could limit the inclusion level in aquafeeds (Pilmer et al., (2022) Using dietary additives to improve palatability of diets containing single-cell protein from methanotrophic bacteria in yellowtail kingfish (Seriola lalandi) diets. Aquaculture Research 53(14): 5006-5017).

[0008] Insect meals have also received attention in recent years due to their advantageous amino acid profile, similar to that of fish meal, and good palatability. Nonetheless, due to legislation constraints, insects cannot be fed on waste but on feed-grade substrates. This means that raw materials that could be directly used in aquafeeds are being fed to insects. Similarly, to SCP, scalability can be an issue and the potential transmission of pathogens to livestock is being investigated (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).

[0009] Another alternative is the use of microalgal biomass produced in bioreactors. This source has an added value with its naturally occurring high levels of health-beneficial long chain polyunsaturated fatty acids. Nonetheless, processing the biomass requires high energy costs and digestibility issues have been found in different 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).

[0010] As such, there is on-going interest in alternative and improved raw materials for use in the production of aquafeed compositions suitable for farmed fish or crustaceans, in order to support the growing aquaculture industry.

[0011] Summary

[0012] Accordingly, the present invention provides an aquafeed composition suitable for a farmed fish or a farmed crustacean, the composition comprising a hemp seed product, wherein the hemp seed product comprises 25% to 100% by weight 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).

[0013] The present invention further provides a method for the production of the aquafeed composition described herein, the method comprising mixing the hemp seed product comprising 25% to 100% by weight protein with one or more additional aquafeed ingredients to form a mix, wherein the hemp seed product is present in the mix in an amount of 5 to 95% by weight.

[0014] In a further aspect the present invention provides a method of farming fish or crustaceans comprising feeding the fish or crustaceans the aquafeed composition as described herein.

[0015] In addition, as described below the present invention also provides the use of a hemp seed product in the manufacture of the aquafeed composition as described herein.

[0016] The aquafeed compositions described herein comprise protein from hemp seed products, e.g. hemp seed meal or hemp seed protein concentrate. The use of this non- conventional protein source in aquafeeds has significant advantages, the hemp seed product being readily digestible and able to support growth. Moreover, the hemp seed product has significant advantages to soy, one of the main protein sources currently used in aquafeeds whose cultivation and use in aquafeeds leads to deforestation and carries a high CO2 footprint (both from farming and international shipping).

[0017] In addition, it is shown herein that the aquafeeds comprising protein from hemp seed are associated with reduced gut inflammation in fish and an overall healthier gut in trials in which aquafeeds comprising hemp seed protein are compared to aquafeeds comprising soya protein concentrate. This surprisingly shows that aquafeeds comprising hemp seed protein are also advantageous for the health of the farmed fish or crustacean.

[0018] In a still further aspect, described herein is an aquafeed composition suitable for a farmed fish or a farmed crustacean comprising hemp seed oil. In particular, the aquafeed composition may comprise from 5 to 50% by weight hemp seed oil. As with the aquafeed compositions described above, the hemp seed oil can be advantageously used in feeds to avoid or reduce the use of fish oil or soy oil.

[0019] Detailed Description

[0020] As noted above, present invention provides an aquafeed composition suitable for a farmed fish or a farmed crustacean, the composition comprising a hemp seed product, wherein the hemp seed product comprises 25% to 100% by weight protein and is present in the composition in an amount of 5 to 100% by weight. In particular, the protein referred to is derived from the hemp seed, i.e. is hemp seed protein.

[0021] The hemp seed product is incorporated in 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, and preferably 20 to 40% by weight, more preferably 25 to 35% by weight. Where the hemp seed product makes up less than 100% of the aquafeed composition, the remainder of the composition is made up of one or more other ingredients suitable for the farmed fish / farmed crustacean for which the composition is intended.

[0022] In particular, the hemp seed product comprised in the aquafeed composition provides at least part of the protein component of the composition. (As discussed further below, additional protein in the composition may come from other ingredients, such as fish, plant, or animal meal.) The hemp seed product itself comprises 25% to 100 % by weight protein or 25% to 99% by weight protein, 25% to 95% by weight protein, 25% to 80% by weight protein, 40% to 80% by weight protein, 50% to 80% by weight protein, 55%% to 80% by weight protein, or 60 to 80% by weight protein. In one example which may be used in aquafeed for farmed salmon, the hemp seed product is hemp seed meal and comprises 55% to 80% by weight protein.

[0023] Assessment of protein content may be performed following the Association of Official Analytical Chemists (AOAC) Official Methods of Analysis (2000) standard procedures. Specifically, protein can be measured using the Kjeldahl method based on analysed nitrogen content (N x 6.25), following sulphuric acid digestion, with an Opsis AB LiquidLINE KjelROC Analyser.

[0024] The above % for protein content can also be on percentages by weight on a dry matter basis.

[0025] The hemp seed product is one which is derived from the hemp seed and comprises hemp seed protein. In particular, the hemp seed product may be hemp seed meal or hemp seed protein concentrate, which are described further below.

[0026] Industrial hemp (Cannabis saliva) is already grown around the world for a number of different uses in the industrial field. Industrial hemp plants have a low concentration of tetrahydrocannabinol (THC), e.g. less that 1% THC. A number of different industrial hemp cultivars (plant genetic varieties) are known and these can be used to provide the hemp seeds from which the hemp seed product is derived. In particular, cultivars are already available that are especially good for producing hemp seeds (as opposed hemp fibre), and these are of particular utility for making the composition of the present invention.

[0027] Hemp seeds comprise an outer hull that is high in fibre, and an inner heart which is high in protein and fats. The hemp seed products for use in the composition described herein can be produced by dehulling the seed to separate off the hulls, or they can be prepared from hemp seeds that retain their hull such that products derived from the hull, e.g. fibre, are retained in the hemp seed product used in the aquafeed composition.

[0028] Hemp seeds with or without their hull can be used to produce the hemp seed meal or hemp seed protein concentrate for the aquafeed composition of the invention. Hemp seed meal (also known as hemp seed cake) can be produced by cold pressing the hemp seed and usually comprises up to 35% by weight protein. Hemp seed protein concentrate is produced from the hemp seed meal (hemp seed cake) by steps that concentrate the protein, such as milling (including sifting / sieving), or solvent / enzyme extraction. These steps can be used to produce a hemp seed product with the desired percentages of protein. In particular, the steps can be used to produce a hemp seed protein concentrate comprising 50% by weight protein and above. The hemp seed protein concentrate may be in the form of flour. Preferably the hemp seed product is hemp seed protein concentrate.

[0029] In some examples, the hemp seed product comprises 20% to 35% by weight protein. In these examples the hemp seed product is derived from hemp seed by cold pressing, without the need for further steps to increase the weight % of protein in the product.

[0030] Hemp seed products produced as described above have an amino acid profile similar to that of soy protein concentrate and are known to include amino acids that are essential for fish / crustacean growth.

[0031] The further components of the aquafeed composition can be selected based on the farmed fish / crustacean to which the aquafeed composition is to be given. Commercial diets for farmed fish / crustaceans are normally formulated on a digestible nutrient and energy basis. In particular, the composition may further comprise one or more of fish meal, soy meal, soy protein concentrate, rapeseed meal, marine meal, crustacean meal, insect meal, single cell proteins, 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 crustacean based on their species are known in the art (e.g. on the website of the Food and Agriculture Organisation of the United Nations - https: / / www.fao.org / fishery / affris / species- profiles / en / ).

[0032] For example, for salmonids such as salmon and trout, dietary requirements generally include 10 essential amino acids, omega-3 fatty acids, energy (supplied primarily from lipids and proteins), 15 essential vitamins, and astaxanthin. Typical diet formulations known in the art can include fish meal, soybean meal, wheat / wheat by-product, vitamins / minerals, and fish or soy oil, where around 40-45% of the feed is protein and around 30 to 40% of the feed is lipid. The hemp seed oil, which can be prepared from hemp seed can be used in the aquafeed formulations described here to provide at least some of the lipid component. Further, one aspect of the invention described herein is an aquafeed composition suitable for a farmed fish or a farmed crustacean comprising the hemp seed oil. In particular, the aquafeed composition may comprise from 5 to 50% by weight hemp seed oil, preferably from 10 to 40% by weight hemp seed oil, more preferably from 25 to 35% by weight hemp seed oil.

[0033] The total protein content of the aquafeed composition may be 40 to 80% by weight, preferably 40 to 70% by weight, and more preferably 50 to 60% by weight. These values can be on a dry matter basis. It may be necessary for fishmeal to be incorporated in the aquafeed composition in order to ensure that the necessary nutrients are present, and accordingly, part of the protein in the aquafeed composition will then come from this fishmeal.

[0034] The aquafeed composition of the invention can be prepared by mixing the hemp seed product with one or more additional ingredients to form a mixture or a mash. The aquafeed may be in the form of a pellet or powder, or may be flaked. Preferably the aquafeed composition is in the form of a pellet.

[0035] The aquafeed composition may be formed by a hot or cold extrusion. In particular, extrusion process technology can be used to provide a product that has good water stability. In general, during extrusion the mixture is forced through a barrel by one or more screws, and then through a die positioned at the end of the barrel. Hot extrusion may be preferred as this enables a higher fat content to be obtained.

[0036] The farmed fish / farmed crustacean are preferably commercially farmed fish / farmed crustaceans. The fish or crustacean for which the aquafeed composition is suitable is not especially limited and can be a fresh water or sea water farmed fish / crustacean. In particular, the farmed fish or farmed crustacean is selected from salmon, Artic charr, carp, trout, sea bass, sea bream, sturgeon, turbot, halibut, kingfish, barramundi, grouper, catfish including shark catfish, marine finfish, eel, flatfish, marine fish, tilapia, herring, rohu, milkfish, cod, tuna (especially yellowfin tuna), sardines, mackerel, lobster (freshwater, brackish or marine), prawn, shrimp, crab (freshwater, brackish or marine), or crayfish (e.g. USA or Australian crayfish). Alternatively, the farmed fish or farmed crustacean is selected from salmon, trout, sturgeon, shrimp, tilapia and carp.

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

[0038] In another embodiment, the farmed fish can be tilapia or pangasius, preferably tilapia, In a further embodiment, the farmed crustacean is a warm-water crustacean, preferably a shrimp or a prawn, more preferably preferably Litopenaeus vannamei. Penaeus monodon (tiger shrimp) or Macrobrachium rossenbergii (giant freshwater prawn), and more preferably Litopenaeus vannamei.

[0039] The aquafeed composition can be fed to the farmed fish / farmed crustaceans over a time period to ensure efficient and healthy growth. In particular, the time period may be at least one week, at least one month, at least six months, at least one year, at least two years or at least 3 years. The time period may start when the fish / crustacean is a juvenile or a young adult and continue until harvesting. The time period may be from one month to 3 years, from one month to three months, from three to six months, from 1 to 2 years. The time period will depend on the species of fish / crustacean, fanning conditions and desired result. For example, to achieve a 4 kg salmon or a 2 kg trout the feeding period may be around 2 years, to achieve a 400 g trout the feeding period may be around 8 months, and for shrimp the feeding period may be around 3 - 6 months.

[0040] In the method of farming described herein the fish / crustacean may be farmed in an open water aquaculture system or in a recirculating aquaculture system. Preferably an open water aquaculture system is used. In particular, where the fish are salmon or trout the fish may be farmed in an open water aquaculture system. Alternatively, where the crustacean is a shrimp, a recirculating aquaculture system can be used.

[0041] The invention will be described further with reference to the following examples.

[0042] EXAMPLES

[0043] Example 1 - Analysis of hemp seed protein concentrate

[0044] The hemp seed meal / hemp seed protein concentrate can be nutritionally characterized (moisture, oil, protein, ash, minerals and amino acid and fatty acid profiles) and antinutritional factors (phytic acid and glycosylates) analysed. The hemp seed product with the desired protein content can be selected. In this example, a hemp seed protein concentrate, which has been produced from a dehulled hemp seed by the steps of cold pressing and milling, is analyzed.

[0045] Table 1. Results of hemp seed protein concentrate analysis

[0046] Example 2 - Testing of aquafeed compositions

[0047] The suitability of the aquafeed compositions described herein can be demonstrated in in vivo testing.

[0048] For example, in a test to determine suitability for feeding Atlantic salmon, a reference composition can comprise 70% fish meal, 10% wheat, 15% fish oil, and 5% vitamin mix (which may include yttrium oxide as an inert marker). A test aquafeed composition may have the same composition as the reference composition but with 30% of the dry mass replaced with the hemp seed product described herein, such as the hemp seed product of Example 1. Alternatively, the reference composition can comprise 40% fish meal, 30% soy meal, 10% wheat, 15% fish oil, and 5% vitamin mix (which may include yttrium oxide as an inert marker), and in the test composition all of the soy meal can be replaced by the hemp seed product described herein, or in the test composition part of the soy meal and part of the fish meal can be replaced by the hemp seed product described herein.

[0049] Palatability testing can be performed to make sure that both the reference feed and the test feed are taken by the test organism.

[0050] An initial trial can be performed in order to test digestibility (such as that described by Betancor et al., (2016) Nutritional Evaluation of an EPA-DHA oil from transgenic Camelina sativa in feeds for post-smolt Atlantic salmon (Salmo salar L.) PLOS One, e0159934). For example, when testing Atlantic salmon a reference composition and the test aquafeed composition can be given for two weeks to different marine water tanks of post-smolt Atlantic salmon of approximately 500g in weight. At the end of the trial all fish in each tank can be euthanized at ~6h after feeding and measured for weight, length and operational welfare indicators. Faecal samples can be collected by stripping based on the methods reported by Austreng (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) for analysis of digestibility. Fish from each tank can be dissected and the anterior and posterior intestine as well as liver collected, fixed in 4 % buffered formalin for histopathological evaluation of the tissues using ImageJ (FIJI) software (Schindelin et al. (2012) Fiji: an open-source platform for biological-image analysis. Nature Methods, 9, 676- 682).

[0051] Full nutritional trials (e.g. of 2.5 months duration) can also be performed using the test and reference compositions to assess the impact of the test composition on fish growth, fillet quality, welfare and health. For example, for Atlantic salmon the trial can be run in post-smolt (marine water) Atlantic salmon, of an approx, weight of 300 g. At the end of the experimental period, fish can be humanely euthanized, measured for length and weight and operational welfare indicators (OWI) recorded. In addition, several samples can be collected: flesh samples can be used for fillet quality (moisture, protein, ash, fat content, fatty acid and amino acid profile, minerals and pigment deposition), samples of liver as well as anterior and posterior intestine can be fixed in 4% buffered formalin for histological evaluation, samples of faeces can be collected for short chain fatty acid and samples of liver and anterior intestine for qPCR. Example 3 - Testing of aquafeed compositions in Atlantic salmon

[0052] Further to the protocols described in Example 2, this example provides testing of aquafeed compositions described herein in Atlantic salmon, and further demonstrates the suitability of hemp seed as a protein source for Atlantic salmon by showing its digestibility and effect on the welfare and performance of the fish.

[0053] Method

[0054] A trial was conducted with post-smolt Atlantic salmon (409±21.4 g). The fish were approximately 1 year old. Nine tanks of fish were used with 40 fish per tank. The three feeds described below were used for 2 weeks after an initial acclimation period, with each feed being fed to 3 tanks of fish, i.e., 120 fish per feed. The feeds were given in excess of appetite by autofeeders twice a day with uneaten feed collected.

[0055] The three feeds contained yttrium as an inert marker. One feed (the FISH feed) containing a high level of fishmeal was used as a control. (In particular, it is known that such feeds are highly digestible.) The second and third feeds were based on the first FISH feed. In the second feed, termed “SPC”, 30% (by weight) of the FISH feed was removed and replaced by soy protein concentration. (It is noted that soy protein is currently the main commercial alternative to fish meal in aquafeeds.) In the third feed, termed “HM”, 30% (by weight) of the FISH feed was removed and replaced by hemp seed meal with a high level of protein. The amounts of the ingredients (g / kg) in each feed are shown below in Table 2, and the composition of the feeds are shown in Table 3. All diets were extruded into 4 mm pellets.

[0056] Table 2. Feed formulation of the three feeds used in the example

[0057] Table 3. Analysed composition of the three feeds used in the example. The % of protein, lipid, dry matter, and ash, in the control, the hempseed meal, and the SPC meal is shown below in Table 4.

[0058] Table 4. Composition of the fish meal, soya protein concentrate and hempseed meal

[0059] The further details of the content of the soya protein concentrate (SPC) and the hempseed meal (HM) are shown in Table 5 below.

[0060] Table 5A and B. Further details of amino acid content (5 A) and element content (5B) of the soya protein concentrate and hempseed meal.

[0061] All composition assessments were done following the Association of Official Analytical Chemists (AOAC) Official Methods of Analysis (2000) standard procedures. Specifically, protein is measured using the Kjeldahl method based on analysed nitrogen content (N x 6.25), following sulphuric acid digestion, with an Opsis AB LiquidLINE KjelROC Analyser. Crude lipid contents determined gravimetrically after Soxhlet lipid extraction (Tecator Soxtec system 2050 Auto Extraction apparatus). Moisture contents were obtained after drying in an oven at 110°C for 24 h and ash contents determined after incineration at 600°C for 16 h.

[0062] As shown by Table 4, the hempseed meal has a protein content of over 60%.

[0063] At the end of the trial all fish were euthanized at ~ 6h after feeding and measured for weight, length and Operational Welfare Indicators (OWI). Faeces were stripped following the methods of Austreng E. 1978. (Aquaculture 13 :265-272). In particular, the faeces were collected from separate fish and were pooled into two samples (A and B) per tank (i.e., each sample comprised the faeces of 20 fish).

[0064] Quantification of protein, lipid and amino acids content was performed to calculate apparent digestibility coefficient (ADC) of the feeds and apparent ingredient digestibility coefficient (AIDC) of hempseed and SPC meal ingredients following Aas et al. 2015 (Nofima Report 32 / 2015: 1-22) and Bureau et al. 1999 (Aquaculture 180(3 -4):345-358), respectively.

[0065] ADC (%) = 100 x ((a- b) / a) where a represents the nutrient to marker ratio in feed and b represents the nutrient to marker ratio in faeces.

[0066] AIDC (%) = ADCREF + ((1 - S) X DR / SDI) X (ADCT - ADCREF) where ADCT is apparent digestibility coefficient of test diet (i.e., either the SPC feed or the HM feed); ADCREF is apparent digestibility coefficient of reference diet (i.e. the FISH feed); DR is % nutrient (or kj / g gross energy) in reference diet mash; DI is % nutrient (or kj / g gross energy) in the test ingredient; s is proportion of test ingredient in test diet mash; 1 - s is proportion of reference diet mash in test diet mash.

[0067] A description of the nutritional biochemical analysis required for these calculations can be found in McMillan et al. 2024 (Aquaculture, Vol. 585, Art. No. 740686) except for amino acid analysis which is described in Glencross et al., 2021 (Aquaculture, Vol. 544, Art. No. 737133).

[0068] The condition factor (k) and specific growth rate (SGR) were calculated based on measurements of fish weight (W) and length (L). In particular, the condition factor (k) was calculated based on the formula k = 100(W / L3).

[0069] The specific growth rate (SGR) calculation followed that of Ricker, 1975 (Bulletin of the Fisheries Research Board of Canada 191, 1-382).

[0070] SGR (% BW / day) = 100 x (es - 1) where g = (In (BWy) - In (BWi)) / (t2 - ti) or g = (In (BWy) - In (BWi)) x (t2- ti)’1 In addition, samples from 6 fish per tank were collected (n=18 per dietary treatment) to evaluate faeces levels of short chain fatty acids. From these same six fish, blood was collected in heparinized syringes, spun at 3000 g for 15 minutes and plasma collected for analysis of circulating levels of short chain fatty acids. In addition, two sections of both anterior and posterior intestine, as well as liver from the same fish were collected in either RNALater stored at 4 °C overnight and kept at -20°C until RNA extraction for the assessment of inflammation through molecular biomarkers. Samples from the same fish / tissues were fixed in 4% buffered formalin for histopathological evaluation of the tissues.

[0071] Short chain fatty analysis in faeces and plasma

[0072] Faecal samples were freeze dried for a minimum of 12 hours (Christ Alpha 1-4 LSC, SciQuip Ltd., Shropshire, United Kingdom). Sigma-Aldrich 13C-short chain fatty acid stool mixture (Merck KGaA, Darmstadt, Germany), acetonitrile (ACN), 3 -Nitrophenylhydrazine hydrochloride solution and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution were added to the faecal and plasma samples. Samples were vortexed, incubated at 40 °C for 50 minutes on ice and centrifuged (2000 *g for 5 minutes). The supernatant was extracted, dried under nitrogen and redissolved in methyl tert-butyl ether (MTBE) solution. Dissolved samples were vortexed and centrifuged (2000 *g for 5 minutes) with the aqueous MTBE layer extracted. The MTBE layer was dried under nitrogen once again and redissolved in equals volumes of ACN and water. The samples were again centrifuged (2000 *g for 5 minutes) with the supernatant extracted and placed in liquid chromatography vials. All samples were analysed using a liquid chromatography-mass spectrometer (Waters Corporation, Massachusetts, United States).

[0073] Quantitative RT-polymerase chain reaction analysis

[0074] Expression of genes of interest was determined by qPCR of all the RNA samples with elongation factor- 1 a (elfl a), hprt and rpl2 used as reference genes (see table below). The cDNA was diluted 20-fold with milliQ water. The efficiency of the primers for each gene was previously evaluated by serial dilutions of cDNA pooled from the samples to guarantee it was > 85 % for all primer pairs. qPCR was performed using a Biometra TOptical Thermocycler (Analytik Jena, Goettingen, Germany) in 96-well plates in duplicate 20 pL reaction volumes containing 10 pL of Luminaris Color Hi Green qPCR Master Mix (Thermo Scientific, Hemel Hempstead, UK), 1 pL of the primer corresponding to the analyzed gene (10 pmol), 3 pL of molecular biology grade water and 5 pL of cDNA (1 / 20 diluted). In addition, amplifications were carried out with a systematic negative control (NTC, no template control) containing no cDNA. Standard amplification parameters contained an UDG pre-treatment at 50 °C for 2 min, an initial denaturation step at 95 °C for 10 min, followed by 35 cycles: 15 s at 95 °C, 30 s at the annealing Tm and 30 s at 72 °C.

[0075] Table 6. Primers used in the qpCR analysis in the present study, including the PCR efficiency and function.

[0076] Histological evaluation

[0077] Transverse sections of liver and midgut from 6 fish per tank were fixed in 4 % buffered neutral-formaldehyde and embedded in paraffin. Four pm sections of the samples were cut on a microtome (Leica RM 2035, Leica Biosystems, Wetzlar, Germany) and subsequently stained with haematoxylin and eosin (H&E) (Betancor et al., 2015). In addition, the slides containing intestinal samples were stained with Alcian Blue / Periodic acid-Schiff (AB-PAS) to differentiate between neutral / mixed and acidic muco-sub stances in goblet cell (Betancor et al., 2016b). Stained slides were scanned (Axio Scan Zl, ZEISS, Oberkochen, Germany) and visualized using QuPath 0.2.3 (Bankhead et al., 2017). Digital images were analysed using ImageJ (FIJI) software (Schindelin et al., 2012).

[0078] Liver micrographs at 20 x original magnification were examined for the presence of lipid intracytoplasmic vacuoles in hepatocytes from each dietary group. The micrographs were converted into black and white 2 bit images, where white corresponds to the area occupied by lipid droplets and the percentage of the area covered by them was measured. In the intestine, 30 measurements were taken per fish of each evaluated parameter including enterocyte height (hE), enterocyte width (nNE) measured as the number of enterocyte nucleus along a distance of 100 pm, circular muscle thickness (CMT), maximum (Lmax) and minimum (Lmin) lengths of the intestine passing through the center of the lumen in a transverse section, and goblet cells (GB, calculated as number of goblet cells per 100000 pm2 of intestinal mucosa). Length measurements are shown in pm.

[0079] Results and Conclusions

[0080] The digestibility results are shown in Table 7 and the fish performance results are shown in Table 8.

[0081] Table 7. Digestibility of diets and experimental ingredients

[0082] The results show that the digestibility of the HM feed containing the hempseed meal was comparable to the control FISH feed diet and the SPC feed which contained the soy protein concentrate. In particular, protein digestibility of the hempseed meal ingredient was higher than that of the SPC meal.

[0083] No significant differences (p > 0.05) in growth or condition were found between the different feed groups at the end of trial. The operational welfare indicators assessed (data not shown) also did not reveal any welfare issues in any of the fish fed any of the feeds.

[0084] Intestinal and faecal short chain fatty acids (SCEA)

[0085] Short chain fatty acids (SCFA) are fatty acids with fewer than 6 carbon atoms that are primarily produced by intestinal microbiota and in general terms are of major importance in gut health. Therefore, a change in their concentrations indicates a change in the intestinal bacterial population.

[0086] The table below shows the SCFA identified in faeces and plasma samples obtained.

[0087] Table 9. Short chain fatty acids levels identified in faeces and plasma of Atlantic salmon fed the three different experimental feeds for a period of two weeks. Values show average ± SD of 18 fish per diet (n = 3 tanks per diet). Differences (p<0.05) are denoted by different superscript letters.

[0088] In general terms, SCFA appeared to be in higher concentrations in the faeces of control fed fish, while the lowest levels appeared in fish fed hempseed. On the contrary, in plasma, fish fed the hempseed treatment displayed the highest circulating levels of SCFA. Therefore, no correlation between the levels of SCFA in plasma and faeces was found, similar to what has been observed for humans (Chen et al., 2022; Mayo-Martinez et al., 2024).

[0089] Nonetheless, when looking at differences in levels of SCFA among treatments, the only difference was observed in plasma valerate levels, where fish fed the hempseed diet displayed significant higher levels. Supplementation of valerate acid in mice proved to have an antiinflammatory role in mouse, downregulating the expression levels of IL-17 and upregulating IL-10 (Luu et al., 2019), potentially indicating a protective effect of hempseed meal towards inflammation by potentiating the production of this SCFA. Histology results

[0090] Quantitative scoring of intracytoplasmic liver vacuolization did not show any differences between the three dietary treatments (see table below), generally observing a homogeneous regular distribution of lipid within the tissue (data not shown). These results indicate that all fish displayed an adequate nutritional status, with acceptable fat storage and no signs of starvation.

[0091] Table 10. Histological parameters evaluated in the liver of Atlantic salmon fed the experimental diets for two weeks. Values show average ± SD of 18 fish per diet (n = 3 tanks per diet)

[0092] Significant differences were observed after the histological evaluation of anterior and posterior intestine (see tables below). Specifically, changes related to goblet cells, such as the total goblet cells count (anterior) as well as the acidic / mixed goblet cells count (%) as well as neutral goblet cells count (%) in the posterior intestine.

[0093] Table 11. Histological parameters evaluated in the anterior intestine of Atlantic salmon fed the experimental diets for two weeks. GB, goblet cells; hE, enterocytes height (pm); nNE, enterocyte width; CMT, circular muscular thickness; Lmax and Lmin, maximum and minimum intestine lengths on a transverse section (pm); CSP, cross sectional perimeter (pm). Values show average ± SD of 18 fish per diet (n = 3 tanks per diet). Differences (p<0.05) are denoted by different superscript letters. the experimental diets for two weeks. GB, goblet cells; hE, enterocytes height (pm); nNE, enterocyte width; CMT, circular muscular thickness; Lmax and Lmin, maximum and minimum intestine lengths on a transverse section (pm); CSP, cross sectional perimeter (pm). Values show average ± SD of 18 fish per diet (n = 3 tanks per diet). Differences (p<0.05) are denoted by different superscript letters.

[0094] It is noted that the neutral cell goblet cell (GB) count for the fish fed the hempseed meal diet is closer to the fishmeal control diet compared to soy. Without wishing to be bound by theory, it is considered that if the soy irritates the gut lining we may expect to see a reduced neutral mucin production.

[0095] In particular, globlet cells (GCs) are specialised intestinal epithelial cells (lECs) that play a crucial role in gut defence. They are distributed throughout the epithelial lining of both the small and large intestines, with a notable abundance in the colon in humans, where a robust mucus barrier is particularly necessary (Tonetti et al., eGastroenterology, 2024; 2:3100098). As noted by Tonetti et al., the apical surface of GCs is characterised by microvilli which significantly increase the surface area available for mucin secretion into the intestinal lumen. Their cytoplasm is distinguished by numerous secretory granules containing mucin precursors highlighting their role in mucin production and secretion. They continuously secrete and renew the mucus layer, physically pushing away pathogens from the gut lining. There are over 20 identified mucins (labelled MUC1 to MUC21), each with slightly different structures and functions. In the intestine, the predominant mucin is MUC2. Deficiency in MUC2 leads to inflammation and increased susceptibility to infection in mice, highlighting its importance in gut health. Mucins also have binding sites for bacteria, further hindering their invasion. Some bacterial species in the gut use components of the mucus layer as an energy source influencing both mucus production and the overall gut microbiome composition.

[0096] Accordingly, the measurement of globlet cell numbers in the present trial can be considered to correspond with a positive functional effect of hemp meal on the production of gut mucous that is beneficial to the fish.

[0097] Gene expression

[0098] The expression of 13 target genes (listed in the methods section above) was analysed both in the anterior and posterior intestine of the control, HEMP and SPC fed fish which overall did not raise any welfare concerns.

[0099] In the table below, showing the gene expression levels of the 13 target genes analysed both in anterior and posterior intestine, it seems that the gene expression is generally up- regulated in the anterior intestine of fish fed the hempseed meal feed while in the posterior intestine up-regulation is observed in SPC-fed animals. Figure 13. Table showing the expression levels of the different genes evaluated in anterior and posterior intestine of Atlantic salmon fed the three experimental diets for a period of two weeks. Expression levels are allocated numbers 1 to 7, where 1 is the least expression and 7 is the most expression.

[0100] It is shown that the hempseed meal diet has an effect on inflammatory process and tolerance of the gut mucosal interface as several genes indicate a positive shift in fish fed this diet as compared to those fed the diet based on soybean meal. In particular, anxal gene is upregulated with the hempseed meal diet in the posterior gut. This gene protects the membranes of cell from inflammation cascaded of specific cytokines. Hsp70 gene upregulation is also notable. Intracellularly localized Hsp70s are an important part of the cell's machinery for protein folding, performing chaperoning functions, and helping to protect cells from the adverse effects of oxidative stress. This would appear to be upregulated in the anterior intestine of salmon fed with the hemp seed meal diet and down regulated in the posterior gut. The implication is that the hemp seed meal diet has supressed the need for this protein to be secreted by posterior gut as less inflammation is occurring in this area compared to in the fish that have been fed the soya protein-based diet. Other notable genes for which a similar positive effect is seen is muc2 gene (particularly prominent in the gut where it is secreted from goblet cells in the epithelial lining into the lumen of the large intestine (posteriors intestine) in fish, occludin. and claudin 15.

[0101] Overall no welfare concerns were identified by the histological, gene expression or SCFA results.

[0102] Accordingly, these results further show that hempseed meal having a high % of protein did not have any impact on the growth and welfare of Atlantic salmon.

[0103] Example 4 - Testing of aquafeed compositions in Atlantic salmon

[0104] Further to the results in Example 3, this example provides further longer-term testing of aquafeed compositions described herein in Atlantic salmon in growth trial.

[0105] Method

[0106] The trial was conducted with post-smolt Atlantic salmon. Following acclimatisation fish were fed either one of two experimental feeds for a period of 2.5 months. A total of twelve cylindroconical tanks were employed in the experimental trial as treatments were run in six duplicates using two feeds.

[0107] A total of 240 fish with an average body weight of 619.3±1.6 g and 37.0±0.7 cm, were individually weighed, measured, evaluated for operational welfare indicators (OWI) and allocated into the experimental tanks (n = 40 fish / tank; 12 tanks in total). The fish were fed daily in excess of expected appetite by autofeeders during the whole period trial.

[0108] At the end of the trial all fish in each tank were euthanized at ~6-12h after feeding and measured for weight, length and OWI. Next, faecal samples were collected by stripping based on the methods reported by Glencross et al. (2005). The right Norwegian quality cut (NQC) from two fish per tank (n= 12 diet) was collected and placed on ice and colour assessed with a digital Salmofan and a colorimeter.

[0109] Two isonitrogenous and isolipidic feeds were formulated to satisfy the known requirements of Atlantic salmon, and produced to contain either a 15% of soy protein concentrate (control) of hempseed meal (HEMP) at the same inclusion rate, as shown in the Table directly below. The composition of the feeds and the composition of the hempseed meal used in the HEMP feed are shown subsequent tables.

[0110] Table 14. Feed formulation of the two feeds used in the example

[0111] Table 15. Analysed chemical composition of the three feeds used in the example

[0112] Table 16. Composition of the hempseed meal used in the HEMP feed.

[0113] Table 17A and B. - Further details of amino acid content (17A) and element content (17B) of the soya protein concentrate and hempseed meal.

[0114] All composition assessments were done as described in Example 3.

[0115] Biometric parameters were calculated using the following equations:

[0116] Feed conversion ratio (FCR) = feed (dry weight) consumed / weight gain (wet weight).

[0117] Fulton’s condition factor (k) = 100 * (W / L3), where W is the fish weight (g) and L is the total length (cm).

[0118] Hepato-somatic index (HSI) = (LW / W) * 100, where LW is the liver weight and W is the somatic weight.

[0119] Specific growth rate (SGR) = 100 * (InWo - InWt) * D-l, where Wo and Wt are the initial and end weights (tanks means, n = 3) of the fish in a specific period, respectively, and D represents the number of feeding days.

[0120] Weight gain (WG, g) = Wt -Wo, where Wo and Wt are the initial and end weights (tanks means, n = 3) of the fish in a specific period.

[0121] Visual assessment of the flesh colour was conducted by taking colorimetric measurements using a colorimeter that measures light reflection from the flesh in comparison to a standard calibration plate. The colour parameters measured were lightness (L*), red-green chromaticity (a*) and yellow-blue chromaticity (b*) as recommended by the International Commission on Illumination (CIE, 1978). The colour measurements were conducted on the left side of each fish fillet. The instrument was placed on the flesh and triplicate measurements were taken at the filleted section. The a* value represents the redness and the b* value represents the yellowness of the flesh. The chroma (Cab*) and hue (H°ab) angle were calculated using the a* and b* values. The chroma is the expression of intensity and clarity of the colour and is expressed by the following equation (Hunt, 1977); Cab*=(a*2 +b*2 )1 . . .Eq.

[0122] 1 The hue angle is used to define the relationship between the redness and the yellowness of the fillet and is calculated by the following equation; if a* >0°, then Hab° = tan-l(b* / a*) . . .Eq.

[0123] 2 if a*. 2) Digital SalmoFan™ was also used to measure salmon flesh colour. In order to do so, the digital sensor device was placed on the NQC and four measures taken at different fillet areas, which in turn provided a SalmoFan™ number.

[0124] Results

[0125] No differences were observed in any of the feed performance parameters at the end of the experimental trial (see Table below). Nonetheless, differences were observed in the FCR, with fish fed the hempseed meal treatment showing a decreased FCR (p = 0.026).

[0126] Table 18. Fish performance and feed utilization of Atlantic salmon fed a commercial control or a feed containing hempseed meal for 13 weeks. K, Fulton’s condition factor; SGR, specific growth rate; FCR, feed conversion ratio; HSI, hepatosomatic index; FI, feed intake. Different superscript letters denote significant differences between the experimental diets (p < 0.05).

[0127] The impact that the two experimental feeds had on fish general welfare was assessed through the scoring of operational welfare indicators, including the integrity of the fins, scale loss, presence of cataracts or anomalies in the operculum. Overall, the fish displayed very low OWI scoring, indicating a good welfare status (data not shown). Nonetheless, fish fed the control feed seemed to have a higher tendency to present cataracts, albeit not significant (p = 0.168).

[0128] Regarding the coloration of the Norwegian quality cut (NQC), no differences were observed in any of the parameters evaluated either with the digital SalmoFan or with the colorimeter. The digital SalmoFan visually assesses colour on a scale 20-34; with 20 representing a low level of colour. In our case the SalmoFan scored was approx. 24 which is an acceptable value for fish this size.

[0129] Table 19. Instrumental colour evaluation of Atlantic salmon Norwegian quality cut (NQC) fed either a commercial control or a hempseed meal containing diet for 13 weeks. The measurements were done employing a colourimeter and a digital SalmoFan at the top of the NQC. Values are means ± standard errors (n=3). L* lightness, a* red / green dimension; b* blue / yellow dimension; c* chroma (Cab*); h* hue (H°ab).

[0130] Conclusions

[0131] The results confirm that hemp seed meal is a highly digestible protein source and that the substitution of soya protein concentrate by hempseed meal did not impact fish performance. The fish fed the hempseed meal containing diet displayed a lower FCR, which we consider can reflect the superior digestibility of this ingredient compared to the soya protein concentrate as found in Example 3. Visual coloration was not impacted by the inclusion of the hempseed meal. No welfare concerns were identified by OWI.

[0132] Atlantic salmon, as relatively sensitive carnivores, are a good model for other salmonid species, such as trout (rainbow trout, brown trout, and sea trout), which have very similar digestibility, gut related physiology, and metabolic outcomes.

[0133] Example 5 - Nutrition trial in Nile tilapia (Oreochromis niloticus)

[0134] A nutrition trial was performed investigating the impact of replacing 30% soyabean protein concentrate with hempseed meal on growth performance of Nile tilapia (Oreochromis niloticus).

[0135] Method

[0136] The trial was performed at a research facility using 8 growing units of 60L capacity, with an initial stocking of 50 juvenile O. nilotucus in each unit (a total of 400 tilapias). It was carried out using a recirculating aquaculture system (RAS) with drum filter, protein skimmer, UV sterilization and biofilter. Water parameters (temperature, oxygen, pH, ammonia, nitrite, nitrate, salinity, and alkalinity) were recorded daily for the duration of the trial. The average water temperature during the entire trial was 28.6°C and oxygen content >5ppm.

[0137] The fish used were previously tested for health status. They had an average body weight at the beginning of the trial of 2.55g, reaching a final overall average body weight of circa 10.73g. Biometry was performed on day 0, day 10 and day 21, by collecting all the fish in the tank. After the biometry, the fish were returned to their respective tanks.

[0138] The trial had a duration of 3 weeks and the fish were fed four times a day until satiation; the feed used was determined daily. Two different extruded feeds, ONCt (control feed containing soya protein concentrate - calculated protein / oil contents of feed: 48.8% / 12.2% by weight) and ONHp (test feed containing hempseed meal - calculated protein / oil contents of feed: 47.5% / 12.3% by weight), were used (see tables below for composition of feeds, soya protein concentrate and hempseed meal). There were 200 fish in each feed group.

[0139] The soya protein concentrate used comprises more than 60% protein and has been subject to significant processing and extraction of anti -nutritional factors (ANFs), and nonstarch polysaccharides (stachyose, rhaffinose) that interfere with digestion in monogastric animals like fish. As a premier product it is more often used for more carnivorous species (salmonids, bass, bream, turbot, eels etc.), while commercial production of tilapia normally uses less processed standard grade soybean meal (47-50% protein). Nevertheless, soya protein concentrate was used in this trial to provide a high standard feed against which to measure the performance of the test diet.

[0140] Table 20. Specification of hempseed meal used in the trial

[0141] Tables 21A and B. Detail of amino acid content (21A) and element content (21B) of the hempseed meal used in the trial.

[0142] Table 22. Composition of soya protein concentrate used in the trial.

[0143] Table 23. Experimental feed used in trial. * due to the difference in oil content between the soya protein concentrate (0.7% by weight) and the hempseed meal (13.3% by weight), soyabean oil was increase in the control diet and wheat flour reduced to create the space for this.

[0144] Statistical analysis

[0145] The data collected during the trial was used to determinate various parameters, using the following formulas: Specific growth rate, SGR (% / day) = (InWtend - InWto / days) x 100

[0146] Where Wtend = weight at end of trial in g and Wto is weight at the beginning of the trial in g.

[0147] Feed Conversion Ratio, FCR = Total feed intake (g) / total body weight gain (g)

[0148] Survival rate (%) = (Final N° fish / Starting N° fish) x 100.

[0149] Condition Factor, K = 100 * W / Z3

[0150] Results of the performance data were analysed using a t-test and Mann Whitney U test, with a significance level of p < 0.05.

[0151] Results

[0152] The results of the final average weight of the fish, SGR, FCR, survival rate and condition factor are presented in the tables below. (Period 1 - up to day 10; period 2 - from day 10 to day 21.) means ± one standard deviation.

[0153] Table 25. Results of specific growth rate (SGR) throughout the experiment. Results are presented as means ± one standard deviation. presented as means ± one standard deviation. 'Mann Whitney U test means ± one standard deviation.

[0154] Table 28. Results of survival throughout the experiment. Results are presented as means as means ± one standard deviation.

[0155] 'Mann Whitney U test presented as means ± one standard deviation.

[0156] Discussion and Conclusions

[0157] Over the period of the experiment, the fish reached an average weight almost four times their body weight (five times in the case of fish fed ONCt). Survival rate showed no significant differences among the treatments.

[0158] Significant differences were obtained when considering average body weight, SGR, FCR, feed intake and K. Body weights, SGR FCR and feed intake showed significant differences after ten days (except FCR), in the second period and at the end of the trial. The condition factor analysis also showed significant differences at the end of the trial. Nevertheless, as noted above, the control feed is a high-quality feed, and the SGR of 5.83 that was achieved with the test diet is very acceptable for this species. Further, it is considered that palatability issues with the test diet may have caused the reduced feed intake. Such issues can be readily resolved while retaining the level of hemp seed meal in the diet, e.g., the organoleptic and olfactory qualities of the feed can be raised by supplements, such as krill meal, mussel meal and feed stimulants. Moreover, it is noted that such issues as palatability and texture of diets appreciably diminish in importance when fish reach larger weights and become less discriminatory.

[0159] Accordingly, it is considered that the trial provides robust evidence that hemp seed meal may support comparable growth to soya bean meal and shows that feeds comprising protein from hemp seed meal can successfully be used as a commercial feed in tilapia and related species such as pangasius.

[0160] Example 6 - Nutrition trial in white leg shrimp (Litopenaeus vannamei

[0161] Nutrition trial investigating the impact of replacing 30% soyabean protein concentrate with the hemp seed meal on growth performance of white leg shrimp (Litopenaeus vannamei).

[0162] Method

[0163] The trial was performed at a research facility using 12 growing units of 500L capacity, with an initial stocking of 50 juvenile L.vannamei in each unit (a total of 600 L.vannamei). It was carried out using a recirculating aquaculture system (RAS) with drum filter, protein skimmer, UV sterilization and biofilter. Water parameters (temperature, oxygen, pH, ammonia, nitrite, nitrate, salinity, and alkalinity) were recorded daily for the duration of the trial. The average water temperature during the entire trial was 29°C±1 and oxygen content >5ppm.

[0164] The shrimp used were obtained from White Panther hatchery and were previously tested for health status through Genies Ltd. They had an average body weight at the beginning of the trial of circa 16.83g. Biometry was performed on day 0, day 15 and day 30, collecting all animals per tank. After the biometry, the shrimp were returned to their respective tanks.

[0165] The trial has a duration of 4 weeks and the shrimp were fed four times a day using a feeding rate of 2.5% body weight during the first period (to day 14) and a feeding rate of 1.8% body weight / day in the second period (day 15 to day 30). Two different dry feeds, LVCt (control feed containing soya protein concentrate - calculated protein / oil contents of feed: 42.6% / 8.4% by weight) and LVHp (test feed containing hempseed meal - calculated protein / oil contents of feed: 41.3% / 8.5% by weight), were used (see table below). There were 300 shrimp in each feed group. As for Example 5, the soya protein concentrate represents a special processed soyabean meal that creates a high standard feed against which to measure the performance of the test diet. Details of the content of the hempseed meal and soya protein concentrate used in the feeds were the same as given above for Example 5.

[0166] Table 30. Experimental feed used in trial * due to the difference in oil content between the soya protein concentrate (0.7% by weight) and the hempseed meal (13.3% by weight), soyabean oil was increase in the control diet and wheat flour reduced to create the space for this.

[0167] Statistical analysis

[0168] The data collected during the trial was used to determinate various parameters, using the following formulas:

[0169] Specific growth rate, SGR (% / day) = (InWtend - InWto / days) x 100

[0170] Where Wtend = weight at end of trial in g and Wto is weight at the beginning of the trial in g.

[0171] Feed Conversion Ratio, FCR = Total feed intake (g) / total body weight gain (g)

[0172] Survival rate (%) = (Final N° fish / Starting N° fish) x 100.

[0173] Results of the performance data were analysed using a t-test and Mann Whitney U test, with a significance level of p < 0.05.

[0174] Results

[0175] The results of the body weight of the shrimp, SGR, FCR, and survival rate are presented in the tables below. (Period 1 - up to day 15; period 2 - from day 15 to day 30.) Table 31. The results of body weight and specific growth rate (SGR) throughout the trial. Results are presented as means + one standard deviation.1Mann Whitney U test

[0176] Table 32. The results of the feed conversion rate (FCR) and survival throughout the trial. Results are presented as means + one standard deviation.1Mann Whitney U test

[0177] Discussion and Conclusions

[0178] The FCR, SGR and survival rate showed no significant differences among the two treatments during the trial, although SGR and FCR values were nominally better when the overall trial results are considered. However, significant differences were obtained when considering the body weight of the shrimps, with significant differences between the two groups tested from Period 2 and considering the overall body weight increase over the trial period.

[0179] Generally, replacing 30% of soybean protein concentrate with hempseed meal increased the shrimp body weight while using the same feed intake, without significantly affecting the FCR, SGR and survival parameters. The results indicate that hemp meal brought about an improvement in shrimp growth when compared to soybean protein concentrate. The results support the conclusion that hemp seed meal can successfully replace the soybean component of the diet and produce advantageous effects on growth rates in shrimp, as well as other cold and warm water crustaceans.

Claims

CLAIMS:

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

2. The aquafeed 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 aquafeed composition according to claim 1 or claim 2, wherein the hemp seed product comprises at least 40% by weight protein.

4. The aquafeed composition according to claim 3, wherein the hemp seed product comprises at least 50% by weight protein, preferably at least 60% by weight protein.

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

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

7. The aquafeed composition according to any 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 aquafeed 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 aquafeed 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 aquafeed composition according to any preceding claim, 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 aquafeed composition according to any preceding claim, wherein the hemp seed product is prepared from dehulled hemp seed or hemp seeds that retain their hull, preferably from dehulled hemp seed.

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

13. The aquafeed composition according to any preceding claim, wherein the farmed fish or farmed crustacean is selected from salmon, Artic chair, carp, trout, sea bass, sea bream, sturgeon, turbot, halibut, kingfish, barramundi, grouper, catfish including shark catfish, marine finfish, eel, flatfish, marine fish, tilapia, herring, rohu, milkfish, cod, tuna, sardines, mackerel, lobster, prawn, shrimp, crab or crayfish.

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

15. The aquafeed composition according to claim 13, wherein the farmed fish is tilapia or pangasius, preferably tilapia.

16. The aquafeed composition according to claim 13, wherein the farmed crustacean is a warm-water crustacean.

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

18. The aquafeed composition according to any preceding claim, wherein the composition is in the form of a powder or a pellet, or wherein the composition is flaked.

19. The aquafeed composition according to claim 18, wherein the composition is in the form of a pellet.

20. The aquafeed composition according to any of claims 1 to 19, wherein the aquafeed composition is produced by extrusion, preferably wherein the aquafeed composition is produced by hot extrusion.

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

22. The method according to claim 21, wherein the hemp seed product comprises the % by weight protein specified by any of claims 2 to 6, and / or the hemp seed product is present in the mix in an amount according to any of claims 7 to 9.

23. The method of claim 21 or 22, comprising extruding the mix through an extruder to form the aquafeed composition, preferably wherein the aquafeed composition is in the form of a pellet.

24. The method of any of claims 21 to 23, wherein the method comprises producing the hemp seed product from hemp seed.

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

26. A method of farming fish or crustaceans comprising feeding the fish or crustaceans the aquafeed composition of any of claims 1 to 20.

27. The method of claim 26, comprising feeding the fish or crustaceans the aquafeed composition for a period of at least one month, and preferably wherein the period is one month to 3 years.

28. The method of claim 26 or claim 27, wherein the fish or crustacean is selected from salmon, Artic chair, carp, trout, sea bass, sea bream, sturgeon, turbot, halibut, kingfish, barramundi, grouper, catfish including shark catfish, marine finfish, eel, flatfish, marine fish, tilapia, herring, rohu, milkfish, cod, tuna, sardines, mackerel, lobster, prawn, shrimp, crab or crayfish.

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

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

31. The method according to claim 28, wherein: (i) the farmed fish is tilapia or pangasius, 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 a shrimp or a prawn, preferably Litopenaeus vannamei, Penaeus monodon (tiger shrimp) or Macrobrachium rossenbergii (giant freshwater prawn), and more preferable Litopenaeus vannamei.

33. The method of any of claims 26 to 32, wherein the fish or crustaceans are farmed in an open water system.

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

35. Use of a hemp seed product comprising 25% to 100% by weight protein in the manufacture of an aquafeed composition, wherein the aquafeed composition is as defined in any of claims 1 to 20.

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