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Aquaculture Research, 2015, 1–11 doi:10.1111/are.12743 Earthworm powder as an alternative protein source in diets for common carp (Cyprinus carpio L.) Tuan Nguyen Ngoc1,2, Johannes Pucher1, Klaus Becker1 & Ulfert Focken1,3 Department of Aquaculture Systems in the Tropics and Subtropics (480b), Universitaet Hohenheim, Stuttgart, Germany Faculty of Fisheries, Vietnam National University of Agriculture, Hanoi, Vietnam Thuenen-Institute of Fisheries Ecology, Ahrensburg, Germany Correspondence: U Focken, Th€ unen-Institute of Fisheries Ecology, Federal Research Institute for Rural Areas, Forestry and Fisheries, Wulfsdorfer Weg 204, D-22926 Ahrensburg, Germany Email: ulfert.focken@ti.bund.de Abstract The increasing need for aquafeed resources and the finite availability of conventional feed resources are making it necessary to search for alternative high-protein resources that are not used as human food The earthworm Perionyx excavatus was tested as a feed ingredient in diets for common carp An experiment was conducted to evaluate the potential of earthworm powder as a replacement for fishmeal In a recirculation aquarium system, triplicate groups of five common carp (Cyprinus carpio L.) were fed a control feed (fishmeal based protein), or experimental diets in which 30% (EW30), 70% (EW70), or 100% (EW100) of fishmeal protein was replaced by earthworm protein Fish growth, feed digestibility and feed utilization were monitored Growth rate, protein efficiency and energy retention in fish were similar (EW30, EW100) or higher (EW70) for diets containing earthworm meal compared to the control diet Protein digestibility in EW30, EW70 and EW100 was higher than in the control diet, but in (EW100), lipid conversion was lower We conclude that earthworm is a suitable partial replacement for fishmeal in feeds for common carp Keywords: alternative feed ingredient, earthworm, fishmeal replacement, common carp Introduction Aquaculture is one of the fastest growing sectors in food production with annual growth rates © 2015 John Wiley & Sons Ltd between 5.1% and 7.4% between 2007 and 2012 (SOFIA 2014) This increasing production requires corresponding increases in aquafeed production (Tacon & Metian 2008) As fishmeal, the traditional source of animal protein for aquafeeds, is finite (Naylor, Hardy, Bureau, Chiu, Elliott, Farrell, Forster, Gatlin, Goldburg, Hua & Nichols 2009), various other protein resources have been studied and applied in aquafeeds such as soybean meal, various other plant proteins and rendered animal products (Hardy 2010; Hern andez, Olvera-Novoa, Hardy, Hermosillo, Reyes & GonzAlez 2010) Apart from the feed resources produced on a large scale and traded internationally, a number of highly nutritive feed resources have been suggested that can be produced on a small scale and mainly utilized and traded locally or regionally These include silk worm pupae meals (Begum, Chakraborty, Zaher, Abdul & Gupta 1994), housefly maggots (Ogunji, Kloas, Wirth, Neumann & Pietsch 2008), snails and termites (Phonekhampheng, Hung & Lindberg 2008) and various earthworm species (e.g Tacon, Stafford & Edwards 1983; Nandeesha, Srikanth, Basavaraja, Keshavanath, Varghese, Bano, Ray & Kale 1988; Paripuranam, Divya, Ulaganathan, Balamurugan & Umamaheswari 2011) The production of earthworm (vermiculture) can be performed either on a small scale (Chakrabarty, Das & Das 2011) or semi-industrially (Sinha, Herat, Agarwal, Asadi & Carretero 2002) So far, the latter has been practiced mainly as a means of organic waste management (Suthar 2009a,b; Yadac & Garg 2009; Sim & Wu 2010) Earthworm meal in feeds for common carp T N Ngoc et al In 1981, earthworm was realized as a potential source of protein for animal feeds (Hartenstein 1981) A number of earthworm species have since been studied in detail for their suitability in different fish species Tacon et al (1983) reported that different earthworm species contain about 50–60% of crude protein (CP) in dry matter (DM) and have low ash content which is a favourable characteristic for an ingredient of fish feed Tacon et al (1983) tested the possibility of partially substituting fishmeal by earthworm (Eisenia foetida, Allolobophora sp and Lumbricus terrestis) in trout diets and reported the latter two species as well suited for the substitution of fishmeal in diets for rainbow trout, for the first species they suggested various processing techniques to improve palatability Nandeesha et al (1988) carried out an experiment in which they replaced fishmeal with earthworm Eudrilus eugeniae in diets for common carp, Cyprinus carpio Dong, Guo, Ye, Song, Huang and Wang (2010) studied the digestibility of various unconventional protein sources in tilapia, including freeze-dried earthworm meal and reported a dry matter digestibility of 98.9 Ỉ 4.65% but did not state which species of earthworm were used Pucher, Ngoc, Yen, Mayrhofer, El-Matboulic and Focken (2014) showed that the tropical earthworm Perionyx excavatus can fully replace fishmeal in supplemental feeds for common carp under semi-intensive production conditions with fish having access also to natural food resources However, in the study by Pucher, Ngoc, et al (2014) it is not possible to estimate to what extent protein from supplemental feeds and protein from natural food resources contributed to the growth of fish For this conclusion, an estimate of the digestibility of supplemental feed is needed To our knowledge, the suitability of P excavatus has not been studied as a feed ingredient for common carp under controlled laboratory conditions in spite of the large global production of 3.7 mio tonnes of this fish species in 2011 (FAO 2011) P excavatus can be produced industrially using organic waste (Sinha et al 2002) or at household level using ruminant manure and other available organic materials (M€ uller, Pucher, Tran, Focken & Kreuzer 2012) The aim of this study was to test under controlled laboratory conditions the digestibility and nutritional quality of P excavatus as a feed ingredient in diets that are used as supplement feed in tropical pond aquaculture of common carp Aquaculture Research, 2015, 1–11 Materials and methods Experimental feeds Earthworm P excavatus was purchased from a small-scale commercial vermiculture facility in suburban Hanoi (Viet Nam) that produces earthworm on a substrate of ruminant manure for use in feeds and traditional medicines The worms were cleaned from soil, frozen, freeze-dried and vacuum-packed for transportation to the University of Hohenheim (Germany) Fishmeal as feed ingredient was purchased from a large feed retailer in Germany (Vereinigte Fischmehlwerke Cuxhaven, Germany) and wheat flour was purchased from the supermarket (Germany) Chemical composition and amino acid composition of these three feed ingredients are shown in Table The earthworm meal used in this trial contained more CP than the fishmeal and had approximately the same gross energy The earthworm material had an amino acid composition that was similar to fishmeal The levels of those amino acids essential for fish, i.e threonine, valine, cystine and methionine, leucine, phenylalanine, histidine and arginine were similar or higher in earthworm meal compared to fishmeal Based on the analyzed chemical and amino acid composition of feed ingredients (Table 1), four iso-nitrogenous and iso-lipidic supplemental diets were formulated at 30% of CP and 10% of crude lipid (CL) The protein content was chosen to be lower than required by fish (NRC Table Chemical composition and amino acid composition of dry feed ingredients of the test diets Feed Earthworm Fish meal Wheat meal CA [% of DM] CP [% of DM] CL [% of DM] GE [MJ kgÀ1 DM] Threonine [% of CP] Valine [% of CP] Cys + Met [% of CP] Isoleucine [% of CP] Leucine [% of CP] Phe + Tyr [% of CP] Histidine[% of CP] Lysine [% of CP] Arginine [% of CP] 7.3 71.3 7.8 21.4 4.5 7.0 3.1 4.2 7.4 7.1 2.4 6.6 6.0 19.1 67.4 12.8 21.6 3.4 4.3 3.0 3.4 6.2 5.4 2.6 6.1 5.2 1.8 15.5 1.8 19.3 2.4 1.9 4.6 2.7 5.9 6.3 2.2 2.1 3.5 CA, crude ash; CL, crude lipid; CP, crude protein; Cys + Met, cystine + methionine; DM, dry matter; GE, gross energy; Phe + Tyr, phenylalanine + tyrosine © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–11 Aquaculture Research, 2015, 1–11 2011) as they were designed to resemble diets used by farmers in semi-intensively managed pond culture of common carp in Vietnam where natural food resources serve as surplus of protein for the common carp Under semi-intensive common carp culture, zooplankton and macro-zoobenthos serve as natural food resources, both being richer in protein than required by common carp (De Silva 1993; based on data by Hepher 1988; NRC 2011) Due to the higher protein supply through natural food resources than required by common carp, lower levels of CP and essential amino acids in supplemental feeds are needed in fish feed (De Silva 1993) Under this pond situation, digestible energy becomes the first limiting factor for fish growth and needs to be supplied to the fish within the supplemental pellet feed (Viola 1989) The Earthworm meal in feeds for common carp T N Ngoc et al diets were not supplemented with crystalline amino acids, as these are typically not available for farm-made aquafeeds in remote rural areas No natural food (or substitute) was offered to the fish as this would have impeded the determination of digestibility of the experimental diets The control diet was composed of fishmeal as the main protein source, wheat meal, sunflower oil and premixes of vitamins and minerals In the three treatment diets (EW30, EW70, and EW100), 30%, 70% or 100% of fishmeal protein was replaced by earthworm protein respectively (see Table 2) The four test feeds had similar contents of crude ash (CA), CP, CL and gross energy (GE) (Table 2) The content of all essential amino acids in the experimental diets increased as the proportion of earthworm increased Titanium dioxide Table Ingredient composition of diets [% of diet DM], proximate composition, gross energy content and essential amino acid composition [% of crude protein] of the test diets in the trial and recommended levels of common carp (recalculated from requirements for common carp given in NRC 2011) Feed Ingredient composition of diets Prox composition Essential amino acid composition Fishmeal [% of diet DM] Earthworm [% of diet DM] Wheat meal [% of diet DM] Sunflower oil [% of diet DM] Mineral* [% of diet DM] Vitamin† [% of diet DM] TiO2 [% of diet DM] CA [% of DM] CP [% of DM] DP [% of DM] CL [% of DM] GE [MJ kgÀ1 DM] DE [% of DM] Threonine [% of CP] Valine [% of CP] Cys + Met [% of CP] Isoleucine [% of CP] Leucine [% of CP] Phe + Tyr [% of CP] Histidine[% of CP] Lysine [% of CP] Arginine [% of CP] Rec 32.0‡ 13.4§ 3.9 3.7 2.6 2.6 3.7 5.3 1.3 5.8 4.5 Control EW30 EW70 EW100 30.3 – 61.7 5.0 2.0 2.0 1.0 8.3 27.8 22.3‡ 9.7 19.4 13.7§ 3.2 4.0 1.4 3.2 6.5 4.0 2.5 5.0 5.0 21.1 8.8 61.6 5.5 2.0 2.0 1.0 8.4 27.7 22.6‡ 9.7 19.4 14.1§ 3.6 4.3 1.8 3.6 6.9 4.0 2.5 5.4 5.4 9.0 20.2 61.7 6.2 2.0 2.0 1.0 8.1 27.5 23.5‡ 9.7 19.4 14.1§ 4.0 5.1 1.8 4.0 7.3 4.4 2.5 5.5 5.5 – 28.7 61.7 6.7 2.0 2.0 1.0 8.2 27.6 23.7‡ 9.7 19.4 13.7§ 4.0 5.8 2.2 4.0 7.6 4.3 2.5 5.4 5.8 CA, crude ash; CL, crude lipid; CP, crude protein; Cys + Met, cystine + methionine; DE, digestible energy; DM, dry matter; DP, digestible protein; GE, gross energy; Phe + Tyr, phenylalanine + tyrosine; Rec., Recommended content of essential amino acids; TiO2, Titanium dioxide *Vitamin premix: retinol palmitate: 500 000 IU kgÀ1; thiamine: g kgÀ1; riboflavin: g kgÀ1; niacin: 25 g kgÀ1; folic acid: g kgÀ1; pyridoxine: g kgÀ1; cyanocobalamine: g kgÀ1; ascorbic acid: 10 g kgÀ1; cholecalciferol: 50 000 IU kgÀ1; a-tocopherol: 2.5 g kgÀ1; menadione: g kgÀ1; inositol: 25 g kgÀ1; pantothenic acid: 10 g kgÀ1; choline chloride: 100 g kgÀ1; biotin: 0.25 g kgÀ1 †Mineral premix (g/k): CaCO3: 336; KH2PO4: 502; MgSO4+7 H2O: 162; NaCl: 49.8; Fe(II) gluconate: 10.9; MnSO4+H2O: 3.12; ZnSO4+7 H2O: 4.67; CuSO4+5 H2O: 0.62; KI: 0.16; CoCl2+6 H2O: 0.08; ammonium molybdate: 0.06; NaSeO3 0.02 ‡Digestible protein calculated based on NRC (2011) for requirement and based on the results of this study (see Table 5) §Digestible energy calculated based on NRC (2011) for requirement and based on the results of this study (see Table 5) © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–11 Earthworm meal in feeds for common carp T N Ngoc et al (TiO2) was added at a level of 1% to each diet as a marker for determining digestibility according to the equation by Bureau, Harris and Cho (1999) Feeds were pelleted by means of a domestic meat grinder (Bosch Comfort plus; Robert Bosch GmbH, Gerlingen, Germany) fitted with a mm die The pellets were dried at 40°C for 36 h and kept in the refrigerator at 8°C Experimental design The trial was carried out for weeks in a recirculation system that consisted of 12 aquaria of 40 L each (four feeds replicates) Water flow through the aquaria was maintained at 6– L minÀ1 Water exchange rate in relation to feed provided accounted for 5.5 m3 kgÀ1 in the first week and 1.9 m3 kgÀ1 in the last week of the experiment Water parameters were maintained at optimal level for common carp (temperature at 25–27°C, dissolved oxygen close to saturation and pH around 7.0–8.0) The photoperiod was set to 12 h light: 12 h dark Five common carp (~8 g) were stocked randomly to each aquarium Carp were fed 16 g kgÀ0.8, five times maintenance requirement of 3.2 g kgÀ0.8 metabolic body mass (Becker, Eckhardt & Struck 1983), equivalent to 4.2% of body mass per day at the beginning and 3.3% of body mass per day at the end of the experiment, divided into five portions fed by means of automatic feeders at 8, 10, 12, 14 and 16 o’clock The pellets sank in the water and were taken up by the fish immediately so that leaching caused by different palatability of feeds can be excluded Fish growth was monitored weekly after 24 h of starvation Fish faeces were collected by siphoning in all aquaria twice a day (at 10 and 16 o’clock) in the last weeks of the trial The collection time was adjusted to be immediately after excretion and was based on observations To ensure that no feed residues remained within the faeces fraction, aquaria were cleaned after feeding 10–15 after cleaning, faeces was siphoned into a cylinder Settled faeces were transferred to test tubes for centrifugation at 4000 g for 10 The top water layer was poured off and the faeces was stored at À20°C before freeze-drying This principal was performed twice daily for weeks To have sufficient amounts for analysis, faeces was pooled over the three replicates and was analyzed in duplicates Aquaculture Research, 2015, 1–11 At the end of the trial, fish were sacrificed to determine final body weight, length of intestine, weight of liver and proximate composition of whole fish Analytical procedure Fish were anesthetized by means of MS222 and were sacrificed by heart puncture For analysis, fish were homogenized and freeze-dried Samples of fish carcasses, feeds, feed ingredients and fish faeces were analyzed for DM and CA according to AOAC (1990) Total nitrogen (TN) were determined using the Kjeldahl method (CP = TN 6.25) CL was determined by extraction with a Soxlet device, and GE with bomb calorimeter (IKA C 7000; Janke & Kunkel IKAAnalysentechnik, Germany) using a benzoic acid standard The amino acid contents of the feed ingredients and feeds were determined by the State Agency for Agricultural Chemistry at Hohenheim according to EU standard methods 98/64/EG and 2000/45/EG All amino acids, except tryptophan, were analyzed by an amino acid analyser (Biochrom 30 & 30+; Laborservice Onken, Greindau, Germany) Tryptophan was analyzed by high-performance liquid chromatography (HPLC) equipped with a fluorescence detector After Kjeldahl digestion, experimental feeds and fish faeces were treated with H2O2 and analyzed for TiO2 by spectrophotometric absorption of 405 nm wave length The quantity of TiO2 (lg mLÀ1 aliquot) was computed by using the following equation (Richter, L€ uckst€ adt, Focken & Becker 2003): TiO2 (lg mLÀ1 aliquot) = 108.1 Abs405 À 0.155 Based on these data, the following factors were calculated: Specific growth rate (SGR) ¼ 100 Â ððln final weight À ln initial weightÞ= days of trialị Condition factor (CF) ẳ fresh body weight [g]=body length [cm]3 ị 100 Hepato-somatic index (HSI) ẳ fresh weight of liver=fresh body weightị 100 â 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–11 Aquaculture Research, 2015, 1–11 Intestine-somatic index (ISI) ¼ Length of intestine=standard body length Feed conversion ratio (FCR) ¼ feed consumption (dry matter)= fish live weight gain Protein productive value (PPV) ¼ 100 Â ðððfinal fish body proteinÞÀ ðinitial fish body proteinÞÞ= ðtotal protein consumedịị Apparent net lipid utilization (ANLU) ẳ 100 ðððfinal fish body lipidÞÀ ðinitial fish body lipidÞÞ= ðtotal lipid consumedịị Apparent net gross energy utilization (ANEU) ẳ 100 ðððfinal fish body gross energyÞÀ ðinitial fish body gross energyÞÞ= ðtotal gross energy consumedÞÞ Apparent digestibility co-efficient of GE (E-ADC) ¼ 100Â ð1 À ðconc of TiO2 in diet=conc of TiO2 in faecesÞ Â ðGE in faeces=GE in dietÞÞ Apparent digestibility co-efficient of CL (L-ADC) ¼ 100Â ð1 À ðconc of TiO2 in diet=conc of TiO2 in faecesÞ Â ðconc of CL in faeces=conc of CL in dietÞÞ Apparent digestibility co-efficient of CP (P-ADC) ¼ 100Â ð1 À ðconc of TiO2 in diet=conc of TiO2 in faecesÞ Â ðconc of CP in faeces=conc of CP in dietÞÞ Statistics All data were tested for normal distribution and homogeneity before statistical analysis Results are presented as means Ỉ SD (standard deviation) Microsoft Excel and the program STATISTICA (version 6, StatSoftâ, Tulsa, OK, USA) were used for data analysis Analysis of Variance (ANOVA) and Tukey post-hoc tests were used to determine any significant differences (P ≤ 0.05) Digestibility data could not be statistically analyzed due to limited sample availability for replication © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–11 Earthworm meal in feeds for common carp T N Ngoc et al Results Feed acceptance, body condition and chemical composition All test diets sank quickly and were eaten very rapidly by the fish Therefore, the nutrient loss by leaching was minimal due to the short time the feed pellets remained in the water and all the feed offered could be assumed as feed intake No difference was observed in the acceptance of the test diets by the fish regardless of the level of earthworm inclusion No mortality occurred in the trial The experimental fish showed no abnormal activity at any time during the trial All the fish behaved normally and had similar and characteristic shape as well as body coloration The morphometric parameters CF, HSI as well as ISI did not show any significant differences (Table 3) Fish fed the control diet had the highest CF and HSI (3.41 Ỉ 0.13 and 2.08 Ỉ 0.19 respectively); fish fed EW70 had the lowest values of these parameters ISI was highest in fish on diet EW100 (6.73 Ỉ 0.65) without showing significant differences to other treatments The proximate carcass composition of common carp are shown in Table Fish in the control feeding group had the highest content of CL (33.7% of DM), followed by the feeding group EW70 (31.3% of DM) EW100 resulted in the lowest CL content in fish with 28.6% of DM The CP and CA of fish carcasses in the trial fluctuated from 58.6% to 61.5% and from 17.4% to 20.7% of DM respectively However, the differences in the chemical composition between fish from the different treatments was not statistically different (Table 4) Fish growth and feed utilization The fish showed a good growth by tripling their body mass within the weeks of the trial Fish growth and feed utilization showed differences between groups Growth of fish reared on diet EW70 had the best growth, significantly different from all other groups (Table 3) The SGR of the control group was lowest with 2.13% da1, fish on EW30 (2.15 Ỉ 0.03% da1) and EW100 (2.18 Ỉ 0.04% da1) showed slightly higher growth, and the SGR of fish on EW70 (2.29 Æ 0.01% dayÀ1) was significantly higher than that of the other three groups (Table 3) As Earthworm meal in feeds for common carp T N Ngoc et al Control IW [g] FW [g] SGR [%] CF HSI [%] ISI [%] 8.1 26.6 2.13 3.41 2.08 6.36 Ỉ Ỉ Ỉ Æ Æ Æ EW30 0.1 1.1b 0.06b 0.13 0.19 0.55 8.1 27.1 2.15 3.34 1.87 6.22 Ỉ Ỉ Ỉ Ỉ Æ Æ Aquaculture Research, 2015, 1–11 EW70 0.0 0.4b 0.03b 0.02 0.05 0.14 8.1 29.1 2.29 3.22 1.83 6.32 EW100 Æ Æ Æ Æ Æ Æ 0.2 0.3a 0.01a 0.13 0.01 0.11 8.0 27.2 2.18 3.19 1.93 6.73 Ỉ Ỉ Æ Æ Æ Æ 0.1 0.5b 0.04b 0.05 0.24 0.65 Table Growth performance factors and morphometric parameters (mean Ỉ standard deviation, n = 3) of groups of five common carp in the trial Values in the same row with different superscript are significantly different at P ≤ 0.05 CF, condition factor; FW, final weight; HSI, hepato-somatic index; ISI, intestine-somatic index; IW, initial weight; SGR, specific growth rate DM [% of FM] CP [% of FM] CL [% of FM] CA [% of FM] GE [MJ kgÀ1 FM] Initial Control 21.4 13.9 3.8 3.4 4.7 23.2 13.5 7.8 2.0 6.1 Æ Æ Æ Æ Æ EW30 0.3a 0.2 0.5 0.1 0.3a 22.7 13.6 7.0 2.2 5.8 Ỉ Ỉ Ỉ Ỉ Æ EW70 0.3a 0.5 0.4 0.2 0.1a 22.7 13.5 7.1 2.2 5.8 Ỉ Ỉ Ỉ Ỉ Ỉ EW100 0.2a 0.3 0.3 0.1 0.0a 21.5 13.1 6.1 2.2 5.3 Ỉ Ỉ Æ Æ Æ 0.3b 0.1 0.4 0.1 0.0b Table Proximate carcass composition (mean Ỉ standard deviation, n = 3) of common carp initial the trial and after feeding the different test diets Values in the same row with different superscript are significantly different at P ≤ 0.05 CA, crude ash; CL, crude lipid; CP, crude protein; DM, dry matter; FM, fresh matter; GE, gross energy Control FCR PPV [%] ANLU [%] ANEU [%] P-ADC [%]* L-ADC [%]* E-ADC [%]* 1.32 28.0 69.6 23.0 80.3 87.8 70.6 Ỉ Ỉ Ỉ Æ 0.08 1.2b 8.2a 2.6a,b EW30 1.26 28.6 60.2 21.0 81.5 85.9 72.6 Ỉ Ỉ Ỉ Ỉ EW70 0.05 1.4b 4.6ab 0.9b 1.22 31.8 64.7 22.5 85.3 83.4 72.7 Ỉ Æ Æ Æ EW100 0.02 1.4a 3.2ab 0.5a 1.27 27.5 50.5 18.0 85.7 75.2 70.5 Ỉ Ỉ Ỉ Ỉ 0.02 0.4b 4.9b 1.1c Table Utilizations of feed, crude protein, crude lipid, and gross energy (mean Ỉ standard deviation, n = 3) and apparent digestibility of crude protein, crude lipid and gross energy of the test diets in common carp Values in the same row with different superscript are significantly different at P ≤ 0.05 *Samples of the three groups per treatment were pooled and data could not be statistically analyzed due to limited sample availability ANEU, apparent net gross energy utilization; ANLU, apparent net lipid utilization; E-ADC, apparent digestibility co-efficient of gross energy; FCR, feed conversion ratio; L-ADC, apparent digestibility co-efficient of crude lipid; P-ADC, apparent digestibility co-efficient of crude protein; PPV, protein productive value was the case for growth, the fish fed EW70 also showed the lowest FCR and better results for PPV (Table 5) Fish fed EW70 showed the highest PPV with proximately 32%, which was significantly higher than that of fish groups fed the other diets (Table 5) However, there was no significant difference between the PPV of the control group, EW30 and EW100 Apparent net lipid utilization (ANLU) was highest in fish fed the control diet (69.6%), and slightly lower in the diets with earthworm inclusion There was no significant difference in ANLU within the earthworm-containing diets (EW30 to EW100), but the difference between control feed and EW100 was significant This group (EW100) also had the lowest energy retention (ANEU) with only 18.0 Ỉ 1.1%, significantly lower than of the other feeding groups Feed conversion ratios (FCRs) in this trial were relatively low The highest FCR was observed in the control group (1.32 Ỉ 0.08), followed by EW100 (1.27 Ỉ 0.02), EW30 (1.26 Ỉ 0.05) while that of EW70 was the lowest (1.22 Ỉ 0.02) There was no significant difference between the control feed group and test groups for this parameter (Table 5) © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–11 Aquaculture Research, 2015, 1–11 Digestibility In general, digestibility of all nutrients in all test diets was high The apparent digestibility of protein in the control feed was 80.3%, which was the lowest protein digestibility in the trial The protein digestibility of feed increased proportionally to the level of inclusion of earthworm meal Thus, feed EW100 had the highest apparent digestibility of CP (85.7%), followed by EW70 (85.3%) and EW30 (81.5%) In contrast, apparent digestibility of lipid was inversely proportional to the amount of earthworm meal in the feed (Table 5) Control feed had the highest lipid digestibility (87.8%), followed by EW30 (85.9%), EW70 (83.4%) and EW100 (75.2%) The lipid digestibility in fully earthworm-based feeds was lower than that of the other test feeds Energy digestibility was almost the same in all diets varying from 70.5% to 72.7% Discussion Experimental design The high content of animal-based protein in the diets (fishmeal and earthworm meal) is not typical in supplemental feeds for pond culture of common carp but in the context of this study, it was necessary to reduce the number of feed ingredients in order to avoid potential interactions between various feed ingredients for the determination of digestibility The protein content of 30% of DM in feeds was chosen to be less than the optimal level for common carp (38%; NRC 2011) and the content of essential of amino acids was chosen to not fully meet the requirements of common carp (NRC 2011) This type of feed was designed in order to be comparable in its nutritional composition to supplemental diets for semi-intensive pond culture in Vietnam (e.g Pucher, Mayrhofer, El-Matbouli & Focken 2014a,b; Tuan 2010) and those used in similar experiments (e.g Khan, Siddiqui & Nazir 1970; Jahan, Watanabe, Kiron & Satoh 2003) According to De Silva (1993) and Viola (1989), digestible energy is the first factor that is limiting fish growth in semi-intensive aquaculture In these systems, digestible energy needs to be supplied by supplemental feeds as high quality protein is available to the fish via natural food resources that are generally rich in proteins of high quality Subsequently, supplemental feeds not need to supply all required nutrients as it is necessary in full feeds © 2015 John Wiley & Sons Ltd, Aquaculture Research, 1–11 Earthworm meal in feeds for common carp T N Ngoc et al for intensive aquaculture Under pond conditions, where natural food is available to the fish, higher growth rates of fish can be expected Feeding trials with similar feeds including natural food resources were conducted after the here described laboratory trial (see Pucher, Ngoc, et al 2014) Nutritional quality of earthworm In our study, the earthworm P excavatus had a higher protein content than that of conventional fishmeal (Hertrampf & Piedad-Pascual 2000) and that of the fishmeal been used in this trial Further, the protein content of P excavatus was higher than in most other earthworm species that were evaluated by Tacon et al (1983), Tacon and Jackson (1985), Hilton (1983), and Stafford and Tacon (1984) Similarly, the content of essential amino acids in P excavates was comparable to or higher than that of conventional fishmeal (Hertrampf & Piedad-Pascual 2000) and that of the fishmeal used in this trial The essential amino acid composition of earthworm differs from species to species (Dynes 2003), culture environment, and substrate quality (Xiang, Zhang, Pan, Qiu & Chu 2006) Compared to the amino acid profile of the earthworm species Lumbricus rubellus, L terrestris, Nicodrilus roseus, N caliginosus, Dendrobawna octaedra, Eisenia nordenskioldi, Octolasium lacteum, Drawida ghilarovi and Hyperiodrilus euryaulos, which were investigated by Pokarzhevskii, Zaboyev, Ganin and Gordienko (1997) and Sogbesan, Ugwumba and Madu (2007), P excavatus showed a profile of essential amino acids that was one of the better profiles fitting to the requirements of common carp Especially, P excavatus showed higher concentration of methionine and cystine (3.1% of CP) than most other earthworm species (