On the Automorphism Groups of Models in ℂ2

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Earthworm powder as an alternative protein source

Tuan Nguyen Ngoc1,2, Johannes Pucher1, Klaus Becker1& Ulfert Focken1,3

1 Department of Aquaculture Systems in the Tropics and Subtropics (480b), Universitaet Hohenheim, Stuttgart, Germany

2 Faculty of Fisheries, Vietnam National University of Agriculture, Hanoi, Vietnam

3 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

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 tradi-tional 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; Hernandez, Olvera-Novoa, Hardy, Hermosillo, Reyes & Gonz Alez 2010) Apart from the feed resources produced on a large scale and traded internationally, a number of highly nutritive feed resources have been sug-gested 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), house-fly maggots (Ogunji, Kloas, Wirth, Neumann & Pietsch 2008), snails and termites (Phonekhamph-eng, Hung & Lindberg 2008) and various earth-worm species (e.g Tacon, Stafford & Edwards 1983; Nandeesha, Srikanth, Basavaraja, Keshav-anath, Varghese, Bano, Ray & Kale 1988; Paripu-ranam, Divya, Ulaganathan, Balamurugan & Umamaheswari 2011)

The production of earthworm (vermiculture) can be performed either on a small scale (Cha-krabarty, 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)

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

differ-ent 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

foet-ida, 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

sug-gested 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

com-mon carp, Cyprinus carpio Dong, Guo, Ye, Song,

Huang and Wang (2010) studied the digestibility

of various unconventional protein sources in

tila-pia, including freeze-dried earthworm meal and

reported a dry matter digestibility of 98.9

4.65% but did not state which species of

earth-worm were used

Pucher, Ngoc, Yen, Mayrhofer, El-Matboulic and

Focken (2014) showed that the tropical

earth-worm Perionyx excavatus can fully replace fishmeal

in supplemental feeds for common carp under

semi-intensive production conditions with fish

hav-ing 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

con-trolled 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

nutri-tional quality of P excavatus as a feed ingredient

in diets that are used as supplement feed in

tropi-cal pond aquaculture of common carp

Materials and methods Experimental feeds

Earthworm P excavatus was purchased from a small-scale commercial vermiculture facility in suburban Hanoi (Viet Nam) that produces earth-worm 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 Univer-sity of Hohenheim (Germany) Fishmeal as feed ingredient was purchased from a large feed retailer

in Germany (Vereinigte Fischmehlwerke Cuxha-ven, Germany) and wheat flour was purchased from the supermarket (Germany) Chemical com-position and amino acid comcom-position of these three feed ingredients are shown in Table 1 The earth-worm 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 fish-meal 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 com-pared to fishmeal Based on the analyzed chemical and amino acid composition of feed ingredients (Table 1), four iso-nitrogenous and iso-lipidic sup-plemental 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 1 Chemical composition and amino acid composi-tion of dry feed ingredients of the test diets

Feed Earthworm Fish meal Wheat meal

CP [% of DM] 71.3 67.4 15.5

GE [MJ kg1DM] 21.4 21.6 19.3 Threonine [% of CP] 4.5 3.4 2.4 Valine [% of CP] 7.0 4.3 1.9 Cys + Met [% of CP] 3.1 3.0 4.6 Isoleucine [% of CP] 4.2 3.4 2.7 Leucine [% of CP] 7.4 6.2 5.9 Phe + Tyr [% of CP] 7.1 5.4 6.3 Histidine[% of CP] 2.4 2.6 2.2 Lysine [% of CP] 6.6 6.1 2.1 Arginine [% of CP] 6.0 5.2 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.

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

pro-tein 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

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 propor-tion of earthworm increased Titanium dioxide

Table 2 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)

Ingredient composition of diets Fishmeal [% of diet DM] 30.3 21.1 9.0 –

Wheat meal [% of diet DM] 61.7 61.6 61.7 61.7 Sunflower oil [% of diet DM] 5.0 5.5 6.2 6.7

DP [% of DM] 32.0 ‡ 22.3 ‡ 22.6 ‡ 23.5 ‡ 23.7 ‡

DE [% of DM] 13.4 § 13.7 § 14.1 § 14.1 § 13.7 § Essential amino acid composition Threonine [% of CP] 3.9 3.2 3.6 4.0 4.0

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; TiO 2 , Titanium dioxide.

*Vitamin premix: retinol palmitate: 500 000 IU kg1; thiamine: 5 g kg1; riboflavin: 5 g kg1; niacin: 25 g kg1; folic acid:

1 g kg1; pyridoxine: 5 g kg1; cyanocobalamine: 5 g kg1; ascorbic acid: 10 g kg1; cholecalciferol: 50 000 IU kg1; a-tocoph-erol: 2.5 g kg1; menadione: 2 g kg1; inositol: 25 g kg1; pantothenic acid: 10 g kg1; choline chloride: 100 g kg1; biotin: 0.25 g kg1.

†Mineral premix (g/k): CaCO 3 : 336; KH 2 PO 4 : 502; MgSO 4 +7 H 2 O: 162; NaCl: 49.8; Fe(II) gluconate: 10.9; MnSO 4 +H 2 O: 3.12; ZnSO 4 +7 H 2 O: 4.67; CuSO 4 +5 H 2 O: 0.62; KI: 0.16; CoC l2 +6 H 2 O: 0.08; ammonium molybdate: 0.06; NaSeO 3 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).

(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 1 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 8 weeks in a

recircu-lation system that consisted of 12 aquaria of 40 L

each (four feeds9 3 replicates) Water flow

through the aquaria was maintained at 6–

7 L min1 Water exchange rate in relation to

feed provided accounted for 5.5 m3kg1 in the

first week and 1.9 m3kg1in 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

ran-domly to each aquarium Carp were fed

16 g kg0.8, five times maintenance requirement

of 3.2 g kg0.8 metabolic body mass (Becker,

Eck-hardt & 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

feed-ers 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 2 weeks of the trial The collection time was

adjusted to be immediately after excretion and was

based on observations To ensure that no feed

resi-dues remained within the faeces fraction, aquaria

were cleaned after feeding 10–15 min after

clean-ing, faeces was siphoned into a cylinder Settled

faeces were transferred to test tubes for

centrifuga-tion at 4000 g for 10 min The top water layer

was poured off and the faeces was stored at

20°C before freeze-drying This principal was

per-formed twice daily for 2 weeks To have sufficient

amounts for analysis, faeces was pooled over the

three replicates and was analyzed in duplicates

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 9 6.25) CL was determined by extrac-tion with a Soxlet device, and GE with bomb calorimeter (IKA C 7000; Janke & Kunkel IKA-Analysentechnik, 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 chromatog-raphy (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 mL1aliquot) was computed by using the fol-lowing equation (Richter, L€uckst€adt, Focken & Becker 2003): TiO2 (lg mL1 aliquot)= 108.1 9 Abs405 0.155

Based on these data, the following factors were calculated:

Specific growth rate (SGR)

¼ 100  ððln final weight  ln initial weightÞ= days of trialÞ

Condition factor (CF)

¼ ðfresh body weight [g]=body length [cm]3Þ

 100 Hepato-somatic index (HSI)

¼ ðfresh weight of liver=fresh body weightÞ

 100

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  ðððfinal fish body proteinÞ

ðinitial fish body proteinÞÞ=

ðtotal protein consumedÞÞ

Apparent net lipid utilization (ANLU)

¼ 100  ðððfinal fish body lipidÞ

ðinitial fish body lipidÞÞ=

ðtotal lipid consumedÞÞ

Apparent net gross energy utilization (ANEU)

¼ 100  ðððfinal fish body gross energyÞ

ðinitial fish body gross energyÞÞ=

ðtotal gross energy consumedÞÞ

Apparent digestibility co-efficient of GE (E-ADC)

¼ 100

ð1  ðconc of TiO2in diet=conc of TiO2in faecesÞ

 ðGE in faeces=GE in dietÞÞ

Apparent digestibility co-efficient of CL (L-ADC)

¼ 100

ð1  ðconc of TiO2in diet=conc of TiO2in faecesÞ

 ðconc of CL in faeces=conc of CL in dietÞÞ

Apparent digestibility co-efficient of CP (P-ADC)

¼ 100

ð1  ðconc of TiO2in diet=conc of TiO2in faecesÞ

 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
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

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 differ-ence was observed in the acceptance of the test diets by the fish regardless of the level of earth-worm inclusion No mortality occurred in the trial The experimental fish showed no abnormal activ-ity at any time during the trial All the fish behaved normally and had similar and character-istic shape as well as body coloration The mor-phometric 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
0.13 and 2.08
0.19 respectively); fish fed EW70 had the lowest values of these parame-ters ISI was highest in fish on diet EW100 (6.73
0.65) without showing significant differ-ences to other treatments

The proximate carcass composition of common carp are shown in Table 4 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 low-est 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 differ-ent 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 8 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% day1, fish

on EW30 (2.15
0.03% day1) and EW100 (2.18
0.04% day1) showed slightly higher

(2.29
0.01% day1) was significantly higher than that of the other three groups (Table 3) As

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

differ-ence between the PPV of the control group, EW30

and EW100

Apparent net lipid utilization (ANLU) was

high-est 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
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
0.08), followed by EW100 (1.27
0.02), EW30 (1.26
0.05) while that of EW70 was the lowest (1.22
0.02) There was no significant difference between the control feed group and test groups for this parame-ter (Table 5)

Initial Control EW30 EW70 EW100

DM [% of FM] 21.4 23.2
0.3 a 22.7
0.3 a 22.7
0.2 a 21.5
0.3 b

CP [% of FM] 13.9 13.5
0.2 13.6
0.5 13.5
0.3 13.1
0.1

CL [% of FM] 3.8 7.8
0.5 7.0
0.4 7.1
0.3 6.1
0.4

CA [% of FM] 3.4 2.0
0.1 2.2
0.2 2.2
0.1 2.2
0.1

GE [MJ kg1FM] 4.7 6.1
0.3 a 5.8
0.1 a 5.8
0.0 a 5.3
0.0 b

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.

Table 4 Proximate carcass compo-sition (mean
standard deviation,

n = 3) of common carp initial the trial and after feeding the different test diets

IW [g] 8.1
0.1 8.1
0.0 8.1
0.2 8.0
0.1

FW [g] 26.6
1.1 b 27.1
0.4 b 29.1
0.3 a 27.2
0.5 b

SGR [%] 2.13
0.06 b 2.15
0.03 b 2.29
0.01 a 2.18
0.04 b

CF 3.41
0.13 3.34
0.02 3.22
0.13 3.19
0.05

HSI [%] 2.08
0.19 1.87
0.05 1.83
0.01 1.93
0.24

ISI [%] 6.36
0.55 6.22
0.14 6.32
0.11 6.73
0.65

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.

Table 3 Growth performance fac-tors and morphometric parameters (mean
standard deviation, n = 3)

of groups of five common carp in the trial

FCR 1.32
0.08 1.26
0.05 1.22
0.02 1.27
0.02

PPV [%] 28.0
1.2 b

28.6
1.4 b

31.8
1.4 a

27.5
0.4 b ANLU [%] 69.6
8.2 a 60.2
4.6 ab 64.7
3.2 ab 50.5
4.9 b

ANEU [%] 23.0
2.6 a,b 21.0
0.9 b 22.5
0.5 a 18.0
1.1 c

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

statisti-cally 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.

Table 5 Utilizations of feed, crude protein, crude lipid, and gross energy (mean
standard deviation,

n = 3) and apparent digestibility of crude protein, crude lipid and gross energy of the test diets in common carp

In general, digestibility of all nutrients in all test

diets was high The apparent digestibility of

pro-tein 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%),

fol-lowed 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

neces-sary to reduce the number of feed ingredients in

order to avoid potential interactions between

vari-ous 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

con-tent 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

avail-able to the fish via natural food resources that are

generally rich in proteins of high quality

Subse-quently, supplemental feeds do not need to supply

all required nutrients as it is necessary in full feeds

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 Fur-ther, 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 (Her-trampf & 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 octae-dra, Eisenia nordenskioldi, Octolasium lacteum, Draw-ida 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 (<2% of CP: Pokarzhevskii et al 1997) These sul-phur-containing amino acids are usually among the most limiting amino acids in aquafeeds We found in our study that P excavatus seems to be a suitable source of essential amino acids as an increasing portion of protein from earthworm resulted in a higher content of almost all essential amino acids in the diets

Perionyx excavatus had a lower lipid content than fishmeal So far, not many publications are available on lipid composition of earthworms, especially no one for P excavatus Paoletti, Bus-cardo, VanderJagt, Pastuszyn, Pizzoferrato, Huang, Chuang, Millson, Cerda, Torres and Glew (2003)

reported that two earthworm species (Andiorrhinus

kuru, Andiorrhinu motto) appeared to contain

insuf-ficient amounts of triacyglycerols which are

impor-tant as energy stores in cells Holmstrup,

Sørensen, Bindesbøl and Hedlund (2007) showed

that the earthworm Dendrobaena octaedra contained

high amounts of long-chain unsaturated fatty

acids (20:n and 18:n) Thus, the composition of

fatty acids in earthworms seems to be complex

and differs from species to species However, the

quality of lipids in earthworm meal should be

con-sidered more in detail in future research as the

results in this study suggest an interaction of

earthworm-protein and lipids in their effect on

growth, digestibility and utilization efficiency

Digestibility

In our study, increased levels of earthworm in the

diets led to proportional reductions of lipid

digest-ibility and CL content in fish Apparent net lipid

utilization (ANLU) showed that full replacement

achieved a significantly lower lipid utilization and

the lowest CL content in fish carcass This might

be caused by the increasing substitution of fish

lipid originating from fishmeal through sunflower

oil in feeds with increasing earthworm meal

con-tent Therefore, substitution by fish oil rather than

by sunflower oil might improve fish growth as

dis-cussed previously (Nandeesha et al 1988) In

future studies, an analysis of the fatty acids in

earthworm P excavatus might give more detailed

insight into the suitability of earthworm meal not

only as an animal protein source but also as a

source of essential fatty acids for fish The

appar-ent digestibility of energy (E-ADC) in groups fed by

the control and EW100 were lowest while the

energy digestibility was highest in the EW70

group Contrarily, the apparent net gross energy

utilization (ANEU) was highest in groups fed the

control and EW70 diet Significantly lowest ANEU

was detected in the EW100 group This pattern

might suggest changes in the utilization of lipids

and proteins in feeds as energy sources for fish

The digestibility of protein increased

proportion-ally with increasing earthworm meal content in

feeds But due to large variation between the

duplicated analyses, any difference in digestibility

could be detected Higher replication might be

ben-eficial to show the significant difference in

earth-worm protein digestibility Even though the

apparent protein digestibility was highest in

EW100, the digested protein contributed to a lower content to the body mass gain as the PPV dropped significantly in the EW100 group com-pared to the EW70 which was the group with highest PPV The PPV in the control group were similar to the EW30 and EW100 groups

Growth performance The increase in earthworm content in feeds improved proportionally the growth performance and feed conversion up to the replacement of 70% fishmeal-protein by earthworm-protein Groups fed

by EW70 showed the highest growth rates (SGR of 2.3% day1) and lowest FCRs (1.22) of all tested feeds The full replacement of fishmeal by earth-worm meal resulted in a drop in growth and feed utilization, even though the test diet EW100 had a higher content of essential amino acids and the protein digestibility of earthworm meal was found

to be highest in the EW100 feed This indicates that the lower growth rate of fish fed EW100 was not directly linked to the quality of the earthworm protein but may have been caused by other factors There seems to be a synergetic effect of combining earthworm and fishmeal or other animal derived proteins sources (e.g natural food resources as shown by Pucher, Ngoc, et al 2014) Earlier stud-ies (Tacon et al 1983; Stafford & Tacon 1984; Nandeesha et al 1988) also reported that total replacement of fishmeal by earthworm meal did not result in the highest growth even though earthworm has a better nutritive profile In the trial by Nandeesha et al (1988), the best feed was the one containing 25% earthworm However, this result could be strongly influenced by the addition

of 5% of sardine oil in the diet as Espe, Lemme, Petri and El-Mowafi (2006) implied that a few per cent of fish oil in the diet could improve palatability

of feeds and lead to higher feed intake and fish growth In our study, fish grew best with 70% of fishmeal-CP replacement This level is equivalent to 20% of DM and agrees with earlier studies (Hilton 1983; Tacon et al 1983; Nandeesha et al 1988) Tacon et al (1983) claimed that the best fish growth could be achieved with lower than 30% fishmeal replacement by earthworm

Hilton (1983) stated that diets containing earth-worm meal reduced feed intake and growth Sev-eral suggestions have been made by researchers to explain why earthworm meal based diets reduce growth rates Tacon et al (1983), Andrews and

Kukulinsky (1975), and Edwards and Lofty (1977)

reported that the coelomic fluid, a yellow fluid in

earthworms, could make feeds unpalatable when

they contained large amounts of earthworm and

could thus decrease feed intake Coelomic fluid

contains specific proteins that affect the immune

system of other animals (Kauschke, Mohrig &

Coo-per 2007) ProCoo-per pre-treatment (e.g heat

treat-ment) of earthworm before using them in fish

diets might improve the palatability of earthworm

meal (Tacon et al 1983; Pucher, Ngoc, et al

2014) In the study of Nandeesha et al (1988),

earthworm was cooked before pelletization

Never-theless, total fishmeal replacement by heat-treated

earthworm did not achieve higher growth than

the feed with 25% earthworm inclusion in the

study by Nandeesha et al (1988) This indicates

that the heat treatment of earthworm by

Nandee-sha et al (1988) did not destroy the

anti-nutri-tional factor Further research is needed to

investigate the existence of potential

anti-nutri-tional factors in certain earthworm species and the

proper pre-treatment to reduce the anti-nutritional

property

There is the possibility, that earthworms been

used in the research trials might be cultured on

waste materials that contain heavy metals,

deter-gents and other undesirable compounds Some

researchers reported accumulation of chemicals

such as fluoride in earthworm living in polluted

soils (Vogel & Ottow 1991) or iron, zinc, lead, and

cadmium (Stafford & Tacon 1984; Paoletti et al

2003; Khwairakpam & Bhargava 2009) Stafford

and Tacon (1984) pointed out that these materials

can contaminate the diets and impair fish

perfor-mance at high levels of earthworm incorporation

Consequently, vermiculture must be well managed

by assuring a high quality of feeds and

environ-ment if earthworm is to be used in animal feeds

In summary, common carp have shown very

good growth response to feeds containing

earth-worm meal as the main protein source For

practi-cal purposes, the use of earthworm meal in mainly

plant-based feeds also needs to be evaluated If

found to be suitable for these feeds, too,

earth-worm meal can become a source of animal protein

for farm-made as well as commercial aquafeeds

Acknowledgments

This study was partly funded by the Deutsche

Forschungsgemeinschaft (DFG) and was carried

out under the umbrella of the Uplands Program (SFB 564) in close collaboration between the Uni-versity of Hohenheim (Germany) and the Hanoi University of Agriculture (Vietnam) A scholarship awarded by the Deutsche Akademische Auslandsd-ienst (DAAD) to Tuan NN is gratefully acknowl-edged Special thanks go to Dr Lawrence for the language editing of this article and to the review-ers for their constructive comments

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