# Partial Replacement of Fishmeal with Duckweed (Spirodela polyrhiza) in Feed for Two Carnivorous Fish Species, Eurasian Perch (Perca fluviatilis) and Rainbow Trout (Oncorhynchus mykiss)

Authors: T. Stadtlander; F. Tschudi; A. Seitz; M. Sigrist; D. Refardt; F. Leiber
Journal: Aquaculture Research
 (2023)

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## Abstract

Two four-week feeding trials were conducted with fingerlings of Eurasian perch (Perca fluviatilis, 3.52 ± 0.08 g) and rainbow trout (Oncorhynchus mykiss, 1.49 ± 0.05 g) fed with graded levels of dried (DWD) and fermented (DWF) duckweed meal (Spirodela polyrhiza). The purpose of these two trials was to evaluate DWD and DWF as replacements for fishmeal. Fishmeal protein was substituted by 12%, 24%, and 35% of duckweed protein and compared to control diets containing 40% (for perch) and 35% (for rainbow trout) fishmeal and no duckweed. The performance of the fish (growth, feed conversion, and protein and lipid utilization) and their whole-body composition were evaluated and compared with the control. While even the lowest inclusion level, regardless of its form (dried or fermented), resulted in significantly reduced performance in Eurasian perch, rainbow trout were able to utilize feed containing duckweed meal considerably well. Compared to the control, at a 12% inclusion level, rainbow trout showed an equal or comparable percent weight gain (PWG; DWD: 377%, DWF: 373%), specific growth rate (SGR; DWD: 4.37%/day, DWF: 4.33%/day), feed conversion ratio (FCR; DWD: 1.11, DWF: 1.12), and protein productive value (PPV; DWD: 21.5%, DWF: 21.2%). Increasing the inclusion levels above 12% of both DWD and DWF resulted in reduced performance in rainbow trout, with the most pronounced effects observed in the DWD35 group. All experimental diets, including control, affected the whole body composition of perch, most notably reducing the lipid content compared to initial fish. Compared to initial, control and DWD rainbow trout increased whole-body protein, lipid, and ash contents. In conclusion, for rainbow trout, fermented and dried S. polyrhiza duckweed meal appears to be a promising feed ingredient when used at a maximum inclusion level of 12%, while for Eurasian perch, it should not be considered as a feed ingredient.


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## Body

## 1. Introduction

Aquaculture is the leading global consumer of fishmeal, despite a significant decrease in its relative inclusion levels in fish and crustacean diets over the last decades [1, 2]. While the fishmeal inclusion level for most grow-out diets has decreased significantly over time, especially the diets for various carnivorous fish species’ fry and fingerlings still contain high amounts of fishmeal with contents as high as 50% being found in practical diets. Replacing fishmeal in the diets of carnivorous fry and fingerlings with a more sustainable protein source that does not compete with human food [3] could improve aquaculture sustainability. Fishmeal is regarded as the gold standard for aquafeeds but is also strongly disputed as being unsustainable. Although producing fishmeal from the trimmings of fish caught for human consumption is playing an increasingly important role in fish feeds [4], the targeted fishmeal production is considered unsustainable and detrimental to natural marine ecosystems [5]. Furthermore, it has been estimated that around 90% of fish caught for reduction to fishmeal are food or prime food grade, and its use for aquaculture directly competes with human food consumption [6]. Plant-based protein sources, such as soybeans, canola/rapeseed, maize, and wheat, have been developed and now serve as the primary protein sources in aquaculture feeds [7].Soy, the primary fishmeal substitute, has high external costs due to habitat loss, eutrophication, acidification, and pesticide and plant protection product uses in Brazil and Argentina [8]. Additionally, soy can be directly consumed by humans, and it contains several antinutritional factors (ANF) such as protease inhibitors, lectins, phytoestrogens, saponins, and phytic acid [9]. Including soybean meal or soy protein concentrate in salmonid diets significantly reduces their specific growth rates [10]. Carnivorous fishes, such as salmonids, sea bass, and sea bream, are among the world’s largest consumers of fishmeal [11], making the search for finding sustainable substitutes for fishmeal an important issue in aquaculture [12]. In recent years, insect meals from different species, particularly those made from the black soldier fly (BSF, Hermetia illucens), have shown great promise as a fishmeal replacement [13]. However, especially in the European Union and Switzerland, their production is only permitted when feed grade substrates are used (EU 2021/1372). This practice may not be sustainable, as it places BSF feed in direct competition with feed for other livestock [14].Another potential protein source for aquaculture feeds is duckweed or water lentil species. These aquatic floating plants, which belong to the family Lemnaceae, have a high protein content ranging between 30 and 45% of dry matter [15–18]. Duckweed species are also known for their high efficiency in nitrogen (N) and phosphorus (P) uptake, with the ability to remove up to 98% of N and P from their growth medium [18–22].For this project, duckweed speciesSpirodela polyrhiza was utilized as feed for two important carnivorous aquaculture species in Switzerland, Eurasian perch (Perca fluviatilis) and rainbow trout (Oncorhynchus mykiss). These fishes have a trophic level of >4 (according to https://www.fishbase.org/, as of March 14, 2022) and were selected for this study for three main reasons: (i) their economic importance in Switzerland, (ii) their high trophic level and thus their high protein requirement, and (iii) the lack of prior research on the use of duckweed in the diet of perch and rainbow trout fingerlings. While both species are highly carnivorous freshwater fish with similar natural food preferences (e.g., both consume insect larvae, insects, and other invertebrates, and as they grow, they are both increasingly piscivorous), they differ in their culture techniques. Rainbow trout culture has been established globally for over a century and is mostly practiced in open flow-through and partly recirculated systems, while perch culture is relatively young and mostly conducted in indoor recirculating aquaculture systems (RAS). Additionally, rainbow trout are among the most extensively studied fishes, and thus their nutritional requirements are well known, whereas perch are not produced in large numbers, and their nutritional requirements have not been well researched [23]. While trout are well suited for cold to medium temperatures, with an optimal temperature of 12–14°C, perch exhibit the best growth rates in warmer waters, with an optimal temperature of around 22°C.We conducted two separate experiments to investigate the use of dried and fermented duckweed as a fish meal protein replacement for fingerlings of both Eurasian perch and rainbow trout. In two similar trials, varying amounts of duckweed were included to replace 12, 24, and 35% of fishmeal protein in the experimental diets. Two differentS. polyrhiza meals, one derived from dried (DWD) and one derived from fermented (DWF) duckweed, were used and evaluated for their effects on growth and whole body proximate composition, as well as feed, protein, and lipid utilization.


## 2. Materials and Methods

### 2.1. Duckweed Production

Duckweed (Spirodela polyrhiza, collection number 9346) was provided by the Landolt Duckweed Collection (Zurich, Switzerland). The fronds were initially grown in a climate chamber, as described by Stadtlander et al. [21]. They were then transferred to a static system with a circular tank with a volume of 1.7 m3 before being mass produced in two 20 m2 circular pools, each of which was placed in a greenhouse. The duckweed production was initiated with an ammonium-nitrogen (NH4-N) concentration of 20 mg l−1 on a modified Hoagland medium (Table 1). When the concentration of NH4-N fell below 0.1 mg l−1, it was increased again to 20 mg·l−1 by supplying additional fertilizer. Fresh biomass was harvested and air-dried on a shaded bench inside the greenhouse to protect it from direct sunlight before being completely dried in a drying chamber. Alternatively, the fresh biomass was fermented using effective microorganisms EM 1 (prepared to EM A according to the manufacturer’s specification; EM Schweiz AG, Switzerland) and Pediococcus pentosaceus (PP100-25, BIOAGRO S.r.l., Italy) as starter cultures. The fermented duckweed was then dried in the same way as the freshly harvested biomass. All dried biomasses were ground to a fine powder and refrigerated at 4°C until use in the fish feed.Table 1
Modified Hoagland medium.
CompoundConcentration (g·l−1)MgSO4·7 H2O246Ca(NO3)2·4 H2O236NH4H2PO4136KNO3101H3BO32.86MnCl2·4 H2O1.82ZnSO4·7 H2O0.22Na2MoO4·2 H2O0.09CuSO4·5 H2O0.09FeCl3·6 H2O0.484EDTA iron(III) sodium salt1.5


### 2.2. Feed Preparation

For both fish species, seven different diets, one control diet, and six test diets were prepared. Dried duckweed (DWD) meal and fermented duckweed (DWF) meal were used to replace 12, 24, and 35% of fishmeal protein in both species. The corresponding treatment groups were C (control, no duckweed), DWD12, DWD24, and DWD35, as well as DWF12, DWF24, and DWF35.The proximate composition of feed ingredients is presented in Table2. The nutritional content of the duckweed differed slightly for perch and rainbow trout due to the different harvest times of the duckweed batches. The diets were formulated according to Table 3 (perch) and Table 4 (rainbow trout) with some minor differences in formulation between species. The nutritional content was based on Fiogbé et al. [24] and Kestemont et al. [25] for perch and NRC [23] for rainbow trout. The diets were designed to be iso-nitrogenous, with a crude protein (CP) content of around 530 g kg−1 for both fish species, a crude lipid (CL) content of around 140 g·kg−1 for perch and 160 g·kg−1 for rainbow trout. The duckweed-free control diet was based on a high percentage of fishmeal (400 g·kg−1 for perch and 350 g·kg−1 for rainbow trout) and a poultry by-product meal (perch) or wheat gluten (rainbow trout) as protein sources. For preparation of the diet, all dry ingredients were thoroughly mixed using a handheld kitchen mixer. Lipid was then added and mixed again thoroughly until no lipid globules remained. Thereafter, water was added, and the dough was mixed and kneaded until the moisture was homogenously distributed. The dough was pelleted through a 2 mm dye in a meat grinder and dried at 40°C for 24 hours. The dry pellets were ground in a mechanical grain mill and sieved to produce final pellet fractions of 0.8–1.0 mm, 1.0–1.2 mm, and 1.2–1.6 mm. The feed was stored at −20°C when not in use.Table 2
Proximate composition of feed ingredients (on dry matter basis).
DM (g·kg−1 FM)CP (g·kg−1 DM)CL (g·kg−1 DM)CA (g·kg−1 DM)NFE (g·kg−1 DM)CF (g·kg−1 DM)GE (kJ·g−1 DM)Fishmeal92967191.52344.30n.d18.8DWD perch95035042.1189.5418.4n.d17.9DWD trout93737232.020229896.115.6DWF perch90027544.4200480.6n.d17.6DWF trout95933144.814238010316.8Wheat gluten93286864.48.6059.93.2023.2PBM94074510695.753.2n.d22.0Wheat flour88714921.416.981322.520.4Potato starch8100.000.001.60998.40.0019.9Alpha-cellulose9500.000.000.00010020.0FM: fresh matter, DM: dry matter, CP: crude protein, CL: crude lipid, CA: crude ash, NFE: nitrogen free extract, CF: crude fiber, GE: gross energy, DWD: dried duckweed, DWF: fermented duckweed, and PBM: poultry by-product meal.Table 3
Feed formulation for perch fry and feed proximate composition (g·kg−1 DM).
ControlDWD12DWD24DWD35DWF12DWF24DWF35Fish meal400.0352.0304.0260.0352.0304.0260.0PBM319.8319.8319.8319.8319.8319.8319.8DWD0.089.8179.6261.90.00.00.0DWF0.00.00.00.0114.3228.5333.3Potato starch147.0115.080.050.095.045.00.9α-cellulose45.935.227.618.532.016.20.0Fish oil67.468.269.169.966.966.566.1Vitamin premix10.010.010.010.010.010.010.0Mineral premix10.010.010.010.010.010.010.0Proximate compositionDry matter (%)92.092.893.493.691.992.592.9Crude protein (% DM)52.852.752.953.053.153.453.5Crude lipids (% DM)14.113.614.013.913.814.114.3Crude ash (% DM)12.513.013.714.313.113.113.3Crude fiber (% DM)2.933.342.893.313.263.243.12NFE (% DM)17.617.316.515.516.816.215.7PBM: poultry by-product meal; NFE: nitrogen free extract (100–CP–CL–CA–CF).Table 4
Feed formulation for rainbow trout fry and feed proximate composition (g·kg−1 DM).
ControlDWD12DWD24DWD35DWF12DWF24DWF35Fish meal350.0308.0266.0227.5308.0266.0227.5Wheat gluten321.2321.2321.2321.2321.2321.2321.2DWD075.6151.2220.6000DWF000085.2170.4248.5Wheat flour77.077.077.077.077.077.077.0α-cellulose126.191.156.123.982.939.70Sunflower oil105.6107.1108.5109.8105.7105.7105.7Vitamin premix10.010.010.010.010.010.010.0Mineral premix10.010.010.010.010.010.010.0Proximate compositionDry matter (%)95.194.494.293.594.794.394.6Crude protein (% DM)52.752.852.952.852.953.253.1Crude lipids (% DM)16.015.515.716.015.615.716.2Crude ash (% DM)9.5710.110.611.19.619.8610.0Crude fiber (% DM)8.526.675.313.646.654.352.33NFE (% DM)13.215.015.516.415.216.918.4NFE: nitrogen free extract (100–CP–CL–CA–CF).


### 2.3. Feeding Trial

The experiments were approved by the animal welfare committee of the canton of Aargau in Switzerland and the cantonal veterinarian authority under permission number AG 75722.


#### 2.3.1. Eurasian Perch—Perca fluviatilis

A total of 780 perch fingerlings (3.52 ± 0.08 g, mean ± SD) were provided by Valperca (Charvonay, Switzerland), and they were acclimatized for one week in 55-l aquaria connected to a flow-through system. During the one-week acclimatization phase, the perch were fed the same feed that was used on the commercial farm (Biomar Inicio plus M; 1.1 mm, 57% CP, 15% CL, 10.4% CA, 0.4% crude fiber (CF)). Afterwards, 220 fish were subdivided into three similar groups, euthanized (150 mg·l−1 MS-222 buffered with 300 mg·l−1 sodium bicarbonate), and used for the initial determination of proximate composition. The remaining 560 fish were then placed into 28 10-l aquaria in groups of 20 fish each (initial stocking density approximately 7 kg·m−3), resulting in four replicates per diet that were randomly allocated. The aquaria were connected to a small recirculating system with a total volume of around 600 l and equipped with individual air stones. Water exchange was carried out daily in the early afternoon, with 200 l of the total water volume being replaced. Water quality (oxygen content and saturation, ammonium, nitrite, and pH) was determined twice per week in the morning. The average water temperature throughout the experiment was 21.9°C and ranged from 20.4 to 23.0°C, the average oxygen concentration was 6.52 mg l−1, the average oxygen saturation was 77.9%, the average ammonium concentration was 0.27 mg l−1, the average nitrite concentration was 0.32 mg l−1, and the average pH was 8.05. During the whole experiment, a 12 h/12 h light/dark lighting regime was applied. Feed allowance was initially set to 5% [24] but was reduced to 3.5% of body weight per day. This was due to low acceptance of the experimental diets, in order to guarantee full consumption of all the feed that was provided. Feeding was carried out for six days a week with no feeding on Sundays to improve precision for the group weighings on Monday. A restricted feeding regime was chosen in order to guarantee equal feed consumption for all groups. Feeding was carried out by hand three times per day during the week and twice per day on Saturday with equally sized portions. At the start of the experiment and every Monday, the fish were group-weighed and the feed amount was adapted to the new group weights.


#### 2.3.2. Rainbow Trout—Oncorhynchus mykiss

A total of 545 rainbow trout fingerlings (1.49 ± 0.05 g, mean ± SD) were purchased from a commercial trout farmer in Switzerland (Pisciculture de Vionnaz, Switzerland), and they were acclimatized for one week in 55-l aquaria connected to a flow-through system. During the acclimatization phase, the rainbow trout were fed the commercial feed that was used in the hatchery (Le Gouessant, Neo Supra, 1.1 mm, 58% CP, 13% CL, 9% CA, 0.5% CF). Afterwards, 120 fish were subdivided into 3 equal groups, euthanized (150 mg·l−1 MS-222 buffered with 300 mg·l−1 sodium bicarbonate), and used for the initial determination of proximate composition. The remaining 420 fish were then placed into 28 10-l aquaria in groups of 15 fish each (initial stocking density around 2.2 kg·m−3), resulting in four replicates per diet that were randomly allocated. The aquaria were connected to the same system that was described for the perch (above), and the feeding and weighing procedures, as well as the lighting conditions and water exchange were identical. Water quality (oxygen content and saturation, ammonium, nitrite, and pH) was determined twice per week in the morning. The average water temperature was 16.8°C and ranged from 15.5 to 17.3°C, the average oxygen concentration was 8.41 mg·l−1, the average oxygen saturation was 90%, the average ammonium concentration was 0.34 mg·l−1, the average nitrite concentration was 0.44 mg·l−1, and the average pH was 8.24. Feeding was carried out as was described for the perch but with a daily feed allowance of 5% of the body weight.


### 2.4. Sampling and Chemical Analysis

For both species, the general sampling schemes were mostly identical. After four weeks of experimental feeding, both experiments were terminated. All fish were euthanized as described above and group-weighed, and the largest and smallest two fish in each aquarium were individually weighed, and their fork length was measured. For the two largest fish in each aquarium, the intestines were excised and weighed to determine the intestine-somatic index (ISI). The liver was subsequently excised and weighed to determine the hepatosomatic index (HSI). To estimate the amount of intestinal lipid in the two largest fish, a 4-step key was used. An estimation of the intestinal fat was made, it ranges from 0–25, 26–50, 51–75, and 75–100%. The lowest category (0–25%) indicated no or very little visible intestinal fat, while the highest category (75–100%) indicated that only fat was visible after opening the abdominal cavity. All fish, including those used for determination of ISI and HSI, as well as the fish sampled at the onset of both experiments, were stored in groups at −20°C until whole body analysis was completed.For whole-body composition analysis, frozen fish were cut with garden scissors while still frozen, autoclaved for 30 minutes at 90°C, and homogenized (Ultra-Turrax T25, IKA Labortechnik, Staufen, Germany). The fish homogenate was frozen at −20°C and lyophilized (Alpha 1-4 LSC, Christ, Osterode am Harz, Germany). Lyophilized fish samples were ground using a coffee mill, and the dried powder was stored at −20°C until proximate composition analysis.The experimental diets and homogenized fish were analyzed for dry matter (DM), crude protein (CP), crude lipids (CL), and crude ash (CA), and the diets were additionally analyzed for crude fiber (CF). In brief, DM was determined by drying samples for five hours at 105°C, CP was determined by the Dumas method (CP =N × 6.25), CL was determined by the Soxhlet method, CA was determined by burning the samples for 5 hours at 550°C in a muffle furnace, and crude fiber was determined according to method 6.1.1 (Verband Deutscher Landwirtschaftlicher Untersuchungs-und Forschungsanstalten) [26]. To determine the CA content of the samples, digestion was performed using boiling sulphuric acid and caustic potash. The resulting residue was filtered, washed, dried, and weighed. The essential amino acid (EAA) determination for the different experimental diets was performed according to the method described by Bidlingmeyer et al. [27]. Feed samples were collected in the last week of both experiments and pooled per treatment and species. The samples were hydrolyzed using hydrochloric acid, and derivatization was carried out using a mix of ethanol, triethylamine, water, and phenyl isothyocyanate (7 : 1 : 1 : 1) at ambient temperature for 20 min. Amino acid determination was carried out with liquid chromatography and an aqueous buffer (0.14 M sodium acetate containing 0.15 ml·l−1 trimethylamine, pH 6.35) and 60% acetonitrile in water as solvents. The nitrogen-free extract was estimated by difference for feed (NFE = 100–CP–CA–CL–CF) and fish (without CF).


### 2.5. Calculations and Statistics

To evaluate the growth and feed utilization response of the fish, the following formulas were used:Weight gain (WG, g): final body weight − initial body weightPercent weight gain (PWG, %): (final body weight − initial body weight)/initial body weight·100Specific growth rate (SGR, % day−1): [(ln final body weight) − (ln initial body weight)]/days of experiment·100Condition factor (K): [(final body weight)/(fork length3)]·100Intestine somatic index (ISI, %): (intestine weight/whole body weight)·100Hepatosomatic index (HSI, %): (liver weight/whole body weight)·100Feed conversion ratio (FCR): total dry feed intake/weight gainProtein productive value (PPV; %): [(final fish protein content − initial fish protein content)/(total feed protein intake)]·100Lipid productive value (LPV; %): [(final fish lipid content − initial fish lipid content)/(total feed lipid intake)]·100Data from both experiments were statistically analyzed using SPSS version 24 (IBM Corporation, Armonk, USA), following Amelchanka et al. [28]. For that, the procedure “compare means > one-way analysis” was used to run a polynomial contrast analysis for all parameters to evaluate the effects of dried and fermented duckweed supplementation on perch and rainbow trout. The contrast analysis was conducted between the control and either the dried (DWD) or fermented (DWF) duckweed groups. For the whole body composition comparison, two contrast analyses were performed for each duckweed type. One analysis compared the whole body composition of the initial fish to that of the final fish, including the control, and the other contrast analysis compared the control and duckweed fed fish. A significance level of p<0.05 was considered for all data which are presented as the mean value of four replicates ± standard deviation, if not stated otherwise.


## 2.1. Duckweed Production

Duckweed (Spirodela polyrhiza, collection number 9346) was provided by the Landolt Duckweed Collection (Zurich, Switzerland). The fronds were initially grown in a climate chamber, as described by Stadtlander et al. [21]. They were then transferred to a static system with a circular tank with a volume of 1.7 m3 before being mass produced in two 20 m2 circular pools, each of which was placed in a greenhouse. The duckweed production was initiated with an ammonium-nitrogen (NH4-N) concentration of 20 mg l−1 on a modified Hoagland medium (Table 1). When the concentration of NH4-N fell below 0.1 mg l−1, it was increased again to 20 mg·l−1 by supplying additional fertilizer. Fresh biomass was harvested and air-dried on a shaded bench inside the greenhouse to protect it from direct sunlight before being completely dried in a drying chamber. Alternatively, the fresh biomass was fermented using effective microorganisms EM 1 (prepared to EM A according to the manufacturer’s specification; EM Schweiz AG, Switzerland) and Pediococcus pentosaceus (PP100-25, BIOAGRO S.r.l., Italy) as starter cultures. The fermented duckweed was then dried in the same way as the freshly harvested biomass. All dried biomasses were ground to a fine powder and refrigerated at 4°C until use in the fish feed.Table 1
Modified Hoagland medium.
CompoundConcentration (g·l−1)MgSO4·7 H2O246Ca(NO3)2·4 H2O236NH4H2PO4136KNO3101H3BO32.86MnCl2·4 H2O1.82ZnSO4·7 H2O0.22Na2MoO4·2 H2O0.09CuSO4·5 H2O0.09FeCl3·6 H2O0.484EDTA iron(III) sodium salt1.5


## 2.2. Feed Preparation

For both fish species, seven different diets, one control diet, and six test diets were prepared. Dried duckweed (DWD) meal and fermented duckweed (DWF) meal were used to replace 12, 24, and 35% of fishmeal protein in both species. The corresponding treatment groups were C (control, no duckweed), DWD12, DWD24, and DWD35, as well as DWF12, DWF24, and DWF35.The proximate composition of feed ingredients is presented in Table2. The nutritional content of the duckweed differed slightly for perch and rainbow trout due to the different harvest times of the duckweed batches. The diets were formulated according to Table 3 (perch) and Table 4 (rainbow trout) with some minor differences in formulation between species. The nutritional content was based on Fiogbé et al. [24] and Kestemont et al. [25] for perch and NRC [23] for rainbow trout. The diets were designed to be iso-nitrogenous, with a crude protein (CP) content of around 530 g kg−1 for both fish species, a crude lipid (CL) content of around 140 g·kg−1 for perch and 160 g·kg−1 for rainbow trout. The duckweed-free control diet was based on a high percentage of fishmeal (400 g·kg−1 for perch and 350 g·kg−1 for rainbow trout) and a poultry by-product meal (perch) or wheat gluten (rainbow trout) as protein sources. For preparation of the diet, all dry ingredients were thoroughly mixed using a handheld kitchen mixer. Lipid was then added and mixed again thoroughly until no lipid globules remained. Thereafter, water was added, and the dough was mixed and kneaded until the moisture was homogenously distributed. The dough was pelleted through a 2 mm dye in a meat grinder and dried at 40°C for 24 hours. The dry pellets were ground in a mechanical grain mill and sieved to produce final pellet fractions of 0.8–1.0 mm, 1.0–1.2 mm, and 1.2–1.6 mm. The feed was stored at −20°C when not in use.Table 2
Proximate composition of feed ingredients (on dry matter basis).
DM (g·kg−1 FM)CP (g·kg−1 DM)CL (g·kg−1 DM)CA (g·kg−1 DM)NFE (g·kg−1 DM)CF (g·kg−1 DM)GE (kJ·g−1 DM)Fishmeal92967191.52344.30n.d18.8DWD perch95035042.1189.5418.4n.d17.9DWD trout93737232.020229896.115.6DWF perch90027544.4200480.6n.d17.6DWF trout95933144.814238010316.8Wheat gluten93286864.48.6059.93.2023.2PBM94074510695.753.2n.d22.0Wheat flour88714921.416.981322.520.4Potato starch8100.000.001.60998.40.0019.9Alpha-cellulose9500.000.000.00010020.0FM: fresh matter, DM: dry matter, CP: crude protein, CL: crude lipid, CA: crude ash, NFE: nitrogen free extract, CF: crude fiber, GE: gross energy, DWD: dried duckweed, DWF: fermented duckweed, and PBM: poultry by-product meal.Table 3
Feed formulation for perch fry and feed proximate composition (g·kg−1 DM).
ControlDWD12DWD24DWD35DWF12DWF24DWF35Fish meal400.0352.0304.0260.0352.0304.0260.0PBM319.8319.8319.8319.8319.8319.8319.8DWD0.089.8179.6261.90.00.00.0DWF0.00.00.00.0114.3228.5333.3Potato starch147.0115.080.050.095.045.00.9α-cellulose45.935.227.618.532.016.20.0Fish oil67.468.269.169.966.966.566.1Vitamin premix10.010.010.010.010.010.010.0Mineral premix10.010.010.010.010.010.010.0Proximate compositionDry matter (%)92.092.893.493.691.992.592.9Crude protein (% DM)52.852.752.953.053.153.453.5Crude lipids (% DM)14.113.614.013.913.814.114.3Crude ash (% DM)12.513.013.714.313.113.113.3Crude fiber (% DM)2.933.342.893.313.263.243.12NFE (% DM)17.617.316.515.516.816.215.7PBM: poultry by-product meal; NFE: nitrogen free extract (100–CP–CL–CA–CF).Table 4
Feed formulation for rainbow trout fry and feed proximate composition (g·kg−1 DM).
ControlDWD12DWD24DWD35DWF12DWF24DWF35Fish meal350.0308.0266.0227.5308.0266.0227.5Wheat gluten321.2321.2321.2321.2321.2321.2321.2DWD075.6151.2220.6000DWF000085.2170.4248.5Wheat flour77.077.077.077.077.077.077.0α-cellulose126.191.156.123.982.939.70Sunflower oil105.6107.1108.5109.8105.7105.7105.7Vitamin premix10.010.010.010.010.010.010.0Mineral premix10.010.010.010.010.010.010.0Proximate compositionDry matter (%)95.194.494.293.594.794.394.6Crude protein (% DM)52.752.852.952.852.953.253.1Crude lipids (% DM)16.015.515.716.015.615.716.2Crude ash (% DM)9.5710.110.611.19.619.8610.0Crude fiber (% DM)8.526.675.313.646.654.352.33NFE (% DM)13.215.015.516.415.216.918.4NFE: nitrogen free extract (100–CP–CL–CA–CF).


## 2.3. Feeding Trial

The experiments were approved by the animal welfare committee of the canton of Aargau in Switzerland and the cantonal veterinarian authority under permission number AG 75722.


### 2.3.1. Eurasian Perch—Perca fluviatilis

A total of 780 perch fingerlings (3.52 ± 0.08 g, mean ± SD) were provided by Valperca (Charvonay, Switzerland), and they were acclimatized for one week in 55-l aquaria connected to a flow-through system. During the one-week acclimatization phase, the perch were fed the same feed that was used on the commercial farm (Biomar Inicio plus M; 1.1 mm, 57% CP, 15% CL, 10.4% CA, 0.4% crude fiber (CF)). Afterwards, 220 fish were subdivided into three similar groups, euthanized (150 mg·l−1 MS-222 buffered with 300 mg·l−1 sodium bicarbonate), and used for the initial determination of proximate composition. The remaining 560 fish were then placed into 28 10-l aquaria in groups of 20 fish each (initial stocking density approximately 7 kg·m−3), resulting in four replicates per diet that were randomly allocated. The aquaria were connected to a small recirculating system with a total volume of around 600 l and equipped with individual air stones. Water exchange was carried out daily in the early afternoon, with 200 l of the total water volume being replaced. Water quality (oxygen content and saturation, ammonium, nitrite, and pH) was determined twice per week in the morning. The average water temperature throughout the experiment was 21.9°C and ranged from 20.4 to 23.0°C, the average oxygen concentration was 6.52 mg l−1, the average oxygen saturation was 77.9%, the average ammonium concentration was 0.27 mg l−1, the average nitrite concentration was 0.32 mg l−1, and the average pH was 8.05. During the whole experiment, a 12 h/12 h light/dark lighting regime was applied. Feed allowance was initially set to 5% [24] but was reduced to 3.5% of body weight per day. This was due to low acceptance of the experimental diets, in order to guarantee full consumption of all the feed that was provided. Feeding was carried out for six days a week with no feeding on Sundays to improve precision for the group weighings on Monday. A restricted feeding regime was chosen in order to guarantee equal feed consumption for all groups. Feeding was carried out by hand three times per day during the week and twice per day on Saturday with equally sized portions. At the start of the experiment and every Monday, the fish were group-weighed and the feed amount was adapted to the new group weights.


### 2.3.2. Rainbow Trout—Oncorhynchus mykiss

A total of 545 rainbow trout fingerlings (1.49 ± 0.05 g, mean ± SD) were purchased from a commercial trout farmer in Switzerland (Pisciculture de Vionnaz, Switzerland), and they were acclimatized for one week in 55-l aquaria connected to a flow-through system. During the acclimatization phase, the rainbow trout were fed the commercial feed that was used in the hatchery (Le Gouessant, Neo Supra, 1.1 mm, 58% CP, 13% CL, 9% CA, 0.5% CF). Afterwards, 120 fish were subdivided into 3 equal groups, euthanized (150 mg·l−1 MS-222 buffered with 300 mg·l−1 sodium bicarbonate), and used for the initial determination of proximate composition. The remaining 420 fish were then placed into 28 10-l aquaria in groups of 15 fish each (initial stocking density around 2.2 kg·m−3), resulting in four replicates per diet that were randomly allocated. The aquaria were connected to the same system that was described for the perch (above), and the feeding and weighing procedures, as well as the lighting conditions and water exchange were identical. Water quality (oxygen content and saturation, ammonium, nitrite, and pH) was determined twice per week in the morning. The average water temperature was 16.8°C and ranged from 15.5 to 17.3°C, the average oxygen concentration was 8.41 mg·l−1, the average oxygen saturation was 90%, the average ammonium concentration was 0.34 mg·l−1, the average nitrite concentration was 0.44 mg·l−1, and the average pH was 8.24. Feeding was carried out as was described for the perch but with a daily feed allowance of 5% of the body weight.


## 2.3.1. Eurasian Perch—Perca fluviatilis

A total of 780 perch fingerlings (3.52 ± 0.08 g, mean ± SD) were provided by Valperca (Charvonay, Switzerland), and they were acclimatized for one week in 55-l aquaria connected to a flow-through system. During the one-week acclimatization phase, the perch were fed the same feed that was used on the commercial farm (Biomar Inicio plus M; 1.1 mm, 57% CP, 15% CL, 10.4% CA, 0.4% crude fiber (CF)). Afterwards, 220 fish were subdivided into three similar groups, euthanized (150 mg·l−1 MS-222 buffered with 300 mg·l−1 sodium bicarbonate), and used for the initial determination of proximate composition. The remaining 560 fish were then placed into 28 10-l aquaria in groups of 20 fish each (initial stocking density approximately 7 kg·m−3), resulting in four replicates per diet that were randomly allocated. The aquaria were connected to a small recirculating system with a total volume of around 600 l and equipped with individual air stones. Water exchange was carried out daily in the early afternoon, with 200 l of the total water volume being replaced. Water quality (oxygen content and saturation, ammonium, nitrite, and pH) was determined twice per week in the morning. The average water temperature throughout the experiment was 21.9°C and ranged from 20.4 to 23.0°C, the average oxygen concentration was 6.52 mg l−1, the average oxygen saturation was 77.9%, the average ammonium concentration was 0.27 mg l−1, the average nitrite concentration was 0.32 mg l−1, and the average pH was 8.05. During the whole experiment, a 12 h/12 h light/dark lighting regime was applied. Feed allowance was initially set to 5% [24] but was reduced to 3.5% of body weight per day. This was due to low acceptance of the experimental diets, in order to guarantee full consumption of all the feed that was provided. Feeding was carried out for six days a week with no feeding on Sundays to improve precision for the group weighings on Monday. A restricted feeding regime was chosen in order to guarantee equal feed consumption for all groups. Feeding was carried out by hand three times per day during the week and twice per day on Saturday with equally sized portions. At the start of the experiment and every Monday, the fish were group-weighed and the feed amount was adapted to the new group weights.


## 2.3.2. Rainbow Trout—Oncorhynchus mykiss

A total of 545 rainbow trout fingerlings (1.49 ± 0.05 g, mean ± SD) were purchased from a commercial trout farmer in Switzerland (Pisciculture de Vionnaz, Switzerland), and they were acclimatized for one week in 55-l aquaria connected to a flow-through system. During the acclimatization phase, the rainbow trout were fed the commercial feed that was used in the hatchery (Le Gouessant, Neo Supra, 1.1 mm, 58% CP, 13% CL, 9% CA, 0.5% CF). Afterwards, 120 fish were subdivided into 3 equal groups, euthanized (150 mg·l−1 MS-222 buffered with 300 mg·l−1 sodium bicarbonate), and used for the initial determination of proximate composition. The remaining 420 fish were then placed into 28 10-l aquaria in groups of 15 fish each (initial stocking density around 2.2 kg·m−3), resulting in four replicates per diet that were randomly allocated. The aquaria were connected to the same system that was described for the perch (above), and the feeding and weighing procedures, as well as the lighting conditions and water exchange were identical. Water quality (oxygen content and saturation, ammonium, nitrite, and pH) was determined twice per week in the morning. The average water temperature was 16.8°C and ranged from 15.5 to 17.3°C, the average oxygen concentration was 8.41 mg·l−1, the average oxygen saturation was 90%, the average ammonium concentration was 0.34 mg·l−1, the average nitrite concentration was 0.44 mg·l−1, and the average pH was 8.24. Feeding was carried out as was described for the perch but with a daily feed allowance of 5% of the body weight.


## 2.4. Sampling and Chemical Analysis

For both species, the general sampling schemes were mostly identical. After four weeks of experimental feeding, both experiments were terminated. All fish were euthanized as described above and group-weighed, and the largest and smallest two fish in each aquarium were individually weighed, and their fork length was measured. For the two largest fish in each aquarium, the intestines were excised and weighed to determine the intestine-somatic index (ISI). The liver was subsequently excised and weighed to determine the hepatosomatic index (HSI). To estimate the amount of intestinal lipid in the two largest fish, a 4-step key was used. An estimation of the intestinal fat was made, it ranges from 0–25, 26–50, 51–75, and 75–100%. The lowest category (0–25%) indicated no or very little visible intestinal fat, while the highest category (75–100%) indicated that only fat was visible after opening the abdominal cavity. All fish, including those used for determination of ISI and HSI, as well as the fish sampled at the onset of both experiments, were stored in groups at −20°C until whole body analysis was completed.For whole-body composition analysis, frozen fish were cut with garden scissors while still frozen, autoclaved for 30 minutes at 90°C, and homogenized (Ultra-Turrax T25, IKA Labortechnik, Staufen, Germany). The fish homogenate was frozen at −20°C and lyophilized (Alpha 1-4 LSC, Christ, Osterode am Harz, Germany). Lyophilized fish samples were ground using a coffee mill, and the dried powder was stored at −20°C until proximate composition analysis.The experimental diets and homogenized fish were analyzed for dry matter (DM), crude protein (CP), crude lipids (CL), and crude ash (CA), and the diets were additionally analyzed for crude fiber (CF). In brief, DM was determined by drying samples for five hours at 105°C, CP was determined by the Dumas method (CP =N × 6.25), CL was determined by the Soxhlet method, CA was determined by burning the samples for 5 hours at 550°C in a muffle furnace, and crude fiber was determined according to method 6.1.1 (Verband Deutscher Landwirtschaftlicher Untersuchungs-und Forschungsanstalten) [26]. To determine the CA content of the samples, digestion was performed using boiling sulphuric acid and caustic potash. The resulting residue was filtered, washed, dried, and weighed. The essential amino acid (EAA) determination for the different experimental diets was performed according to the method described by Bidlingmeyer et al. [27]. Feed samples were collected in the last week of both experiments and pooled per treatment and species. The samples were hydrolyzed using hydrochloric acid, and derivatization was carried out using a mix of ethanol, triethylamine, water, and phenyl isothyocyanate (7 : 1 : 1 : 1) at ambient temperature for 20 min. Amino acid determination was carried out with liquid chromatography and an aqueous buffer (0.14 M sodium acetate containing 0.15 ml·l−1 trimethylamine, pH 6.35) and 60% acetonitrile in water as solvents. The nitrogen-free extract was estimated by difference for feed (NFE = 100–CP–CA–CL–CF) and fish (without CF).


## 2.5. Calculations and Statistics

To evaluate the growth and feed utilization response of the fish, the following formulas were used:Weight gain (WG, g): final body weight − initial body weightPercent weight gain (PWG, %): (final body weight − initial body weight)/initial body weight·100Specific growth rate (SGR, % day−1): [(ln final body weight) − (ln initial body weight)]/days of experiment·100Condition factor (K): [(final body weight)/(fork length3)]·100Intestine somatic index (ISI, %): (intestine weight/whole body weight)·100Hepatosomatic index (HSI, %): (liver weight/whole body weight)·100Feed conversion ratio (FCR): total dry feed intake/weight gainProtein productive value (PPV; %): [(final fish protein content − initial fish protein content)/(total feed protein intake)]·100Lipid productive value (LPV; %): [(final fish lipid content − initial fish lipid content)/(total feed lipid intake)]·100Data from both experiments were statistically analyzed using SPSS version 24 (IBM Corporation, Armonk, USA), following Amelchanka et al. [28]. For that, the procedure “compare means > one-way analysis” was used to run a polynomial contrast analysis for all parameters to evaluate the effects of dried and fermented duckweed supplementation on perch and rainbow trout. The contrast analysis was conducted between the control and either the dried (DWD) or fermented (DWF) duckweed groups. For the whole body composition comparison, two contrast analyses were performed for each duckweed type. One analysis compared the whole body composition of the initial fish to that of the final fish, including the control, and the other contrast analysis compared the control and duckweed fed fish. A significance level of p<0.05 was considered for all data which are presented as the mean value of four replicates ± standard deviation, if not stated otherwise.


## 3. Results

Analysis of diet proximate compositions revealed comparable nutrient levels between all diets for the same fish species (Tables3 and 4). Essential amino acid composition and total EAA showed relatively little variation in the perch diets (Table 5), while the amounts of all EAAs in the rainbow trout diets tended to be lower with higher duckweed concentration and generally in the diets that contained fermented duckweed (DWF), with the most pronounced reduction in DWF35 (Table 6). While most EAA in the rainbow trout diets remained at or above the required level, lysine was below the required levels in all diets and methionine was below the required levels in the DWF diets.Table 5
Essential amino acids in the different perch diets (% DM).
ControlDWD12DWD24DWD35DWF12DWF24DWF35ReqArginine3.183.113.363.063.353.082.961.8Histidine1.041.021.061.001.111.051.02Leucine3.243.173.443.213.453.373.27Isoleucine1.811.751.881.761.901.851.78Lysine3.893.733.743.473.653.503.192.2Methionine0.910.840.830.750.910.840.840.8Phenylalanine + tyrosine2.792.732.832.732.952.972.92Threonine1.751.701.891.681.821.721.661.2Valine2.322.272.492.312.492.412.34Sum20.9320.3221.5219.9721.6320.7919.98Bold numbers indicate values below the required level according to the National Research Council for European sea-bass (Dicentrarchus labrax) (rightmost column). Requirements for Eurasian perch are not published.Table 6
Essential amino acids in the different rainbow trout diets (% DM).
ControlDWD12DWD24DWD35DWF12DWF24DWF35ReqArginine2.102.242.172.131.881.891.731.5Histidine1.031.060.981.010.970.990.890.8Leucine3.333.413.183.253.083.192.961.5Isoleucine1.821.871.831.811.641.691.521.1Lysine1.992.271.901.761.861.851.462.4Methionine0.800.780.740.700.610.640.570.7Phenylalanine + tyrosine3.313.473.373.232.883.102.831.8Threonine1.431.471.371.411.211.271.131.1Valine2.162.232.172.171.972.031.891.2Sum17.9718.817.7117.4716.116.6514.98Bold numbers indicate values below the required level according to the National Research Council [23] (rightmost column).


### 3.1. Eurasian Perch—Perca fluviatilis

Feed acceptance for perch was low at the start of the experiment, and only after 2-3 days were all experimental diets accepted well by the fish. As this was the case with all diet types, this cannot be attributed to the inclusion of duckweed. Once feeds were accepted, they were consumed within two minutes of feeding with a tendency toward slower consumption in the higher concentrated duckweed diets. Perch reared on diets that contained duckweed had significantly reduced performance, including growth, feed conversion, and nutrient utilization (Table7). The reduction in performance and feed and nutrient utilization was more pronounced in perch fed fermented duckweed compared to perch fed dried duckweed. The results show clearly that the performance reduction increased with increasing the duckweed inclusion level, independent of duckweed type (Table 7 and Figure 1). The performance reduction was already seen in perch fed with DWD12, which was the best performing duckweed diet. The PWG was only 86.5% compared to 111% in the control. The worst performing duckweed diet, DWF35, resulted in just 35.4% PWG. Similarly, SGR, FCR, PPV, and LPV were inferior in DWF35 (0.86% day−1, 4.60, 6.03%, and 9.13%, respectively) compared to control (SGR: 2.13, FCR: 1.53, PPV: 20.0%, and LPV: 11.7%, respectively). Perch fed with the highest inclusion level of fermented duckweed also showed a marked increase in liver weights and a reduction in survival rates. The most obvious change in perch whole body composition between the initial fish and fish of all experimental groups including the control was a significant decrease in CL and an increase in CA (Table 8). When comparing the two duckweed types with the control, no significant trend was observed for moisture, CP, CL, or CA. Perch fed with DWF35 and DWD35 showed higher mortalities (21.2% and 15%, respectively) compared to the other duckweed groups and the control diets.Table 7
Dietary effects on growth, feed conversion, protein and lipid utilization, and body condition parameters in Eurasian perch. Values are averages of four replicates.
Perch fed withp valueDried duckweed (DWD)Fermented duckweed (DWF)ControlDWD12DWD24DWD35DWF12DWF24DWF35SEMDWDDWFIBW (g)3.533.463.553.513.503.513.550.02——FBW (g)7.306.456.055.206.255.694.680.18<0.001<0.001WG (g)3.762.992.501.692.752.181.130.17<0.001<0.001PWG (%)11186.575.050.280.664.435.45.09<0.001<0.001SGR (% day−1)2.131.781.591.161.681.390.860.09<0.001<0.001FCR (g·g−1)1.531.792.102.881.862.424.600.240.0110.011PPV (%)20.016.712.911.515.311.46.030.88<0.0010.010LPV (%)11.71.915.16a−8.17−1.45−0.63−9.131.360.033<0.001K1.571.511.541.461.521.411.450.020.8430.019ISI (%)7.677.558.127.708.106.928.130.120.5750.691HSI (%)1.421.401.331.401.321.271.500.030.5500.036Visceral fat (%)46.925.025.021.925.033.325.02.670.0260.460Survival (%)93.893.885.092.595.092.578.81.860.4220.023IBW: initial body weight; FBW: final body weight; WG: weight gain; PWG: percentage weight gain; SGR: specific growth rate; FCR: feed conversion ratio; PPV: protein productive value; LPV: lipid productive value;K: condition factor; ISI: intestinal-somatic index; HSI: hepatosomatic index.Figure 1
Growth of Eurasian perch (a) and rainbow trout (b) fingerlings fed with control diet (black, solid) and diets including dried (turquoise) and fermented (orange) duckweed to replace different proportions of fish meal protein (12%: solid, 24%: dashed, and 35%: dotted). Values are mean ± SD (n = 4).
(a)(b)Table 8
Initial and final whole-body composition of Eurasian perch (N = 4).
Final fish fed withp value∗Initial fishControlDWD12DWD24DWD35DWF12DWF24DWF35SEMDWDDWFMoisture (%)73.574.274.772.574.275.674.375.70.330.908 (0.577)0.023 (0.967)Crude protein (g·100 g−1 FM16.417.217.017.716.116.417.216.10.170.802 (0.238)0.417 (0.154)Crude lipids (g·100 g−1 FM)6.184.503.654.452.943.363.943.450.19<0.001 (0.507)<0.001 (0.457)Crude ash (g·100 g−1 FM)3.564.004.104.604.293.984.354.370.0650.001 (0.208)<0.001 (0.009)∗Contrast analysis between initial and final fish = p value; contrast analysis between control and duckweed fed fish = (p value).


### 3.2. Rainbow Trout—Oncorhynchus mykiss

Immediately after the experiment started, the rainbow trout accepted all experimental feeds, including those with the highest inclusion levels of both duckweed types, and all feed was eaten within the first minute after feeding. Similarly to perch, rainbow trout performance, including growth, feed, and nutrient utilization was influenced by feeding both duckweed types. However, compared to perch, the effects were less pronounced, and the lowest inclusion levels of both duckweed types did not produce any effects when compared to the control diets, while the highest inclusion levels triggered the largest effects (Table9 and Figure 1). Rainbow trout fed with either DWD35 or DWF35 showed the lowest growth (PWG: 296% and SGR: 3.93% day−1 for DWD35 and PWG: 324% and SGR: 4.10% day−1 for DWF35, respectively), highest FCR (1.26 for DWD35 and 1.20 for DWF35, respectively), lowest PPV (18.8% for DWD35 and 19.8% for DWF35, respectively) and LPV (21.3% for DWD35 and 22.3% for DWF35, respectively), and the lowest visceral fat reserves (47.9% for both DWD35 and DWF35). There was no significant effect on survival rates by feeding duckweed, regardless of the duckweed type. However, in diets with the highest inclusion levels of both DWD and DWF, the survival rates were slightly reduced. Compared to the initial fish, moisture was reduced in all experimental groups including the control, while CP was only higher in the control and DWD12 groups (Table 10). Crude lipids increased in all experimental groups, although contrast analysis only showed a significant effect for DWD-fed rainbow trout. Comparing the effects of the diets on whole body composition, the most remarkable effect was a decreasing CP content with an increased duckweed composition, while no clear effects for CL and CA could be determined (Table 10).Table 9
Dietary effects on growth, feed conversion, protein and lipid utilization, and body condition parameters in rainbow trout (N = 4).
Rainbow trout fed withp valueDried duckweed (DWD)Fermented duckweed (DWF)ControlDWD12DWD24DWD35DWF12DWF24DWF35SEMDWDDWFIBW (g)1.471.511.471.471.531.481.480.009——FBW (g)6.816.996.355.816.936.416.220.0970.0010.033WG (g)5.355.484.884.345.414.934.740.0920.0010.023PWG (%)3643773332963733373246.61<0.0010.033SGR (% day−1)4.394.374.173.934.334.184.100.037<0.0010.012FCR (g·g−1)1.111.111.161.261.121.191.200.012<0.0010.023PPV (%)21.421.520.418.821.220.019.80.22<0.0010.016LPV (%)23.723.822.821.323.522.422.30.20<0.0010.020K1.271.231.241.181.251.271.200.0110.0510.277ISI (%)16.016.015.916.416.616.717.70.160.5780.008HSI (%)2.432.422.422.312.532.552.600.0350.5950.162Visceral fat (%)66.758.350.047.960.452.147.91.840.0030.004Survival (%)10010010095.010010096.70.600.0580.391IBW: initial body weight; FBW: final body weight; WG: weight gain; PWG: percentage weight gain; SGR: specific growth rate; FCR: feed conversion ratio; PPV: protein productive value; LPV: lipid productive value;K: condition factor; ISI: intestinal-somatic index; HSI: hepatosomatic index.Table 10
Initial and final whole-body composition of rainbow trout (N = 4).
Initial fishFinal fish fed withp value∗ControlDWD12DWD24DWD35DWF12DWF24DWF35SEMDWDDWFMoisture (%)79.671.470.674.476.073.274.076.60.670.001 (0.033)0.005 (0.045)Crude protein (g·100 g−1 FM13.615.315.513.312.313.913.512.00.320.022 (0.011)0.051 (0.014)Crude lipids (g·100 g−1 FM)4.897.958.436.476.247.717.456.610.320.007 (0.122)0.092 (0.449)Crude ash (g·100 g−1 FM)1.541.862.162.072.081.841.901.730.0470.031 (0.168)0.074 (0.603)∗Contrast analysis between initial and final fish = p value; contrast analysis between control and duckweed fed fish = (p value).


## 3.1. Eurasian Perch—Perca fluviatilis

Feed acceptance for perch was low at the start of the experiment, and only after 2-3 days were all experimental diets accepted well by the fish. As this was the case with all diet types, this cannot be attributed to the inclusion of duckweed. Once feeds were accepted, they were consumed within two minutes of feeding with a tendency toward slower consumption in the higher concentrated duckweed diets. Perch reared on diets that contained duckweed had significantly reduced performance, including growth, feed conversion, and nutrient utilization (Table7). The reduction in performance and feed and nutrient utilization was more pronounced in perch fed fermented duckweed compared to perch fed dried duckweed. The results show clearly that the performance reduction increased with increasing the duckweed inclusion level, independent of duckweed type (Table 7 and Figure 1). The performance reduction was already seen in perch fed with DWD12, which was the best performing duckweed diet. The PWG was only 86.5% compared to 111% in the control. The worst performing duckweed diet, DWF35, resulted in just 35.4% PWG. Similarly, SGR, FCR, PPV, and LPV were inferior in DWF35 (0.86% day−1, 4.60, 6.03%, and 9.13%, respectively) compared to control (SGR: 2.13, FCR: 1.53, PPV: 20.0%, and LPV: 11.7%, respectively). Perch fed with the highest inclusion level of fermented duckweed also showed a marked increase in liver weights and a reduction in survival rates. The most obvious change in perch whole body composition between the initial fish and fish of all experimental groups including the control was a significant decrease in CL and an increase in CA (Table 8). When comparing the two duckweed types with the control, no significant trend was observed for moisture, CP, CL, or CA. Perch fed with DWF35 and DWD35 showed higher mortalities (21.2% and 15%, respectively) compared to the other duckweed groups and the control diets.Table 7
Dietary effects on growth, feed conversion, protein and lipid utilization, and body condition parameters in Eurasian perch. Values are averages of four replicates.
Perch fed withp valueDried duckweed (DWD)Fermented duckweed (DWF)ControlDWD12DWD24DWD35DWF12DWF24DWF35SEMDWDDWFIBW (g)3.533.463.553.513.503.513.550.02——FBW (g)7.306.456.055.206.255.694.680.18<0.001<0.001WG (g)3.762.992.501.692.752.181.130.17<0.001<0.001PWG (%)11186.575.050.280.664.435.45.09<0.001<0.001SGR (% day−1)2.131.781.591.161.681.390.860.09<0.001<0.001FCR (g·g−1)1.531.792.102.881.862.424.600.240.0110.011PPV (%)20.016.712.911.515.311.46.030.88<0.0010.010LPV (%)11.71.915.16a−8.17−1.45−0.63−9.131.360.033<0.001K1.571.511.541.461.521.411.450.020.8430.019ISI (%)7.677.558.127.708.106.928.130.120.5750.691HSI (%)1.421.401.331.401.321.271.500.030.5500.036Visceral fat (%)46.925.025.021.925.033.325.02.670.0260.460Survival (%)93.893.885.092.595.092.578.81.860.4220.023IBW: initial body weight; FBW: final body weight; WG: weight gain; PWG: percentage weight gain; SGR: specific growth rate; FCR: feed conversion ratio; PPV: protein productive value; LPV: lipid productive value;K: condition factor; ISI: intestinal-somatic index; HSI: hepatosomatic index.Figure 1
Growth of Eurasian perch (a) and rainbow trout (b) fingerlings fed with control diet (black, solid) and diets including dried (turquoise) and fermented (orange) duckweed to replace different proportions of fish meal protein (12%: solid, 24%: dashed, and 35%: dotted). Values are mean ± SD (n = 4).
(a)(b)Table 8
Initial and final whole-body composition of Eurasian perch (N = 4).
Final fish fed withp value∗Initial fishControlDWD12DWD24DWD35DWF12DWF24DWF35SEMDWDDWFMoisture (%)73.574.274.772.574.275.674.375.70.330.908 (0.577)0.023 (0.967)Crude protein (g·100 g−1 FM16.417.217.017.716.116.417.216.10.170.802 (0.238)0.417 (0.154)Crude lipids (g·100 g−1 FM)6.184.503.654.452.943.363.943.450.19<0.001 (0.507)<0.001 (0.457)Crude ash (g·100 g−1 FM)3.564.004.104.604.293.984.354.370.0650.001 (0.208)<0.001 (0.009)∗Contrast analysis between initial and final fish = p value; contrast analysis between control and duckweed fed fish = (p value).


## 3.2. Rainbow Trout—Oncorhynchus mykiss

Immediately after the experiment started, the rainbow trout accepted all experimental feeds, including those with the highest inclusion levels of both duckweed types, and all feed was eaten within the first minute after feeding. Similarly to perch, rainbow trout performance, including growth, feed, and nutrient utilization was influenced by feeding both duckweed types. However, compared to perch, the effects were less pronounced, and the lowest inclusion levels of both duckweed types did not produce any effects when compared to the control diets, while the highest inclusion levels triggered the largest effects (Table9 and Figure 1). Rainbow trout fed with either DWD35 or DWF35 showed the lowest growth (PWG: 296% and SGR: 3.93% day−1 for DWD35 and PWG: 324% and SGR: 4.10% day−1 for DWF35, respectively), highest FCR (1.26 for DWD35 and 1.20 for DWF35, respectively), lowest PPV (18.8% for DWD35 and 19.8% for DWF35, respectively) and LPV (21.3% for DWD35 and 22.3% for DWF35, respectively), and the lowest visceral fat reserves (47.9% for both DWD35 and DWF35). There was no significant effect on survival rates by feeding duckweed, regardless of the duckweed type. However, in diets with the highest inclusion levels of both DWD and DWF, the survival rates were slightly reduced. Compared to the initial fish, moisture was reduced in all experimental groups including the control, while CP was only higher in the control and DWD12 groups (Table 10). Crude lipids increased in all experimental groups, although contrast analysis only showed a significant effect for DWD-fed rainbow trout. Comparing the effects of the diets on whole body composition, the most remarkable effect was a decreasing CP content with an increased duckweed composition, while no clear effects for CL and CA could be determined (Table 10).Table 9
Dietary effects on growth, feed conversion, protein and lipid utilization, and body condition parameters in rainbow trout (N = 4).
Rainbow trout fed withp valueDried duckweed (DWD)Fermented duckweed (DWF)ControlDWD12DWD24DWD35DWF12DWF24DWF35SEMDWDDWFIBW (g)1.471.511.471.471.531.481.480.009——FBW (g)6.816.996.355.816.936.416.220.0970.0010.033WG (g)5.355.484.884.345.414.934.740.0920.0010.023PWG (%)3643773332963733373246.61<0.0010.033SGR (% day−1)4.394.374.173.934.334.184.100.037<0.0010.012FCR (g·g−1)1.111.111.161.261.121.191.200.012<0.0010.023PPV (%)21.421.520.418.821.220.019.80.22<0.0010.016LPV (%)23.723.822.821.323.522.422.30.20<0.0010.020K1.271.231.241.181.251.271.200.0110.0510.277ISI (%)16.016.015.916.416.616.717.70.160.5780.008HSI (%)2.432.422.422.312.532.552.600.0350.5950.162Visceral fat (%)66.758.350.047.960.452.147.91.840.0030.004Survival (%)10010010095.010010096.70.600.0580.391IBW: initial body weight; FBW: final body weight; WG: weight gain; PWG: percentage weight gain; SGR: specific growth rate; FCR: feed conversion ratio; PPV: protein productive value; LPV: lipid productive value;K: condition factor; ISI: intestinal-somatic index; HSI: hepatosomatic index.Table 10
Initial and final whole-body composition of rainbow trout (N = 4).
Initial fishFinal fish fed withp value∗ControlDWD12DWD24DWD35DWF12DWF24DWF35SEMDWDDWFMoisture (%)79.671.470.674.476.073.274.076.60.670.001 (0.033)0.005 (0.045)Crude protein (g·100 g−1 FM13.615.315.513.312.313.913.512.00.320.022 (0.011)0.051 (0.014)Crude lipids (g·100 g−1 FM)4.897.958.436.476.247.717.456.610.320.007 (0.122)0.092 (0.449)Crude ash (g·100 g−1 FM)1.541.862.162.072.081.841.901.730.0470.031 (0.168)0.074 (0.603)∗Contrast analysis between initial and final fish = p value; contrast analysis between control and duckweed fed fish = (p value).


## 4. Discussion

Duckweed is a promising plant-based protein source for fish feeds, and its application as a feed component in diets for herbivorous or omnivorous fish such as carp or tilapia has been studied repeatedly, testing different duckweed species and concentrations [29–35]. However, according to our knowledge, duckweed has not been tested as fishmeal substitute for Eurasian perch, and only two studies have reported on the utilization of duckweed in rainbow trout. When rainbow trout fry received feed with only smaller proportions of fishmeal being replaced by duckweed (1/16 and 1/8), only slightly reduced feed conversion ratios were observed that did not differ between the duckweed inclusion levels [21]. The current study reports significantly differing performances between how the two carnivorous species Eurasian perch and rainbow trout utilize dried and fermented duckweed S. polyrhiza.


### 4.1. Perch (P. fluviatilis) Trial

The initial low feed acceptance of the experimental diets in perch confirms observations in previous experiments by us (not published). Adaptation to new feeds, especially when they have different physical properties (e.g., floating versus sinking), needs more time in perch compared to other fishes.When fed with diets containing dried and fermented duckweed, perch exhibited a significantly increased FCR irrespective of the level of duckweed inclusion and, especially at higher levels of duckweed inclusion, a reduction of growth. This effect was stronger with feed containing fermented duckweed (DWF) than with feed containing dried duckweed (DWD). Growth curves showed a clear pattern, with control-fed fish performing best, followed by DWD12-fed perch, and, finally, with perch fed either DWD35 or DWF35 performing worst (Figure1). The low growth performance of perch fed with duckweed was also reflected in a significantly decreased condition factor K of perch fed with increasing levels of DWF but not in those fed with DWD, where K was not influenced by DWD levels. Survival rates were significantly reduced in fish fed with the highest concentrations of both duckweed types (DWD35 and DWF35). This was confirmed by daily visual observations that revealed higher intraspecific aggression of larger and more dominant individuals towards smaller and subordinate individuals. All mortalities occurred either due to fish being killed during periods when no observation took place or due to being severely injured and euthanized by us according to Swiss animal welfare regulations. No apparent nutrient deficiency signs were observed, and perch fingerling behavior was similar to earlier experiments where the strongest fish showed intensive agonistic behavior towards weak and smaller conspecifics. However, considering the highest mortalities occurred in both DWD35 and DWF35, the diets with the highest duckweed content, a connection with nutrient content, availability, or digestibility, appears reasonable. The most pronounced effect of the experimental diets on body composition was the significant decrease in whole-body lipids in all treatments when compared to the fish at the onset of the experiment. The experimental diet was formulated according to the nutritional requirements of perch (P. fluviatilis) [24, 25]. In this study, the lipid content of the feed was set to 14%, which was based on the observation that even though a content of 18% led to the highest growth in perch fry, it also led to increased lipid droplets in the hepatocytes [25].The significantly reduced whole body lipids and the low, sometimes negative lipid productive values (LPV) suggest that, independently of treatment, CL and energy or the physiological availability, i.e., the digestibility, was too low in the formulated perch diets. Up to now, no study has evaluated the effects of fishmeal replacement by any plant protein onP. fluviatilis. For the closely related North American yellow perch (P. flavescens), a fish meal replacement of 50% and 100% by untreated soybean meal led to significantly reduced performance, while fish meal replacement by an ANF-reduced soybean variant in the same concentrations did not show similar strong effects [36].


### 4.2. Rainbow Trout (O. mykiss) Trial

Trout readily accepted the experimental diets, which confirmed previous observations with experimental diets containing insect meal or duckweed [21, 37].The results clearly show that rainbow trout utilized feed containing duckweed better than perch, and the new diet did not compromise growth, FCR, or nutrient utilization at low to moderate levels of duckweed inclusion. Only at high inclusion levels was survival reduced. Like in perch, this was confirmed by daily observations which revealed intraspecific aggression of larger and dominant individuals towards the smallest individuals in the corresponding tanks. Condition factorsK, ISI, and HSI were, except for ISI in trout fed DWF35, not significantly influenced by duckweed, which renders a treatment-related mortality unlikely. Both types of duckweed, fermented and dried, had a significant effect on PPV, which was lowest in the diets with the highest inclusion levels (DWD35 and DWF35), while the lowest inclusion levels (DWD12 and DWF12) showed similar PPVs to the control group. The same picture was observed for the lipid productive value (LPV), in which increasing duckweed levels resulted in decreasing LPVs for both duckweed types, although this effect was somewhat less pronounced in DWF groups.As rainbow trout is among the commercially most important freshwater fishes, several studies reporting on replacing fishmeal protein with plant-based proteins have been published. These include, among others, soybean, lupine, rapeseed/canola, sunflower seeds, and pumpkin seeds [38–42]. As in perch, however, almost no studies exist that tested duckweed as a fishmeal replacement in rainbow trout fry or fingerlings or even in other salmonids. One study included 6.25 and 12.5% concentrations of duckweed (S. polyrhiza) in rainbow trout fry feed [21], but the investigation did not strictly exchange fishmeal for duckweed on protein basis. There was only a small but significant increase in feed conversion, but no decrease in SGR was observed (a tendency was observed, though). Most interestingly, both groups of duckweed-fed trout showed similar performance, independent of dietary duckweed concentration [21]. One study reported the effects of partial fishmeal and soybean substitution by duckweed (Lemna minor) on grow-out rainbow trout. Only in diets with the highest inclusion level (280 g kg−1) did they find a severe reduction in performance, resulting in the conclusion that up to 20% of fishmeal and soybean protein could be substituted by duckweed [43].


### 4.3. Duckweed as a Fishmeal Replacement

The success of fishmeal replacement depends on several factors, the more important ones being the concentration of fishmeal in the control diet, its relative replacement level, and the quality of the plant ingredient to which the protein concentration, amino acid composition, and the amount of ANFs contribute. While several ANFs are known for typical plant-based proteins such as soybean or canola/rapeseed [9], only few ANFs such as oxalic acid have been reported for Lemna gibba and L. minor [44, 45] and cyanides, tannins, and phytic acid for S. polyrhiza [46]. This does not exclude the potential presence of other ANFs such as protease inhibitors, thus warranting further study concerning the presence and concentrations of ANFs in different duckweed species.The amino acid compositions reported for different duckweed species are usually comparable to those of soybean or lupine [47]. The EAA profiles for both feeds showed comparatively little differences. The most striking of these was a generally higher EAA concentration in the perch diets and a higher lysine content in the perch feeds compared to the rainbow trout feeds. This difference is likely caused by the difference in the secondary protein source in perch feeds (poultry by-product meal) compared to rainbow trout feed (wheat gluten), although this contradicts the usually higher lysine content in wheat gluten meal compared to poultry by-product meals [23]. While for Eurasian perch, according to the authors’ knowledge, no EAA requirements have been published up to date, the EAA requirements for rainbow trout are mostly known. A deficit in lysine is reported to result in reduced growth and feed efficiency but could also cause certain health issues such as caudal fin erosion in trout [23]. Similarly, a deficiency of methionine leads to reduced growth and feed efficiency. Therefore, it could be assumed that, especially in diets containing higher levels of DWF, a lysine and methionine deficiency might have contributed to the reduced performance in rainbow trout. However, while rainbow trout performed rather well despite a presumed lysine and methionine deficiency in all diets, including the control, perch showed very poor performance, although no EAA deficiency appeared to exist and EAA concentrations were generally higher compared to rainbow trout. Although the true EAA requirements are not known for perch, the decreasing growth performance with increasing dietary DWD or DWF inclusion levels points to other factors significantly influencing and contributing to the poor performance in perch.Up to now, different duckweed species have been used with varying success as a feed component or fishmeal replacement in several fish species. Most of the studies have been conducted in fish of the lower trophic levels belonging to the families of cichlids [31–33] and cyprinids [29, 30, 48]. The responses and performance of the different fish species depended strongly on the experimental setup and duckweed inclusion levels, as well as on duckweed protein content and quality. Feeding Labeo rohita with increasing levels of raw and fermented duckweed as a fishmeal replacement in formulated feeds resulted in similar or improved performance compared to the control diet [29]. The authors furthermore reported an apparent protein digestibility for dried and fermented duckweed of between 82.3 and 94.4%. For Nile tilapia (Oreochromis niloticus), duckweed protein digestibility was reported to range between 75.9 and 79%, depending on the inclusion level of duckweed, and the digestibility decreased with higher inclusion levels [32]. The authors also reported that dried and fresh duckweed inclusion of 20 or 40% led to a reduction of growth by 5–13%. Nevertheless, the authors highlighted that neither the reduction in growth and feed conversion, nor in the digestibility, was significant enough to discourage the use of duckweed in tilapia feeds, especially in places where fishmeal-based diets are scarce or too expensive. Replacing up to 30% of fishmeal protein by S. polyrhiza did not show any significant negative effects on performance traits when fed to fingerling Nile tilapia for 56 days [33]. Similarly, de Matos et al. [31] did not find any significant reduction in growth or increase in FCR after feeding L. valdiviana pellets (39.9% CP) to red tilapia fry (O. mossambicus × O. niloticus) for 83 days compared to commercial compound feed (53.3% CP). Nevertheless, almost no studies exist evaluating duckweed as a fishmeal substitute in diets for carnivorous fishes other than rainbow trout. One study tested fermented duckweed (Lemna minor) in diets for juvenile barramundi (Lates calcarifer) in dietary concentrations of 15, 25, 35, and 45%. They found that barramundi fed with up to 35% fermented L. minor meal performed equal to control fish with barramundi fed 25% fermented duckweed meal even performing slightly better [49]. Generally, it is probably safe to assume that digestibility and nutrient availability are higher in fish of lower trophic levels that are more omnivorous or herbivorous compared to perch and rainbow trout. The results obtained in this study for Eurasian perch point towards a low utilization efficiency of duckweed for, up to now, unknown reasons, although the generally low lipid utilization in perch diets appears to be an important factor. Rainbow trout, on the other hand, appears to be reasonably able to utilize low to medium duckweed inclusion levels, independent of duckweed type, dried or fermented. For perch, neither DWD nor DWF seem to be an interesting feed ingredient, although the performance was somewhat worse in DWF-fed fish compared to DWD-fed fish. For rainbow trout, however, both DWD and DWF could be used in concentrations equaling those in DWD12 and DWF12. Higher concentrations start to reduce performance. Contrary to perch, the lowest performing group in rainbow trout was the one with the highest dried duckweed concentration, DWD35, although the differences to control were comparatively low. One factor contributing to the general performance of fish feeds is the feed manufacturing process. Our diets were cold extruded, which is not nearly as efficient in improving digestibility and nutrient availability compared to sophisticated extrusion processes [50, 51]. State-of-the-art feed production will likely cause beneficial effects to both fish species, which will allow higher inclusion levels.


## 4.1. Perch (P. fluviatilis) Trial

The initial low feed acceptance of the experimental diets in perch confirms observations in previous experiments by us (not published). Adaptation to new feeds, especially when they have different physical properties (e.g., floating versus sinking), needs more time in perch compared to other fishes.When fed with diets containing dried and fermented duckweed, perch exhibited a significantly increased FCR irrespective of the level of duckweed inclusion and, especially at higher levels of duckweed inclusion, a reduction of growth. This effect was stronger with feed containing fermented duckweed (DWF) than with feed containing dried duckweed (DWD). Growth curves showed a clear pattern, with control-fed fish performing best, followed by DWD12-fed perch, and, finally, with perch fed either DWD35 or DWF35 performing worst (Figure1). The low growth performance of perch fed with duckweed was also reflected in a significantly decreased condition factor K of perch fed with increasing levels of DWF but not in those fed with DWD, where K was not influenced by DWD levels. Survival rates were significantly reduced in fish fed with the highest concentrations of both duckweed types (DWD35 and DWF35). This was confirmed by daily visual observations that revealed higher intraspecific aggression of larger and more dominant individuals towards smaller and subordinate individuals. All mortalities occurred either due to fish being killed during periods when no observation took place or due to being severely injured and euthanized by us according to Swiss animal welfare regulations. No apparent nutrient deficiency signs were observed, and perch fingerling behavior was similar to earlier experiments where the strongest fish showed intensive agonistic behavior towards weak and smaller conspecifics. However, considering the highest mortalities occurred in both DWD35 and DWF35, the diets with the highest duckweed content, a connection with nutrient content, availability, or digestibility, appears reasonable. The most pronounced effect of the experimental diets on body composition was the significant decrease in whole-body lipids in all treatments when compared to the fish at the onset of the experiment. The experimental diet was formulated according to the nutritional requirements of perch (P. fluviatilis) [24, 25]. In this study, the lipid content of the feed was set to 14%, which was based on the observation that even though a content of 18% led to the highest growth in perch fry, it also led to increased lipid droplets in the hepatocytes [25].The significantly reduced whole body lipids and the low, sometimes negative lipid productive values (LPV) suggest that, independently of treatment, CL and energy or the physiological availability, i.e., the digestibility, was too low in the formulated perch diets. Up to now, no study has evaluated the effects of fishmeal replacement by any plant protein onP. fluviatilis. For the closely related North American yellow perch (P. flavescens), a fish meal replacement of 50% and 100% by untreated soybean meal led to significantly reduced performance, while fish meal replacement by an ANF-reduced soybean variant in the same concentrations did not show similar strong effects [36].


## 4.2. Rainbow Trout (O. mykiss) Trial

Trout readily accepted the experimental diets, which confirmed previous observations with experimental diets containing insect meal or duckweed [21, 37].The results clearly show that rainbow trout utilized feed containing duckweed better than perch, and the new diet did not compromise growth, FCR, or nutrient utilization at low to moderate levels of duckweed inclusion. Only at high inclusion levels was survival reduced. Like in perch, this was confirmed by daily observations which revealed intraspecific aggression of larger and dominant individuals towards the smallest individuals in the corresponding tanks. Condition factorsK, ISI, and HSI were, except for ISI in trout fed DWF35, not significantly influenced by duckweed, which renders a treatment-related mortality unlikely. Both types of duckweed, fermented and dried, had a significant effect on PPV, which was lowest in the diets with the highest inclusion levels (DWD35 and DWF35), while the lowest inclusion levels (DWD12 and DWF12) showed similar PPVs to the control group. The same picture was observed for the lipid productive value (LPV), in which increasing duckweed levels resulted in decreasing LPVs for both duckweed types, although this effect was somewhat less pronounced in DWF groups.As rainbow trout is among the commercially most important freshwater fishes, several studies reporting on replacing fishmeal protein with plant-based proteins have been published. These include, among others, soybean, lupine, rapeseed/canola, sunflower seeds, and pumpkin seeds [38–42]. As in perch, however, almost no studies exist that tested duckweed as a fishmeal replacement in rainbow trout fry or fingerlings or even in other salmonids. One study included 6.25 and 12.5% concentrations of duckweed (S. polyrhiza) in rainbow trout fry feed [21], but the investigation did not strictly exchange fishmeal for duckweed on protein basis. There was only a small but significant increase in feed conversion, but no decrease in SGR was observed (a tendency was observed, though). Most interestingly, both groups of duckweed-fed trout showed similar performance, independent of dietary duckweed concentration [21]. One study reported the effects of partial fishmeal and soybean substitution by duckweed (Lemna minor) on grow-out rainbow trout. Only in diets with the highest inclusion level (280 g kg−1) did they find a severe reduction in performance, resulting in the conclusion that up to 20% of fishmeal and soybean protein could be substituted by duckweed [43].


## 4.3. Duckweed as a Fishmeal Replacement

The success of fishmeal replacement depends on several factors, the more important ones being the concentration of fishmeal in the control diet, its relative replacement level, and the quality of the plant ingredient to which the protein concentration, amino acid composition, and the amount of ANFs contribute. While several ANFs are known for typical plant-based proteins such as soybean or canola/rapeseed [9], only few ANFs such as oxalic acid have been reported for Lemna gibba and L. minor [44, 45] and cyanides, tannins, and phytic acid for S. polyrhiza [46]. This does not exclude the potential presence of other ANFs such as protease inhibitors, thus warranting further study concerning the presence and concentrations of ANFs in different duckweed species.The amino acid compositions reported for different duckweed species are usually comparable to those of soybean or lupine [47]. The EAA profiles for both feeds showed comparatively little differences. The most striking of these was a generally higher EAA concentration in the perch diets and a higher lysine content in the perch feeds compared to the rainbow trout feeds. This difference is likely caused by the difference in the secondary protein source in perch feeds (poultry by-product meal) compared to rainbow trout feed (wheat gluten), although this contradicts the usually higher lysine content in wheat gluten meal compared to poultry by-product meals [23]. While for Eurasian perch, according to the authors’ knowledge, no EAA requirements have been published up to date, the EAA requirements for rainbow trout are mostly known. A deficit in lysine is reported to result in reduced growth and feed efficiency but could also cause certain health issues such as caudal fin erosion in trout [23]. Similarly, a deficiency of methionine leads to reduced growth and feed efficiency. Therefore, it could be assumed that, especially in diets containing higher levels of DWF, a lysine and methionine deficiency might have contributed to the reduced performance in rainbow trout. However, while rainbow trout performed rather well despite a presumed lysine and methionine deficiency in all diets, including the control, perch showed very poor performance, although no EAA deficiency appeared to exist and EAA concentrations were generally higher compared to rainbow trout. Although the true EAA requirements are not known for perch, the decreasing growth performance with increasing dietary DWD or DWF inclusion levels points to other factors significantly influencing and contributing to the poor performance in perch.Up to now, different duckweed species have been used with varying success as a feed component or fishmeal replacement in several fish species. Most of the studies have been conducted in fish of the lower trophic levels belonging to the families of cichlids [31–33] and cyprinids [29, 30, 48]. The responses and performance of the different fish species depended strongly on the experimental setup and duckweed inclusion levels, as well as on duckweed protein content and quality. Feeding Labeo rohita with increasing levels of raw and fermented duckweed as a fishmeal replacement in formulated feeds resulted in similar or improved performance compared to the control diet [29]. The authors furthermore reported an apparent protein digestibility for dried and fermented duckweed of between 82.3 and 94.4%. For Nile tilapia (Oreochromis niloticus), duckweed protein digestibility was reported to range between 75.9 and 79%, depending on the inclusion level of duckweed, and the digestibility decreased with higher inclusion levels [32]. The authors also reported that dried and fresh duckweed inclusion of 20 or 40% led to a reduction of growth by 5–13%. Nevertheless, the authors highlighted that neither the reduction in growth and feed conversion, nor in the digestibility, was significant enough to discourage the use of duckweed in tilapia feeds, especially in places where fishmeal-based diets are scarce or too expensive. Replacing up to 30% of fishmeal protein by S. polyrhiza did not show any significant negative effects on performance traits when fed to fingerling Nile tilapia for 56 days [33]. Similarly, de Matos et al. [31] did not find any significant reduction in growth or increase in FCR after feeding L. valdiviana pellets (39.9% CP) to red tilapia fry (O. mossambicus × O. niloticus) for 83 days compared to commercial compound feed (53.3% CP). Nevertheless, almost no studies exist evaluating duckweed as a fishmeal substitute in diets for carnivorous fishes other than rainbow trout. One study tested fermented duckweed (Lemna minor) in diets for juvenile barramundi (Lates calcarifer) in dietary concentrations of 15, 25, 35, and 45%. They found that barramundi fed with up to 35% fermented L. minor meal performed equal to control fish with barramundi fed 25% fermented duckweed meal even performing slightly better [49]. Generally, it is probably safe to assume that digestibility and nutrient availability are higher in fish of lower trophic levels that are more omnivorous or herbivorous compared to perch and rainbow trout. The results obtained in this study for Eurasian perch point towards a low utilization efficiency of duckweed for, up to now, unknown reasons, although the generally low lipid utilization in perch diets appears to be an important factor. Rainbow trout, on the other hand, appears to be reasonably able to utilize low to medium duckweed inclusion levels, independent of duckweed type, dried or fermented. For perch, neither DWD nor DWF seem to be an interesting feed ingredient, although the performance was somewhat worse in DWF-fed fish compared to DWD-fed fish. For rainbow trout, however, both DWD and DWF could be used in concentrations equaling those in DWD12 and DWF12. Higher concentrations start to reduce performance. Contrary to perch, the lowest performing group in rainbow trout was the one with the highest dried duckweed concentration, DWD35, although the differences to control were comparatively low. One factor contributing to the general performance of fish feeds is the feed manufacturing process. Our diets were cold extruded, which is not nearly as efficient in improving digestibility and nutrient availability compared to sophisticated extrusion processes [50, 51]. State-of-the-art feed production will likely cause beneficial effects to both fish species, which will allow higher inclusion levels.


## 5. Conclusion

The observed effects differ remarkably between the two carnivorous fish species Eurasian perch (P. fluviatilis) and rainbow trout (O. mykiss). While both species are at a trophic level of 4 or above and are considered to prey on similar organisms, they reacted differently to a graded inclusion of dried and fermented duckweed in their diets. In general, perch could not utilize duckweed well, independent of the duckweed type or inclusion level, and exhibited both reduced growth and an increased feed conversion ratio. On the contrary, trout were able to utilize both dried and fermented duckweed up to an inclusion level of 12% fishmeal protein replacement without significant growth reduction or increased feed conversion and up to 24% with only slight performance reductions. This study suggests that duckweed is an interesting future protein source for rainbow trout but not for Eurasian perch. Future work should focus on practical trials using extruded diets, determination of duckweed digestibility in trout or other salmonids, and presence and concentrations of important ANFs.



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*Source: 6680943-2023-03-16.xml*