A comparative study on the use of Pennisetumclandestinum and Moringa oleifera as protein sourcesin the diet of the herbivorous Tilapia rendalli

S. N. Hlophe • N. A. G. Moyo

Received: 28 August 2013 / Accepted: 22 December 2013� Springer Science+Business Media Dordrecht 2014

Abstract The efficacy of kikuyu grass and moringa leaves as protein sources in Tilapia

rendalli (11.5 ± 1 g) diets was compared. Nine diets (30 % CP 20 MJ kg-1) were for-

mulated by substituting fishmeal (FM) for kikuyu leaf meal (KLM) and moringa leaf meal

(MLM). A control diet contained 10 % FM and no leaf meal. FM was replaced at 25, 50,

75 and 100 % by KLM in diets: KLM 25, KLM 50, KLM 75 and KLM 100, and then by

MLM in diets: MLM 25, MLM 50, MLM 75 and MLM 100, respectively. Maize and

gluten meals were adjusted accordingly. Each diet was fed to triplicate groups of fish for

60 days. The best growth (SGR and TGC) was in the control group. There was no sig-

nificant (P [ 0.05) decrease in SGR and TGC when KLM replaced up to 50 % FM. There

was a significant (P \ 0.05) decrease when MLM replaced [25 % FM. Kikuyu diets had

no effect on villi height. A trend towards shorter villi was evident with increasing MLM.

Hepatocyte degradation was higher in fish fed moringa-based diets. Anti-nutrients (tannins

and polyphenols) in moringa may have contributed to the poor growth, irritation of the

enterocytes and hepatotoxic effects. These results show that replacing up to 25 % FM with

KLM is effective in reducing the costs without negatively impacting the growth perfor-

mance or health of T. rendalli. Adding MLM even at the lowest level (25 %) was

expensive and resulted in compromised growth and health.

Keywords Digestibility � Growth � Histology � Intestine � Liver

Introduction

Aquaculture is the fastest-growing food-producing sector. For this sector to maintain its

current average growth rate of 8–10 % per year (Tacon et al. 2011), the supply of feed

S. N. Hlophe (&) � N. A. G. MoyoAquaculture Research Unit, Faculty of Science and Agriculture, School of Agricultural andEnvironmental Sciences, University of Limpopo, Turfloop Campus, Private Bag X1106,Sovenga 0727, South Africae-mail: hlophesam@gmail.com

123

Aquacult IntDOI 10.1007/s10499-013-9744-4

inputs (particularly protein source) must grow at a similar rate. However, production of

fishmeal (FM) is decreasing at an average rate of 1.7 % per year (Tacon et al. 2011). This

in turn increases the price of FM, making its inclusion in aqua feeds unsustainable. Thus,

for the aquaculture industry to continue to develop, sustainable substitutes to FM are a

prerequisite.

Accordingly, researchers have focused on the use of cheap, locally available plant

protein sources. An appreciable amount of work has been done on the use of different leaf

meals as inexpensive sources of protein in fish feeds [cassava (Ng and Wee 1989); sweet

potato (Adewolu 2008); alfalfa (Yousif et al. 1994); moringa (Richter et al. 2003); Leu-

caena leucocephala (Bairagi et al. 2004)]. There is need to continually search for new

sources of protein in fish diets because competition from other sectors has led to the

commercialisation of some of the widely used leaf meals (moringa, cassava).

Replacement of FM in fish diets without reducing the growth performance would result

in more profitable aquaculture enterprises. In this study, the potential for kikuyu grass

(Pennisetum clandestinum) as a protein source in tilapia diets is compared with that of

moringa (Moringa oleifera) leaves. Kikuyu grass is a course-textured grass with a rapid

growth rate, and it is commonly used as forage, lawn or turf (Marais et al. 1987). This grass

has relatively high protein content and a good amino acid profile (Marais 2001; Flukerson

et al. 2007). The amino acid profile in KLM (Hlophe and Moyo 2011) is comparable to the

amino acid requirement in tilapia (Jauncey et al. 1983). There is very little work done on

the potential of this plant as a source of protein in fish diets. On the contrary, an appre-

ciable amount of work has been done on the use of moringa leaf meal as a replacement for

FM (Madalla 2008; Afuang et al. 2003; Richter et al. 2003). Moringa is also known as the

miracle tree. It has been used traditionally to cure a number of diseases (Foidl et al. 2001).

The leaves have high protein content and are rich in carotenoids, ascorbic acid and iron

(Makkar and Becker 1997). There has been an upsurge of interest in moringa in recent

years in South Africa. The government has encouraged farmers to grow moringa trees in a

drive to decrease malnutrition in many rural areas of South Africa.

In this study, the efficacy of leaf meals from the two plants as protein sources in the diet

of a herbivorous fish Tilapia rendalli is explored. T. rendalli is a commonly cultured fish in

South Africa. It is preadapted to utilising plant-based diets and is widely known for its

voracious appetite for macrophytes and ability to change its diet to whatever resources are

available (Meschiatti and Arcifa 2002; Weliange et al. 2006). The negative effects caused

by plant-based practical diets on the digestive morphology and growth performance have

been extensively studied in carnivorous fish (Collins et al. 2012; Caballero et al. 2003;

Krogdahl et al. 2000). However, most of the research on FM replacement in the diets of

herbivorous fish has focussed solely on growth performance. The adverse effects of plant-

based diets on the digestive physiology have been overlooked. The aim of this study was to

investigate the effects of different dietary inclusion levels of kikuyu and moringa leaf

meals on the growth performance, digestive morphology (liver and intestine histology) and

haematological parameters in Tilapia rendalli.

Materials and methods

Fish and feed preparation

A group of 405 Tilapia rendalli fingerlings (12 ± 1.0 g) obtained from Flag Boshielo

Dam, South Africa, were transferred to the University of Limpopo Aquaculture Research

Aquacult Int

123

Unit, South Africa, and kept in 200-L tanks to be acclimatised for 1 month. The fish were

fed at maintenance requirement with a standard commercial tilapia diet containing 34 %

protein, 3 % lipid and a gross energy content of 17 MJ kg-1 dry matter.

Fresh kikuyu grass (lawn) was harvested from the Aquaculture Research Unit,

University of Limpopo. Moringa leaves were purchased from the Patient Wellness

Centre, Lebowakgomo, Limpopo Province, South Africa. Kikuyu grass and moringa

leaves were dried under a shade and milled using a hammer mill. The proximate and

amino acid composition of kikuyu leaf meal (KLM), moringa leaf meal (MLM) and

fishmeal used is given in Table 1. Nine isonitrogenous (crude protein 30 %) and iso-

energetic (gross energy 20 MJ kg-1) diets were formulated by substituting fishmeal for

KLM and MLM. Ingredients were purchased from a local feed manufacturer (Wille

Pille, Mokopane, South Africa). The control diet did not contain any plant leaf meal, but

10 % FM (Table 2). In the KLM diets, FM was substituted at 25, 50, 75 and 100 %

with KLM in diets designated as KLM 25, KLM 50, KLM 75 and KLM 100,

respectively. Similarly, in diets designated as MLM 25, MLM 50, MLM 75 and MLM

100, fishmeal was replaced with MLM at 25, 50, 75 and 100 %, respectively. Maize

meal and maize gluten meal were adjusted to regulate protein and energy levels.

Chromic oxide (Cr2O3) was added in each diet (0.5 %) as an inert marker. The diets

were formulated using the Winfeed 3, EFG Software (Natal).

Table 1 Proximate and amino acid composition of kikuyu, moringa leaf meals and fishmeal used

Components KLM MLM Fishmeal Dietary amino acidrequirements for tilapia

Dry matter (%) 93.63 92.00 90.50

Protein (%) 26.84 32.09 64.80

Energy (MJ kg-1) 17.99 18.30 12.90

Ash (%) 9.50 7.17 21.10

Fat (%) 4.38 1.99 5.60

Fibre (%) 18.58 10.10 0.00

Methionine (%) 0.45 0.33 2.95 0.4

Arginine (%) 1.44 1.20 5.82 1.13

Tyrosine (%) 0.80 0.99 3.4 1.79

Histidine (%) 0.51 0.58 2.43 0.42

Threonine (%) 0.93 0.80 4.31 1.17

Isoleucine (%) 1.12 0.87 4.68 0.80

Leucine (%) 2.33 2.05 7.60 1.35

Phenylalanine (%) 1.34 1.39 4.21 1.00

Lysine (%) 1.42 1.10 7.57 1.51

Valine (%) 1.56 1.10 5.25 0.88

Cysteine (%) 0.43 0.25 0.91 0.53

Polyphenols (%) 0.64 1.34 –

Tannins (%) 0.54 0.58 –

Amino acid requirements for tilapia adopted from Jauncey et al. (1983)

Aquacult Int

123

Ta

ble

2In

gre

die

nts

(%)

and

pro

xim

ate

com

po

siti

on

and

app

aren

td

iges

tib

ilit

yco

effi

cien

tfo

rp

rote

ino

fex

per

imen

tal

die

ts

FM

repla

ced

(%)

Co

ntr

ol

KL

M2

5K

LM

50

KL

M7

5K

LM

10

0M

LM

25

ML

M5

0M

LM

75

ML

M1

00

02

55

07

51

00

25

50

75

10

0

KL

M–

7.5

01

5.0

02

2.5

03

0.0

0–

––

–

ML

M–

––

––

7.2

71

4.7

22

1.7

02

9.0

0

Fis

hm

eal

10

.62

7.7

35

.25

2.7

7–

8.0

05

.31

2.7

00

.00

So

yb

ean

mea

l7

.32

7.3

27

.32

7.3

27

.32

7.0

07

.00

7.0

07

.00

Can

ola

mea

l1

8.0

01

8.0

01

8.0

01

8.0

01

8.0

01

8.7

11

8.7

11

8.7

11

8.7

1

Su

nfl

ow

erm

eal

16

.80

16

.80

16

.80

16

.80

16

.80

16

.80

16

.80

16

.80

16

.80

Mai

zeg

lute

n1

1.6

01

2.0

01

2.0

01

2.0

01

2.0

01

1.6

01

1.6

01

1.6

01

1.6

0

Wh

eat

mid

dli

ng

s2

.17

2.1

72

.17

2.1

72

.17

5.0

05

.00

5.0

05

.00

Mai

zem

eal

28

.56

23

.45

18

.37

13

.30

8.2

51

8.0

81

3.2

58

.73

4.3

9

Can

ola

oil

1.0

01

.00

1.0

01

.00

1.0

01

.00

1.0

01

.00

1.0

0

Min

eral

pre

mix

a1

.00

1.0

01

.00

1.0

01

.00

1.0

01

.00

1.0

01

.00

Vit

amin

pre

mix

b1

.00

1.0

01

.00

1.0

01

.00

1.0

01

.00

1.0

01

.00

Bin

der

2.0

02

.00

2.0

02

.00

2.0

04

.00

4.0

04

.00

4.0

0

Ch

rom

ico

xid

e0

.50

0.5

00

.50

0.5

00

.50

0.5

00

.50

0.5

00

.50

Dry

mat

ter

(%)

94

.69

92

.40

92

.32

91

.32

91

.40

94

.65

95

.22

95

.47

94

.84

Cru

de

pro

tein

(%)

30

.03

29

.87

29

.96

29

.71

30

.07

29

.84

29

.98

30

.48

30

.38

En

erg

y(M

Jk

g-

1)

20

.21

20

.34

20

.48

20

.53

20

.47

20

.58

20

.42

20

.26

20

.36

Fat

(%)

3.9

44

.65

4.5

24

.31

3.9

33

.60

3.7

83

.84

3.3

5

Cru

de

fib

re(%

)7

.04

8.0

78

.88

10

.31

12

.91

7.0

37

.59

7.8

88

.54

Pro

tein

AD

C(%

)8

2.1

80

.97

9.6

77

.17

6.0

79

.07

6.5

74

.17

3.2

aM

iner

alp

rem

ix(g

kg

-1):

KH

2P

O4

,5

02

;M

gS

O4.

7H

2O

,1

62

;N

aCl,

49

.8;

CaC

O3,

33

6;

Fe(

II)

glu

con

ate,

10

.9;

Mn

SO

4.

H2O

,3

.12;

Zn

SO

4.

7H

2O

,4

.67

;Cu

SO

4.

5H

2O

,0

.62

;K

I,0

.16;

Co

Cl 2

.6

H2O

,0

.08;

amm

oniu

mm

oly

bd

ate,

0.0

6;

NaS

eO3,

0.0

2b

Vit

amin

pre

mix

(go

rIU

kg

-p

rem

ix);

thia

min

e,5

;ri

bo

flav

in,

5;

nia

cin

,2

5;

foli

cac

id,

reti

no

lp

alm

itat

e,5

00

,00

0IU

;1;

py

rid

ox

ine,

5;

cyan

oco

bal

amin

,5;

chole

calc

ifer

ol;

50

,00

0IU

;a-

toco

ph

ero

l,2

.5;

men

adio

ne,

2;

inosi

tol,

25

;p

anto

then

icac

id,

10

;as

corb

icac

id,

10

;ch

oli

ne

chlo

rid

e,1

00

;b

ioti

n,

0.2

5

Aquacult Int

123

Experimental design

A completely randomised design was used in this experiment. Twenty-seven rectangular

(1.5 m3) fibreglass tanks in a greenhouse were used. Aged water was filled up to the 1 m3

mark in each tank (experimental unit). The tanks were connected to a recirculating system.

An Elektror side channel blower Model: D7300 (Karl W. Miller) and an air stone were

used to blow air in each tank. Water quality parameters were measured daily, water

temperature ranged between 25 and 28 �C, the amount of dissolved oxygen was

6.4 ± 0.5 mg L-1, pH ranged from 7 to 8.2, and photoperiod was natural.

Diet allocation and feeding

Each diet was randomly allocated to triplicate groups of T. rendalli (12.00 ± 1 g) stocked

at 15 fish per tank. All fish were hand-fed their allocated diet to apparent satiation (until

one or two pellets remain uneaten for 2 min). Feed consumed was recorded daily. Feed

was offered three times daily at 0900, 1300 and 1700 h for 60 days. Faecal samples were

siphoned from each tank three times per day (2 h after each feeding). Faecal samples from

the same dietary treatments were pooled (excess water drained) and stored in a freezer

(-20 �C) until enough samples were collected for analysis. At the end of the experiment,

fish were weighed individually for the determination of growth performance. Blood

samples were drawn through a caudal venous puncture from five fish per tank (15 fish per

treatment) for haematological analysis. The same fish were killed for histological analysis

of intestines and the liver.

Proximate composition of experimental diets and faeces

All the diets and ingredients (Table 2) were analysed for dry matter, crude protein, crude

lipid, crude fibre and gross energy following the procedures stipulated by the Association

of Official Analytical Chemists (AOAC International 2012). Dry matter was determined by

drying each sample at 105 �C for 24 h. A LECO FP2000 Nitrogen Analyser was used to

determine the nitrogen content of the dry matter using the Dumas combustion method.

Protein content was calculated as percentage nitrogen 9 6.25. Lipid content was assessed

by Soxhlet extraction of dried samples with petroleum ether at 50 �C. Gross energy was

determined using a DDS isothermal CP 500 bomb calorimeter. Crude fibre was determined

as loss on ignition of dried lipid-free residues after digestion with 1.25 % H2SO4 and

1.25 % NaOH. To estimate the amino acid concentrations in FM, KLM and MLM, samples

were hydrolysed with 6 N HCl at 110 �C for 24 h. For the determination of methionine and

cysteine, samples were treated with performic acid to oxidise methionine to methionine

sulphone and cysteine to cysteic acid (Kasprowicz-Potocka et al. 2013). All samples were

then hydrolysed at 1,108 �C in vacuo in 6 M-HCl for 22 h and analysed in an automatic

AA analyser (4151 Alpha Plus Amino Acid Analyser; Pharmacia LKB Biochrom Ltd,

Cambridge, UK). Anti-nutrients, polyphenols and tannins in KLM and MLM were

determined using the spectrophotometric methods as described by Makkar et al. (1993).

Growth performance parameters

Specific growth rate (SGR) was calculated according to Winberg (1956) as:

SGR ¼ InWf�InW0

t� 100; where Wf = final body weight (g), W0 = initial body weight (g),

Aquacult Int

123

In = natural logarithm (log)-10 and t = feeding period (days). Thermal-unit growth

coefficient (TGC) was calculated according to Iwama and Tautz (1981) as: TGC ¼

100� FBW0:333�IBW0:333

PT�C�days

where FBW = final body weight (g/fish); IBW = initial body

weight (g/fish); and T = temperature. Feed utilisation was estimated as feed conversion

ratio ðFCR) ¼ food consumed (g)weight gained (g)

and protein efficiency ratio ðPER) ¼weight gained (g)protein intake (g)

: Determination of the apparent digestibility coefficient (ADC; %) for

protein was performed using the indirect method of Cho et al. (1982) using Cr2O3 as an

inert marker: ADC %ð Þ¼ 100 1� % Cr2O3 in diet% Cr2O3 in faeces

� �� % protein in faeces

% protein in diet

� �� �:

Histological analysis

Liver samples and 1-cm segments from the small intestine were obtained from the mid-

intestine of fifteen fish per dietary treatment. The samples were submerged in 10 % neutral

buffered formalin for 24 h. Thereafter, the tissues were dehydrated according to standard

histological techniques in graded ethanol series and embedded in paraffin wax for his-

tology. Sections (3–5 lm) were cut and mounted on glass slides before staining with

haematoxylin and eosin. Slides were then examined and photographed under light trin-

ocular microscopy at 4009 (Leica Microsystems model DM750, Leica, Bannockburn, IL,

USA). Images were captured with a DVC digital camera (Digital Video Camera Company,

Austin, TX) mounted on a BH-2.

Villi heights and widths (lm) from the intestine samples were measured on Image J

(1.46) software. Twenty measurements were taken from each slide. The number of goblet

cells per segment was counted according to Baeverfjord and Krogdahl (1996). The number

of goblet cells was expressed as the total number of phenotypically mature goblet cells

(based on the intensity of staining, the size of the apical region and morphology) per 100

epithelial cells. McFadzen et al.’s (1997) criteria were used to categorise the differences in

(liver) hepatocyte condition. The classification of the samples was based on the poorest

liver observed. Each specimen was assigned one of three grades (1–3), a healthy specimen

scoring 1 to degraded liver scoring 3 (Table 3). The hepatocyte degradation mean value of

liver samples from each treatment was the mean score of all samples in that treatment.

Table 3 Histological criteria used for scoring of T. rendalli hepatocytes

Tissue Grade

1 (Healthy) 2 (Intermediate) 3 (Degraded)

Liver nuclei Lightly granular, small anddistinct

Abundant granules,enlarged or indistinctnucleoli

Small dark and pyknotic

Cytoplasm Structured: Varied texture,scattered granules witheosin-positive patches

Homogenous: Granularslight variability instaining property

Hyaline: Lacking texture, darksmall and often separated thecell boundary

Vacuolation Vacuoles follow cell boundaryand encroach on the nucleus

Small occasionalvacuoles

Poor vacuolation, cells shrinkleading to large sinusoidalspaces

Adapted from McFadzen et al. (1997)

Aquacult Int

123

Haematological analysis

On termination of the experiment, blood samples were taken from 15 fish per dietary

treatment for haematological analysis. Blood was drawn through caudal venous puncture

into lavender-top EDTA tubes (with anticoagulant) for total blood count. Total blood count

was done on a Systemex, XT-1800i blood analyser. For glucose determination, blood was

drawn into glucose tubes with sodium fluoride, and for the determination of protein and

blood urea nitrogen (BUN), blood was drawn into serum tubes with acid citrate dextrose.

Analysis for plasma glucose was done on a 600: UniCel DxC General Chemistry Analyzer.

Cost analysis

Assuming that all other operating costs remained constant and the cost of ingredients was

the only variable cost, the following economic indicators as used by Bahnasawy et al.

(2003) were calculated:

Incidence cost ¼ cost of feed

quantity of fish produced (kg)

Profit index ¼ local market value of fish

cost of feed

Data analysis

Normality and homogeneity of variance were confirmed using the Shapiro–Wilk normality

test (SAS 2008). Data on the growth performance, feed utilisation, histological and hae-

matological parameters were subjected to a one-way analysis of variance (ANOVA).

Significance was accepted at probabilities \0.05. All data were analysed on the general

linear model (GLM) procedure of SAS (2008).

Results

Growth performance and protein apparent digestibility

The control diet was readily consumed, and fish in this treatment group had the highest

feed intake (Table 4a, b). Inclusion of KLM in the diet led to a decrease in feed intake; this

decrease was not significantly different (P [ 0.05) compared with the control (Table 4a).

In the moringa diets, a reduction in feed intake was also observed with increasing MLM in

the diet. Fish fed MLM 100 had significantly lower feed intake than the control (Table 4b).

T. rendalli fed the control diet had the best growth performance (TGC 0.07; SGR

2.12 %/day). Fish fed kikuyu diets showed better growth performance than those fed

moringa diets at all replacement levels. SGR and TGC were significantly (P \ 0.05)

reduced when KLM replaced more than 50 % of FM (Table 4a and Fig. 1, respectively).

On the other hand, MLM could only replace 25 % of FM without a decrease in SGR and

TGC. As a consequence of the low feed intake, increasing MLM replacement above 25 %

resulted in significantly lower (P \ 0.05) SGR (Table 4b) and TGC (Fig. 1). Fish fed

MLM 100 had the poorest growth performance.

The FCR values also showed that KLM is a better FM substitute than MLM. The best

FCR was recorded in fish fed the control diet (1.59 ± 0.08). The FCR of fish fed KLM 25

Aquacult Int

123

Table 4 Growth performance of T. rendalli fed (a) kikuyu-based diets and (b) moringa-based diets

Control KLM 25 KLM 50 KLM 75 KLM 100

(a)

IBW (g) 11.5 ± 0.5 12.0 ± 1.0 12.0 ± 1.0 11.9 ± 2.1 11.5 ± 1.5

FBW (g) 41.1 ± 1.9a 38.5 ± 1.5a 36.6 ± 2.0a 30.5 ± 1.8b 28.3 ± 1.0b

Feed intake* 0.8 ± 0.2a 0.8 ± 0.1a 0.7 ± 0.2a 0.7 ± 0.1a 0.6 ± 0.1a

SGR(% day-1) 2.1 ± 0.2a 2.0 ± 0.4a 1.9 ± 0.2a 1.6 ± 0.1b 1.5 ± 0.4b

FCR 1.6 ± 0.1e 1.8 ± 0.3c 2.1 ± 0.1b 2.6 ± 0.3a 2.8 ± 0.2a

PER 2.1 ± 0.9a 1.9 ± 0.9a 1.6 ± 0.1b 1.3 ± 0.2c 1.2 ± 0.3c

ADC CP (%) 82.1 80.9 79.6 77.1 76.0

Incidence cost 0.02 0.02 0.02 0.02 0.01

Profit index 1.27 1.32 1.37 1.42 1.48

Control MLM 25 MLM 50 MLM 75 MLM 100

(b)

IBW (g) 11.5 ± 0.5 12.0 ± 1.0 11.6 ± 1.5 11.7 ± 2.2 12.1 ± 1.0

FBW (g) 41.1 ± 1.9a 35.4 ± 0.6a 29.0 ± 1.2b 25.2 ± 1.3c 22.0 ± 0.5d

Feed intake* 0.8 ± 0.2a 0.8 ± 0.2a 0.7 ± 0.2a 0.6 ± 0.1a 0.5 ± 0.1b

SGR (% day-1) 2.1 ± 0.2a 1.8 ± 0.1a 1.5 ± 0.1b 1.3 ± 0.1c 1.0 ± 0.1d

FCR 1.6 ± 0.1e 2.0 ± 0.1d 2.3 ± 0.1c 2.7 ± 0.1b 3.7 ± 0.1a

PER 2.1 ± 0.9a 1.7 ± 0.1b 1.5 ± 0.1c 1.3 ± 0.1d 0.9 ± 0.1e

ADC CP (%)# 82.1 79.0 76.5 74.1 73.2

Incidence cost 0.02 0.02 0.03 0.03 0.03

Profit index 1.27 1.12 1.04 0.97 0.91

Values are mean of triplicate determinations ± standard deviations. Values in the same rows with the samesuperscripts are not significantly different (P [ 0.05)

* Feed intake (g/fish/day)# Faecal samples from each dietary treatment were pooled; therefore, statistical analysis was not determined

a

aa

b

c

a

a

b

bc

c

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08

0 25 50 75 100

TG

C

% FM replacement

KLM MLM

Fig. 1 Growth rate (TGC) of T.rendalli (average initialweight = 11.50 g/fish) fedKLM- and MLM-based diets

Aquacult Int

123

(1.77 ± 0.28) was not significantly different from the control (P [ 0.05). Increasing KLM

to replace more than 25 % of FM led to FCR values that were significantly higher

(P \ 0.05) than the control (Table 4a). When MLM replaced FM, the FCR values were

higher than those of KLM-based diets (Table 4b). Even at the lowest inclusion (25 %), the

FCR of fish fed MLM 25 was significantly higher (P \ 0.05) than that of the control.

Similarly, the values recorded for PER and ADC (protein) show that KLM protein is

utilised better than MLM protein. In the KLM-based diets, the PER of fish fed KLM 25

(1.88 ± 0.91) was not significantly different (P [ 0.05) from that of fish fed the control

diet (2.10 ± 0.94). Only when KLM replaced C 50 % of FM was the PER significantly

lower than the control. On the contrary, PER of fish fed MLM-based diets was significantly

lower (P \ 0.05) than that of the control even at the lowest inclusion level (MLM 25:

1.69 ± 1.05). The poorest PER (0.91 ± 0.11) was recorded in fish fed MLM 100

(Table 4b). ADC (protein) was highest (82.09 %) in fish fed the control diet. ADC

decreased when FM was replaced with increasing levels of either KLM or MLM. The ADC

for protein was higher in KLM-based diets (Table 4a) compared with the MLM-based diets

(Table 4b) at all inclusion levels.

Effect of plant diets on histology

The villi length and width in T. rendalli were not affected by the increasing levels of KLM

in the diet (Table 5). However, the goblet cell number increased with increasing KLM

levels (Fig. 2), but this increase was not statistically significant (P [ 0.05). There were no

degenerative changes observed in the intestine of T. rendalli fed lower levels of KLM

(KLM 25 and KLM 50). In the MLM diets, there was an apparent trend towards shorter

villi and increased number of goblet cells in fish fed higher levels of MLM (Fig. 3).

However, there were no significant differences (P [ 0.05) in these parameters among the

dietary treatments (Table 5).

Liver histology showed centrally located lightly granular nuclei with distinct boundaries

of the hepatocytes and normal vacuolation in T. rendalli fed the control diet (Fig. 4). This

group was assigned a hepatocyte degradation mean value of 1.33 (Table 6). Fish fed KLM

25 showed a slight decrease in vacuolisation (Fig. 4). The alterations were, however, not

Table 5 Histological values of the midintestine of T. rendalli fed different inclusion levels of kikuyu andmoringa leaf meals

Percentage of fishmeal replaced by the plant meals in the diet

0 25 50 75 100 P value

Villi length (lm)

KLM 488.6 ± 12 481.3 ± 15 476.8 ± 10 484.2 ± 18 479.9 ± 11 0.44

MLM 479.8 ± 20 472.0 ± 15 469.7 ± 10 461.0 ± 18 0.17

Villi width (lm)

KLM 68.2 ± 11 72.7 ± 21 66.9 ± 23 65.4 ± 21 69.3 ± 17 0.75

MLM 69.7 ± 16 71.9 ± 19 68.5 ± 10 71.8 ± 14 0.52

Goblet cells (per 100 epithelial cells)

KLM 397 ± 18 381 ± 12 402 ± 22 400 ± 13 423 ± 24 0.34

MLM 386 ± 16 416 ± 17 429 ± 20 447 ± 18 0.14

KLM kikuyu leaf meal, MLM moringa leaf meal

Aquacult Int

123

Fig. 2 Histological sections (H & E–stained) of the intestine from the midgut of T. rendalli fed kikuyu-based diets. lm lumen, lp lamina propria: arrows point to—mv microvilli, gc goblet cells. Scale bar 10 lm

Fig. 3 Histological sections (H & E–stained) of the intestine from the midgut of T. rendalli fed moringa-based diets. lm lumen, lp lamina propria: arrows point to—mv microvilli, gc goblet cells. Scale bar 10 lm

Aquacult Int

123

significantly different (P [ 0.05) from the control (Table 6). Increasing the level of KLM

in the diet resulted in increased degradation of the hepatocytes. Fish fed KLM 75 and KLM

100 showed an increased number of pyknotic nuclei, reduced vacuolation and increased

sinusoidal space (Fig. 4). The hepatocyte degradation mean values (1.90 and 2.02,

respectively) were significantly higher (P \ 0.05) than the control (1.33 Table 6).

Hepatocyte histology in T. rendalli fed the moringa-based diets displayed greater

degradation when compared to the kikuyu diets at the same inclusion levels. Fish fed MLM

25 showed granular cytoplasm and increased sinusoids (Fig. 5). The hepatocyte degra-

dation score (1.80) was significantly greater (P \ 0.05) than that of the control (1.33:

Table 6). When the level of MLM in the diet increased, the degree of hepatocyte degra-

dation also increased. In MLM 75 and MLM 100 treatments, the hepatocyte degradation

values were 2.43 and 2.63, respectively (Table 6). These hepatocytes were irregularly

shaped with nucleus pushed to the periphery of the cells; increased sinusoidal space and

fatty degeneration were also observed (Fig. 5).

Fig. 4 Histological sections (HE stained) of the liver of T. rendalli fed kikuyu-based diets. Arrows point to:hp hepatocytes, vc vacuoles, sn sinusoids, kc Kupffer cells. Scale bar 10 lm

Table 6 Hepatocyte degradation scores of T. rendalli fed different inclusion levels of kikuyu and moringaleaf meals (100 hepatocytes per treatment)

Percentage of fishmeal replaced by the plant meals in the diet

0 25 50 75 100

Hepatocyte degradation score

KLM 1.33a 1.63a 1.87ab 1.90bc 2.20c

MLM 1.33a 1.80ab 2.27b 2.43c 2.63d

Values in the same row with the same superscripts are not significantly different (P [ 0.05)

Aquacult Int

123

Effect of plant diets on haematological parameters

No trend was observed in the white blood cells (WBC) with increasing leaf meal inclusion.

WBC count was not significantly different (P [ 0.05) in all KLM levels in the diet

(Table 7a). The red blood cells (RBC) and haematocrit (HCT) decreased with increasing

KLM, and these were also not significantly different (P [ 0.05) between treatments. There

was no apparent trend in the plasma glucose of T. rendalli fed all KLM diets when

compared to the control. Plasma protein and blood urea nitrogen (BUN) were not signif-

icantly (P [ 0.05) affected by all levels of KLM inclusion in diet (Table 7a). In the

moringa-based diets, WBC increased with increasing MLM inclusion and were signifi-

cantly higher (P \ 0.05) in T. rendalli fed MLM75 and MLM100 (Table 7b). The RBC

and HCT were significantly lower in MM100. A decrease in plasma glucose was also

observed with increasing MLM levels; this decrease was not significantly different

(P [ 0.05) in all treatments. Plasma protein and blood urea nitrogen (BUN) were not

significantly (P [ 0.05) affected by MLM inclusion in the diet (Table 7b).

The economic analysis of the experiment demonstrated that adding KLM in T. rendalli

diets led to lower incidence cost and increased the profit index when compared to the

control (Table 4a). The inclusion of MLM on the other hand led to higher incidence costs

and lower profit index than the control (Table 4b).

Discussion

The growth performance of T. rendalli decreased as the level of both plant meals in the diet

increased. These results are consistent with several studies on the replacement of FM with

plant-based protein (Garduno-Lugo and Olvera-Novoa 2008; Sheeno and Sahu 2006; Luo

Fig. 5 Histological sections (HE stained) of the liver of T. rendalli fed moringa-based diets. Arrows pointto: hp hepatocytes, vc vacuoles, sn sinusoids, kc Kupffer cells. Scale bar 10 lm

Aquacult Int

123

et al. 2012). At all inclusion levels, the growth parameters measured were poorer in the

moringa-based diets than in the kikuyu-based diets. MLM had higher protein content than

KLM. Conventionally, plants with high protein content result in superior growth perfor-

mance and are preferred as FM replacers in fish diets. The merit of a protein source,

however, is dependent not only on the amount of protein but also on the amino acid

composition and the biological availability thereof (El-Sayed 2006). All diets in this study

had the same protein content (30 %). The essential amino acid composition of the two leaf

meals was largely similar. Lysine and methionine are the most limiting amino acids in fish

feed (El-Sayed 2006). Their levels were marginally higher in kikuyu grass than in moringa

leaves. The energy content also plays an important role in the nutritional quality of a diet as

it can spare dietary protein from energy metabolism, thereby increasing its utilisation for

growth (Wang et al. 2006; Schulz et al. 2008; Ahmadr 2008). All diets in this study were

formulated to be isoenergetic.

Table 7 The effect of the kikuyu (a)- and moringa (b)-based diets on haematological parameters of T.rendalli (n = 15)

Control KM25 KM50 KM100

(a)

WBC (103ll) 264.41 ± 7.5 255.11 ± 8.2 244.19 ± 5.9 260.03 ± 4.5

RBC (106ll) 1.49 ± 0.24a 1.37 ± 0.20a 1.23 ± 0.32a 1.16 ± 0.15a

HTC(L/L) 0.26 ± 0.14a 0.25 ± 0.21a 0.24 ± 0.13a 0.16 ± 0.11a

Hb (g/dL) 6.00 ± 1.1a 6.60 ± 2.1a 5.20 ± 1.3a 6.60 ± 1.4a

MCV (fL) 181.81 ± 3.4a 182.48 ± 5.2a 195.12 ± 6.1a 139.70 ± 7.2a

MCH (pg) 42.00 ± 2.1a 48.18 ± 3.5a 42.30 ± 2.4a 56.90 ± 2.2a

MCHC (g/dL) 23.08 ± 4.1a 26.40 ± 5.7a 21.67 ± 2.6a 41.25 ± 8.1a

Glucose (g/dL) 10.20 ± 2.1a 13.40 ± 2.3a 12.40 ± 1.5a 8.00 ± 1.4a

Total protein (g/L) 32.00 ± 2.5a 28.00 ± 2.1a 27.00 ± 1.0a 26.00 ± 2.2a

BUN 1.00 ± 0.11a 1.20 ± 0.12a 1.50 ± 0.21a 1.10 ± 0.13a

Control MM25 MM50 MM100

(b)

WBC (103ll) 264.41 ± 7.5a 268.75 ± 9.2a 271.14 ± 4.2b 298.91 ± 7.3b

RBC (106ll) 1.49 ± 0.24a 1.44 ± 0.11a 1.43 ± 0.25a 1.27 ± 0.18b

HTC(L/L) 0.26 ± 0.14a 0.27 ± 0.21a 0.27 ± 0.15a 0.12 ± 0.05b

Hb (g/dL) 6.00 ± 1.1a 5.60 ± 1.2a 6.80 ± 1.5a 4.70 ± 1.2a

MCV (fL) 181.81 ± 3.4a 187.50 ± 3.8a 181.21 ± 4.5a 169.06 ± 6.1a

MCH (pg) 42.00 ± 2.1a 38.90 ± 3.1a 45.64 ± 2.4a 56.20 ± 3.5a

MCHC (g/dL) 23.08 ± 4.1a 20.74 ± 1.3a 25.19 ± 2.5a 39.17 ± 3.4a

Glucose (g/dL) 10.20 ± 2.1a 9.80 ± 1.9a 8.50 ± 1.3a 8.00 ± 1.2a

Total protein (g/L) 30.00 ± 2.5a 27.00 ± 1.5a 26.00 ± 2.0a 26.00 ± 1.5a

BUN 1.00 ± 0.11a 1.60 ± 0.21a 1.00 ± 0.14a 1.20 ± 0.18a

Values are mean of five determinations ± standard deviations

WBC white blood cells, RBC red blood cells, HCT haematocrit, Hb haemoglobin, MCV mean corpuscularvolume, MCH mean corpuscular haemoglobin, MCHC mean corpuscular haemoglobin concentration,Glucose plasma glucose; BUN blood urea nitrogen

Aquacult Int

123

High fibre levels in fish diets have also been reported to reduce nutrient digestibility

(Azaza et al. 2008). The amount of fibre in the diets increased with increasing levels of leaf

meals. KLM had higher fibre content than MLM, and kikuyu-based diets had higher fibre

levels. This did not seem to have a major impact in hindering nutrient utilisation as the

kikuyu-based diets were utilised better than the moringa-based diets.

The presence of anti-nutritional factors in the diet may reduce the availability or

digestibility of dietary nutrients (Francis et al. 2001). Thus, the poor growth performance

observed in moringa-based diets may also be attributed to the higher concentration of anti-

nutrients in MLM. MLM had higher concentrations of tannins and phenolics than KLM.

Tannins hamper the digestive process by binding digestive enzymes of feed components

such as proteins and also reduce the absorption of essential vitamins (Francis et al. 2001).

Phenolic substances are known to form phenolic–protein–enzyme complexes that reduce

protein digestibility and amino acid availability. Other anti-nutrients such as saponins,

phytate, alkaloids and trypsin inhibitors have been found in moringa leaves (Ogbe and

Affiku 2011; Richter et al. 2003) and in kikuyu grass albeit at lower concentrations (Marais

2001). Saponins have surface-active components that lead to the damage of biological

membranes, resulting in increased permeability of the mucosal cells (Krogdahl et al. 1995;

Bureau et al. 1998). This may eventually inhibit active nutrient transport (Johnsen et al.

1990).

Feed intake decreased with increasing leaf meal inclusion in the diet and was lower in

fish fed moringa-based diets than in fish fed kikuyu diets. The low feed intake observed is

an important factor that may also be responsible for the poor growth performance in fish

fed moringa diets. Saponins and tannins have an astringent/bitter taste (Jansman 1993;

Francis et al. 2001; Makkar 2003; Hostettmann and Marston 2005) which could have

lowered palatability resulting in poor feed intake. Reduced feed intake with increasing

moringa leaf meal levels in the diet was also reported by Afuang et al. (2003) in Nile

tilapia.

The adverse effects caused by the anti-nutrients found in diets with higher levels of

plant meals are supported by the decrease in apparent digestibility of protein as the level of

KLM and MLM in the diet increased. The apparent protein digestibility was lower in

moringa-based diets. Several authors (Ray and Das 1994; Bairagi et al. 2004) who used

leaf meals to replace fishmeal also reported a decrease in protein digestibility with

increasing leaf meal inclusion. Olivera-Novoa et al. (2002) attributed the decrease in

protein digestibility to a decline in the absorption of nutrients. The protein digestibility

values obtained in the moringa-based diets are lower than those reported by Madalla

(2008) where protein digestibility in O. niloticus was 91 % in the control and 84 % when

moringa leaf meal provided 60 % of dietary protein feed. Digestibility is species specific,

and there are no studies available to compare the digestibility in T. rendalli fed moringa or

kikuyu leaf meals. It is important to note that the apparent digestibility values reported in

this study may be overestimated because of the method of faecal collection used (Cho et al.

1982). The protein digestibility of kikuyu-based diets in this study fell within limits

regarded as high, i.e. 95–75 % (Cho and Kaushik 1990).

The intestinal villi displayed a trend towards shorter villi with increased MLM level in

the diet. In the kikuyu-based diets, however, there was no apparent trend in the villi height.

The shorter villi found in fish fed more MLM resulted in lower efficiency of the absorptive

process (Da Silva et al. 2012; Caballero et al. 2003) as a result of the reduced absorption

surface area. This may also explain the poor growth performance of fish fed these diets.

Aquacult Int

123

There was an increase in the number of goblet cells in the villi when the amount of leaf

meal in the diet increased. The abundance of goblet cells was higher in fish fed moringa-

based diets than in fish fed kikuyu-based diets. The increase in the number of goblet cells

may be an indication of increased irritation as these cells produce mucous lining the brush

border which acts as a lubricant and provides protection against chemical and mechanical

damage (Marchetti et al. 2006).

The liver is a good indicator of the nutritional status because of its role in metabolising

products of the digestive system. Replacing fishmeal with plant meals led to a decrease in

vacuolisation of the hepatocytes, displacement of the nuclei and sinusoidal reduction. The

decrease in the number and size of vacuoles and the reduction in nuclei size as fishmeal in

the diet was replaced by higher levels of moringa (75–100 %) indicate poor nutritional

status of the fish (Mcfadzen et al. 1997). This corresponds with the poor protein digest-

ibility, low PER values and the resultant poor growth observed in T. rendalli fed higher

levels of moringa leaf meal in the diet.

In the present study, RBC, Hb and HCT of T. rendalli decreased when both KLM and

MLM levels increased in the diet. This decrease, however, was only significant when

[75 % FM was replaced with MLM. This decrease further confirms the signs of nutritional

stress caused by the reduced bioavailability of proteins. According to Qiang et al. (2013)

when dietary protein levels are low, physiological stress is induced, and this increases

damage to the liver leading to reduced RBC and Hb concentration. Similarly, Sakthivel

(1988) and Abdel-Tawwab et al. (2010) reported a decrease in RBC and Hb in fish fed low

protein levels in the diet. The WBC increased with increasing levels of both KLM and

MLM, with higher levels observed in the MLM diets. This increase was significant in fish

fed MLM 100. The replacement of FM with KLM and MLM did not have a significant

effect on plasma protein and BUN of T. rendalli. However, plasma protein levels were

lower in fish fed high MLM levels. The low plasma protein observed in fish fed high levels

of MLM may be a consequence of decreased protein absorption emanating from the shorter

villi and poor protein digestibility recorded for these fish.

Kikuyu-based diets had a higher profit index and lower incidence cost. Thus, in spite of

the reduced growth performance, cost–benefit analysis indicates that KLM diets are eco-

nomically superior to the FM-based control. Adding MLM on the other hand leads to lower

profit index and a higher incidence cost. This is because of the higher cost of moringa

leaves compared with FM. Moringa has been commercialised, and its inclusion may not be

sustainable in fish diets at least not in South Africa where claims on its human health

benefits are on the rise.

Kikuyu grass is a promising FM substitute in the diets of tilapias that are herbivorous in

nature. It has an adequately balanced amino acid profile, relatively high protein and energy

levels. KLM can replace up to 25 % FM and MLM\25 % FM without adverse effects on

growth performance, intestine and liver histology or haematological parameters. Further-

more, unlike moringa that is expensive, there was no financial value associated with this

common lawn grass. Investigations into methods of destroying anti-nutritional factors in

KLM may provide further insights to enhancing its utilisation. It is recommended that

further experiments be carried out at different concentrations of tannins and polyphenols to

determine their specific effect on growth performance of T. rendalli.

Acknowledgments We extend our utmost appreciation to Mr. G. Geldenhuys for technical support andMs. L.P. Moela who helped with fish husbandry. The authors acknowledge the National Research Foun-dation and the Aquaculture Research unit for funding this study.

Aquacult Int

123

References

Abdel-Tawwab M, Ahmad MH, Khattab YAE et al (2010) Effect of dietary protein level, initial bodyweight, and their interaction on the growth, feed utilization, and physiological alterations of Niletilapia, Oreochromis niloticus (L.). Aquaculture 298:267–274

Adewolu MA (2008) Potentials of Sweet Potato (Ipomoea batatas) leaf meal as dietary ingredient forTilapia zilli fingerlings. Pakistan J Nutr 7:444–449

Afuang W, Siddhuraju P, Becker K (2003) Comparative nutritional evaluation of raw, methanol extractedresidues and methanol extracts of moringa (Moringa oleifera Lam.) leaves on growth performance andfeed utilization in Nile tilapia (Oreochromis niloticus L.). Aquac Res 34(13):1147–1159

Ahmadr MH (2008) Response of African catfish, Clarias gariepinus, to different dietary protein and lipidlevels in practical diets. J World Aqua Soc 39(4):541–548

AOAC International (2012) 2nd Revision, 18th Edition, Association of Analytical Communities, Gai-thersburg, MD, USA

Azaza MS, Mensi F, Ksouri J et al (2008) Growth of Nile tilapia (Oreochromis niloticus L.) fed with dietscontaining graded levels of green algae ulva meal (Ulva rigida) reared in geothermal waters ofsouthern Tunisia. J Appl Ichthyol 24:202–207

Baeverfjord G, Krogdahl A (1996) Development and regression of soybean meal induced enteritis inAtlantic salmon, Salmo salar L., distal intestine: a comparison with the intestines of fasted fish. J FishDis 19:375–387

Bahnasawy MH, Abdel-Baky TE, Gamal A et al (2003) Growth performance of Nile tilapia (Oreochromisniloticus) fingerlings raised in an earthen pond. Arch Pol Fish 11:277–285

Bairagi A, Sarkar Ghash K, Sen SK et al (2004) Evaluation of the nutritive value of Leucaena leucocephalaleaf meal, inoculated with fish intestinal bacteria Bacillus subtilis and Bacillus circulans in formulateddiets for rohu, Labeo rohita (Hamilton) fingerlings. Aquac Res 35:436–446

Bureau DP, Harris AM, Cho CY (1998) The effects of purified alcohol extracts from soy products on feedintake and growth of Chinook salmon (Oncorhynchus tshawytscha) and rainbow trout (Oncorhynchusmykiss). Aquaculture 161:27–43

Caballero MJ, Izquierdo MS, Kjørsvik E et al (2003) Morphological aspects of intestinal cells from giltheadseabream (Sparus aurata) fed diets containing different lipid sources. Aquaculture 225:325–340

Cho CY, Kaushik SJ (1990) Nutritional energetic in fish: energy and protein utilization in rainbow trout(Salmo gairdneri). World Rev Nutr Diet 61:132–172

Cho CY, Slinger SJ, Bayley HS (1982) Bioenergetics of salmonid fishes: energy intake, expenditure andproductivity. Comp Biochem Physiol B 73(1):25–41

Collins SA, Desai AR, Mansfield GS et al (2012) The effect of increasing inclusion rates of soybean, peaand canola meals and their protein concentrates on the growth of rainbow trout: concepts in dietformulation and experimental design for ingredient evaluation. Aquaculture 344–349:90–99

Da Silva MR, Natali MRM, Hahn NS (2012) Histology of the digestive tract of Satanoperca pappaterra(Osteichthyes, Cichlidae). Biol Sci 34:319–326

El-Sayed AFM (2006) Tilapia culture. CABI Publishers, OxfordshireFlukerson WJ, Neal JS, Clark CF et al (2007) Nutritive value of forage species grown in the temperate

climate of Australia for dairy cows: grasses and legumes. Livest Sci 107:253–264Foidl N, Makkar HPS, Becker K (2001) The potential of Moringa oleifera for agricultural and industrial

uses. In: Fuglie LJ (ed) The Miracle Tree. CTA and CWS, Dakar, pp 45–77Francis G, Makkar HPS, Becker K (2001) Antinutritional factors present in plant-derived alternate fish feed

ingredients and their effects in fish. Aquaculture 199:197–227Garduno-Lugo M, Olvera-Novoa MA (2008) Potential of the use of peanut (Arachis hypogaea) leaf meal as

a partial replacement for fish meal in diets for Nile tilapia (Oreochromis niloticus L.). Aquac Res39:1299–1306

Hlophe SN, Moyo NAG (2011) The effect of different plant diets on the growth performance, gastricevacuation rate, and carcass composition of Tilapia rendalli. Asian J Anim Vet Adv 6(10):1001–1009

Hostettmann K, Marston A (2005) Chemistry and pharmacology of natural products. Cambridge UniversityPress, Saponins

Iwama GK, Tautz AF (1981) A simple growth model for salmonids in hatcheries. Can J Fish Aquat Sci38:649–656

Jansman AJM (1993) Tannins in feedstuffs for simple stomached animals. Nutr Res Rev 6(1):209–236Jauncey K, Tacon AGJ, Jackson AJ (1983) The quantitative essential amino acid requirements of Ore-

ochromis (Sarotherodon) mossambicus. In: Fishelson L, Yaron Z (eds) Proceedings of the internationalsymposium on Tilapia in Aquaculture. Tel Aviv University, Tel Aviv, pp 328–337

Aquacult Int

123

Johnsen PB, Zhou H, Adams MA (1990) Gustatory sensitivity of the herbivore Tilapia zillii to amino acids.J Fish Biol 36(4):587–593

Kasprowicz-Potocka M, Walachowska E, Zaworska A et al (2013) The assessment of influence of differentnitrogen compounds and time on germination of Lupinus angustifolius seeds and chemical compositionof final products. Acta Soc Bot Pol 82(3):199–206

Krogdahl A, Roem A, Baeverfjord G (1995) Effects of soybean saponin, raffinose and soybean alcoholextract on nutrient digestibilities, growth and intestinal morphology in Atlantic salmon. In: SvennevigN, Krogdahl A (eds) Quality in aquaculture, Proceedings of international conference aquaculture ‘95and the satellite meeting Nutrition and Feeding of Cold Water Species, Trondheim, Norway. Aquacult.Soc. Spec. Publ. No. 23, Gent, Belgium, pp 118–119

Krogdahl A, Bakke-Mckellep AM, Roed KH et al (2000) Feeding Atlantic salmon Salmo salar L. soybeanproducts: effects on disease resistance (furunculosis), and lysozyme and IgM levels in the intestinalmucosa. Aquac Nutr 6:77–84

Luo Z, Liu C-X, Wen H (2012) Effect of dietary fish meal replacement by canola meal on growth per-formance and hepatic intermediary metabolism of genetically improved farmed tilapia strain of Niletilapia, Oreochromis niloticus, reared in fresh water. J World Aqua Soc 43:670–678

Madalla N (2008) Novel feed ingredients for Nile Tilapia (Oreochromis niloticus L.). Thesis, University ofSterling

Makkar HPS (2003) Effects and fate of tannins in ruminant animals, adaptation to tannins, and strategies toovercome detrimental effects of feeding tannin-rich feeds. Small Rum Res 49(3):241–256

Makkar HPS, Becker K (1997) Nutrients and antiquality factors in different morphological parts of theMoringa oleifera tree. J Agric Sci 128:311–322

Makkar HPS, Blummel M, Borowy NK et al (1993) Gravimetric determination of tannins and their cor-relations with chemical and protein precipitation methods. J Sci Food Agric 61:161–165

Marais JP (2001) Factors affecting the nutritive value of Kikuyu grass (Pennisetum clandestinum): a review.Tropic Grassl 35:65–84

Marais JP, Figenischou DL, Dennison C (1987) The accumulation of nitrate in Kikuyu grass (Pennisetumclandestinum Hochst). SA J Plant Soil 4:82–88

Marchetti L, Capacchietti M, Sabbieti MG et al (2006) Histology and carbohydrate histochemistry of thealimentary canal in the rainbow trout Oncorhynchus mykiss. J Fish Biol 68:1808–1821

McFadzen IRB, Coombs SH, Halliday NC (1997) Histological indices of the nutritional condition ofsardine, Sardina pilchardus (Walbaum) larvae of the north coast of Spain. J Exp Mar Biol Ecol212:239–258

Meschiatti AJ, Arcifa MS (2002) Early life stages of fish and the relationships with zooplankton in a tropicalBrazilian reservoir: Lake Monte Allegro. Braz J Biol 62:41–50

Ng WK, Wee KL (1989) The nutritive value of cassava leaf meal in pelleted feed for Nile tilapia. Aqua-culture 83:45–58

Ogbe AO, Affiku JP (2011) Proximate study, mineral and anti-nutrient composition of Moringa oleiferaleaves harvested from Lafia, Nigeria: potential benefits in poultry nutrition and health. J MicrobiolBiotechnol Food Sci 1(3):296–308

Olivera-Novoa MA, Olivera-Castillo L, Martinez-Palacios CA (2002) Sunflower seed meal as a proteinsource in diets for Tilapia rendalli (Boulenger, 1896) fingerlings. Aquac Res 33:223–229

Qiang J, Yang H, Wang H et al (2013) Interacting effects of water temperature and dietary protein level onhematological parameters in Nile tilapia juveniles, Oreochromis niloticus (L.) and mortality underStreptococcus iniae infection. Fish Shellfish Immunol 34:8–16

Ray AK, Das L (1994) Apparent digestibility of some aquatic macrophytes in rohu, Labeo rohita (Ham-ilton), fingerlings. J Aquac Tropics 9:335–342

Richter N, Siddhuraju P, Becker K (2003) Evaluation of nutritional quality of moringa (Moringa oleiferaLam.) leaves as an alternative protein source for Nile tilapia (Oreochromis niloticus L.). Aquaculture217:599–611

Sakthivel M (1988) Effects of varying dietary protein level on the blood parameters of Cyprinus carpio.Proc Indian Nati 97:363–366

SAS (2008) Proprietary software release 9.2. Statistical Analysis Systems Institute, Inc., Cary, NorthCarolina, USA

Schulz C, Huber M, Ogunji J, Rennert B (2008) Effects of varying dietary protein to lipid ratios on growthperformance and body composition of juvenile pike perch (Sander lucioperca). Aquac Nutr14:166–173

Sheeno TP, Sahu NP (2006) Use of freshwater aquatic plants as a substitute of fishmeal in the diet of Labeorohita fry. J Fish Aqua Sci 1:126–135

Aquacult Int

123

Tacon AGJ, Hasan MR, Metian M (2011) Demand and supply of feed ingredients for farmed fish andcrustaceans. Trends and prospects. FAO Fisheries and aquaculture Technical paper 564

Wang Y, Guo JL, Li K et al (2006) Effects of dietary protein and energy levels on growth, feed utilizationand body composition of cuneate drum (Nibea miichthioides). Aquaculture 252:421–428

Weliange WS, Amarasinghe US, Moreau J et al (2006) Diel feeding periodicity, daily ration and relativefood consumption in some fish populations in three reservoirs of Sri Lanka. Aquat Living Resour19:229–237

Winberg GG (1956) Rate of metabolism and food requirements of fishes. J Fish Res Bd Can Trans Ser194:1–202

Yousif OM, Alhadhrami GA, Passaraki M (1994) Evaluation of dehydrated alfalfa and saltbush (Atriplex)leaves in diets for Tilapia (Oreochromis aureus L.). Aquaculture 126:341–347

Aquacult Int

123