The aquaculture industry remains the leading global consumer of fish meal (FM), even despite its decreasing use in fish and shellfish feed in recent decades [1]. Traditionally, FM has accounted for about 68% of the global supply of protein for aquafeed formulation, mostly due to its favorable amino acid profile, but also the contents of essential fatty acids, nucleotides, phospholipids, minerals, as well as water- and fat-soluble vitamins. All of this contributes to increased palatability and digestibility of feed [2]. However, FM is becoming decreasingly available because it originates from catch fisheries, where the overexploitation of wild stocks along with ongoing climate change (e.g., increasing frequency of the El Niño phenomenon) not only limit the final yield, but also drive the cost of FM. Therefore, it is essential for the future development of aquaculture to search for long-term sustainable, alternative sources of protein, and incorporate them in produced feed [3]. They can come from both plants and animals (e.g., insects [4]), and there are also possibilities to use bacteria or fungi [5]. The relevance of such dietary ingredients, however, depends on their chemical composition and how it matches the nutritional demands of each species. Nevertheless, there are other risks that must be accounted for to enable the production of safe, sustainable and functional aquafeed [5].

At the turn of the millennium, the search concentrated mainly on ingredients derived from terrestrial plants [6], and significant progress in terms of incorporating them in fish feed had already been made before 2006, especially for omnivores [7]. Back then, they were the preferred replacement for FM due to their much lower prices. Unfortunately, dietary plant protein can impair the health and overall performance of fish, mainly due to anti-nutritional factors contained therein. It also upsets the balance of macronutrients and trace elements in which FM is rich [7]. Naturally, in order to achieve sustainable and efficient fish production, it is crucial to formulate diets that result in high growth rates and good health but also have low environmental impacts. However, this is especially difficult when substituting FM with unconventional and novel ingredients [8].

Industrial hemp (Cannabis sativa) is becoming a significant part of agriculture worldwide, with an expected compound annual growth rate of 16.1% through 2034 [9]. Its cultivated area in the European Union alone increased by 75% between 2015 and 19. In terms of environmental impact, hemp is valued for its prominent CO₂ binding capacity, but also for preventing soil erosion, enhancing local biodiversity, and having low pesticide requirements. Hemp fiber is used in textiles, construction, biofuel production, and many new, innovative applications, while its seeds are used for human and animal consumption. In the context of aquaculture, the inclusion of hemp seeds in striped bass (Morone saxatilis) feed yielded promising results [10], and there were also benefits from the use of hemp protein and oil for other commercial species, such as cobia (Rachycentron canadum), common carp (Cyprinus carpio), and Nile tilapia (Oreochromis niloticus) [11,12,13].

Hence, it should be acknowledged that industrial hemp is currently among the plants which are of greatest interest to aquafeed producers. Following that line of reasoning, the purpose of this study was to test the feasibility of using hemp meal (HM) as an alternative source of protein in an extruded feed for the European perch (Perca fluviatilis), which is an omni-carnivorous freshwater species [14] that shows high promise in the context of European aquaculture diversification [15,16,17,18].

Due to its alleged beneficial properties, industrial hemp has recently attracted the attention of feed producers, and the European Food Safety Authority’s (EFSA) Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) opinionated that hemp seed (and cake) can be used in animal nutrition [24]. In fact, many relevant studies were already carried out on livestock, e.g., broiler chickens [25], but there is little available data when it comes to the use of hemp products in fish diets. Hemp seeds are indeed a rich source of essential amino acids (especially arginine), and contain significant amounts of vitamin E, magnesium, phosphorus, potassium, iron, or zinc. By weight, hemp flour has high carbohydrate (50%) and protein (35%), and low fat (10%) contents, while further processed hemp meal (post oil extraction) can contain up to 70% protein [26]. Cannabis sativa seeds also contain cannabinoids, alkaloids, polyphenols and flavonoids, all of which contribute to their high antioxidant, anti-inflammatory, antimicrobial and immunomodulatory properties. Hemp seed-based diets improve animal memory and expression levels of anti-aging genes, while their polyunsaturated and omega-3 fatty acids contribute to lower cholesterol levels, preventing heart disease [27].

Dietary HM inclusion at up to 30% did not significantly affect the survival or growth rates of the European perch in the current study; however, the rearing indices (SGR, FER, PER) peaked for the 20% HM diet. In comparable experiments performed on juvenile fish, similar improvements were found for common carp fed a 10% hemp cake diet [12], striped bass given a 13% HM diet (20% FM replacement) [10], and cobia fed a 37.5% HM diet (40% FM replacement) [11]. In contrast, a 20% HM diet slightly restricted the growth of hybrid striped bass (Morone chrysops × M. saxatilis), but its proximate composition of muscles was unaffected [28], exactly as in the studied P. fluviatilis. Intriguingly, perch fed with the 20–30% FM feeds were characterized by higher VSI and K indices, and lower HSI. In the cobia study [11], the HSI and fat contents (muscle and whole-body) were raised due to the 37.5% HM diet, while protein and ash contents were reduced. In comparison, striped bass which were given a 13% HM diet showed slightly higher HSI and whole-body protein, and lower fat content [10]. All these contradictory results suggest that dietary HM affects basic body parameters of fish in a species-specific manner, rather than following a consistent pattern, implying that the underlying mechanisms are more complicated than initially presumed. However, HM processing, such as defatting, could also be the reason for such differing outcomes.

Biochemical analysis of blood serum is a preliminary diagnostic tool, which allows us to assess the physiological and nutritional state of fish [29]. Serum proteins have a wide range of functions, which is why total protein is a very revealing blood parameter [30], whereas triglyceride and cholesterol levels also reflect metabolic alterations [31]. Meanwhile, activity spikes of enzymes such as ALT, AST, or ALP point towards impaired liver function [32]. In this context, it appears that dietary inclusion of HM neither improved nor worsened the condition of European perch, as it did not have any significant effects on the studied plasma parameters [13,33,34].

Imbalanced nutrition and/or anti-nutritional factors in the diet change the morphology of intestinal mucosa of fish, impeding nutrient digestion and absorption, which can be assessed using histological methods. Common negative markers include: (a) height decreases in intestinal folds and supranuclear areas of enterocytes, (b) the widening of lamina propria, (c) an increase in the numbers of mucosal cells, and (d) an infiltration of immune cells into the epithelium [35]. In the studied perch juveniles, intestinal folds became longer in both the anterior and posterior sections in the two groups fed diets with 10% and 20% HM inclusion. In consequence, their mucosal surface likely became augmented, which usually improves feed utilization by enhancing the breakdown and pickup of nutrients [36,37]. Similar changes were observed in the posterior intestine of sharpsnout sea bream (Diplodus puntazzo) fed diets containing up to 48% pea protein concentrate [38]. This is truly important, because shortening of mucosal folds in the posterior intestine would have indicated an inflammatory state, as that part of the tract has additionally prominent immune functions [36]. In contrast, intestinal fold height was unaffected by high inclusion levels of soybean protein in the diets of rainbow trout (Oncorhynchus mykiss) [39], meagre (Argyrosomus regius) [40], and gilthead seabream (Sparus aurata) [41]. Meanwhile, even though the absorptive supranuclear area of enterocytes in the anterior intestine was much diminished in the HM20-30 groups, it was improved in the posterior intestine in groups HM10 and HM30. The latter observation opposes the results of the rainbow trout study [39], as well as those of a feeding experiment on Siberian sturgeon (Acipenser baerii) with lupin-containing diets [42]. Furthermore, shorter ASH was also found in our previous study on perch juveniles fed a FM diet containing 46% protein [43], which ultimately was deemed the most adequate of the tested compositions, with the current dietary formulations completed in accordance with those conclusions. Hence, it can be stated that the results of histological measurements in the intestine were generally favorable for the HM-containing feeding groups.

Histological measurements of hepatocytes are indirect markers of the liver’s condition, with changes in cellular area being a result of lipid and/or glycogen accumulation levels [44], and with larger nuclei being an implication of improved total gene transcription rates [45]. In this context, the changes which occurred in the studied perch livers due to dietary HM inclusion were not severe; however, the changes in hepatocyte nuclear and cellular areas trended negatively in the HM20 group. Notably, the control group HM0 had the highest level of accumulated glycogen, while the lipid levels were significantly reduced only in the HM30 group. In comparison, hepatocyte glycogen storage increased in yellow perch (Perca flavescens) when a wheat gluten protein-based diet was supplemented with a lysine-glycine dipeptide, yet diminished (and lipid storage too) when both amino acids were added freely to the diet [46].

Increased expression of genes encoding pro-inflammatory cytokines, such as il1b and il6, relates to the activation of innate immunity in response to, e.g., infections, but also commonly occurs in fish fed low-FM diets [47]. However, in our study, there were no differences in the expression of il1b, while the il6 expression was significantly reduced in the anterior (groups HM20-30) and also slightly in the posterior (HM10-30) parts of the intestines of fish fed diets with lower FM contents than the control group HM0. These observations are very consistent with il1b and il6 levels reported in the anterior intestine of gilthead seabream offered a diet based on a mixture of plant meals [47]. On the other hand, diets based on raw or fermented soybeans brought an increase in the expression of pro-inflammatory cytokines in the posterior intestine of turbot (Scophthalmus maximus) [48]. Meanwhile, the expression of slc15a1b, encoding the membrane-bound peptide transporter PEPT1b, which plays an important role in intestinal absorption [49], was raised in the posterior intestine in perch groups fed with diets containing 20-30% HM. Similarly, this gene’s expression increased in both the anterior and posterior intestine of rainbow trout four hours after feeding with a low FM diet [50], although a plant meal-based diet slightly reduced the expression of this gene in the anterior intestine of gilthead seabream [47]. Meanwhile, the expression of two genes important for the metabolism of fatty acids, slc27a4 and pparaa [51,52], decreased in the anterior intestines of fish from groups HM20-30, suggesting that their absorption rate of lipids diminished due to the higher HM inclusion in their diets. In comparison, the expression of both these genes increased in the pyloric caeca of Atlantic salmon (Salmo salar) post-smolts offered a diet with plant meal and supplementary soy saponin [53]. Moreover, there were no differences between perch groups in both intestinal and hepatic expression of the fatty acid desaturase-encoding gene fads, contrasting with the results of studies on the gilthead seabream and Atlantic cod (Gadus morhua), both of which were given diets with 100% vegetable oils [54,55]. Furthermore, a diet composed entirely of plant protein and oil increased the intestinal and hepatic expression of fads in sea bass (Dicentrarchus labrax) [56]. In perch livers, the only significant difference among the studied genes occurred for the expression of pparaa, which was raised in groups HM20-30. In reference, although the hepatic pparaa levels of the Atlantic salmon fed with a plant-based diet were not altered, its slc27a4 expression did increase [53], similar to the trend observed in our study in the HM30 group.

In the liver, antioxidative enzymes such as GPX and SOD, as well as the metabolic-oriented ALP, are negative markers of inflammation and stress [57,58]. For example, in common carp, increasing dietary supplementation levels with a crude leaf extract of C. sativa reversely lowered the hepatic activity of ALP [59], while the dietary addition of lactic acid reduced the activity of GPX [60]. Hence, as the hepatic ALP and GPX activities decreased in perch groups fed with the 20–30% HM-containing diets, and SOD remained relatively constant throughout, it can be concluded that the dietary addition of HM was beneficial in terms of this organ’s homeostasis.

In summary, based jointly on the results of all performed analyses, it was concluded that the dietary addition of hemp meal did not impede the development of juvenile European perch and had no profoundly negative effects on the homeostasis of its digestive organs. Undeniably, further research is still required to assess the effects of such dietary plant protein on this generally carnivorous species, for instance when administered during its different life stages. Different feed components (e.g., lipid sources) should also be tested in combination with the hemp meal. Nevertheless, the trends observed within the data suggest that the 20% inclusion level of hemp meal in the feed (approx. 30% fish meal replacement rate) was the most adequate for the species among the tested dietary formulations. It is worth noting that this approach could also potentially reduce feed costs, as demonstrated for intensively cultured common carp [61]. It also aligns with a broader trend of using agricultural and food industry by-products as feed ingredients (i.e., following the principles of circular economy), and would also reduce the pressure exerted on wild fish stocks [62]. However, the use of such raw materials requires strict quality control (e.g., nutritional profile, assessment of antinutrients and chemical contaminants), as their composition and safety can vary significantly depending on their origin and processing technology [63].

Conceptualization, W.C., P.G., K.P.-Ż., D.Ż., S.K., J.K. and M.K.; Methodology, D.A.-U., M.W. (Maciej Wójcik), M.W. (Małgorzata Woźniak), and J.K.; Formal analysis, W.C., D.A.-U., M.W. (Maciej Wójcik), J.B., H.B., K.K., J.M., A.S., H.S. and J.Ś.; Investigation, W.C., D.A.-U., J.B., H.B., K.K., J.M., A.S., H.S., M.W. (Małgorzata Woźniak), J.Ś. and K.P.-Ż.; Resources, J.K., M.K. and S.K.; Data curation, W.C., D.A.-U., R.K. and D.Ż.; Writing—original draft preparation, W.C., D.A.-U. and M.K.; Writing—review and editing, R.K.; Visualization, D.A.-U. and R.K.; Supervision, M.K.; Project administration, J.K. and M.K.; Funding acquisition, P.G., J.K. and M.K. All authors have read and agreed to the published version of the manuscript.

The animal study protocol was approved by the Local Ethical Committee for Animal Experiments in Olsztyn (Poland) (LKE 34/2022, dated 8 November 2022).

The raw data supporting the conclusions of this article will be made available by the authors on request.

The authors declare no conflicts of interest.

This study was a part of the project titled “Diversification of the productive function of earthen ponds based on semi-intensive rearing of Perca fluviatilis—PROPERCH” (project no. 00002-6521.1-OR1400004/17/20), founded by the European Union through the Operational Program “Fisheries and Sea (2014–2020)”, The Agency for Restructuring and Modernization of Agriculture (ARMA) of Poland.