Evaluation of raw and hydrothermically p (1)

Aquaculture Nutrition
2011 17; e164–e173

doi: 10.1111/j.1365-2095.2009.00745.x

..............................................................................................

B R Doshi School of Biosciences, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India

Hydrothermically processed Prosopis juliflora (PJ) seed meal as
a supplementary diet for Labeo rohita is found to be rich in
protein (330 g kg)1) having antinutritional factors in permissible limits and containing essential amino acids adequately
except lysine, methionine and cysteine. Ten iso-nitrogenous
and iso-energetic diets with crude, soaked and autoclaved
seed meal at 20%, 35% and 50% replacement of fish meal were
tested (D1–D9, respectively). The growth of fish (weight gain,
specific growth rate, feed conversion ratio and protein
efficiency ratio) fed diet D4 (soaked seed meal at 20%
replacement) was higher among the test diets, but lower than
reference diet (RD). Diets with 50% seed meal resulted in
lowering of growth, carcass composition, digestive enzyme

activity and digestibility compared to test diets at 20% and
35% inclusion levels in the respective groups. Hydrothermically processed seed meal improved the growth compared to
unprocessed one, though not up to RD level. This could be
because of amino acid imbalance and presence of non-starch
polysaccharides in seed meal. Looking to the easy availability
and its nutritional quality, processed PJ seed meal can be
incorporated in carp diet at lower inclusion level.
KEY WORDS: antinutritional factors, diets, growth, hydrothermically processed, Labeo rohita, Prosopis juliflora seed meal

Received 7 May 2009, accepted 23 October 2009
Correspondence: Dr Sujata S. Bhatt, B R Doshi School of Biosciences,
Sardar Patel University, Vallabh Vidyanar 388120, Gujarat, India.
E-mail: bhatt.sujata@gmail.com

The formulation of feed with high nutritional quality and its
availability as an alternative to fish meal are known to be very

important aspects of intensive aquaculture. Emphasis has
been given for the development of cost-effective feed using
plant proteins. Oil seed by-products and legume seeds as plant

protein sources in aqua feed have shown promising results,
but the presence of antinutritional factors (ANFs) and deficiency of essential amino acids (EAAs) are known to adversely
affect their use in complete replacement of fish meal (Tacon
1993, 1997). However, processing technologies have helped in
removal of ANFs and improvement in the nutritional quality
of feed to some extent (Wee 1991; Fagbenro 1999).
Legume seeds can form an important component of fish
feed because they are rich in protein, lipid and carbohydrates. Soybeans have been widely used as plant protein
source in feed because of their high protein content in spite
of the presence of ANFs and low level of certain EAAs
(Wilson & Poe 1985; Robaina et al. 1995). Prosopis juliflora (PJ) (mesquite) is a leguminous tree and grows in arid
and semi-arid regions of the world (Batista et al. 2002). In
Gujarat (India), PJ trees grow extensively in semi-dried
regions (Shukla et al. 1990). Mesquite pods form an
important feed source for livestock in many areas of world
(Riveros 1992). Because of their palatability and nutritional
value, pods of PJ are largely used for feeding dairy and
beef cattle with good nutritional and economic results
(Silva et al. 2007). Products from this plant have also been
used for human consumption in bread, biscuits, sweeties,

syrup and liquors (Van Den Eynden et al. 2003). Information available on PJ mainly deals with PJ pods and
leaves (Lyon et al. 1988), whereas not much is reported
about use and nutritive value of PJ seeds. Recently, effect
of prosopis seed meal on the growth performance of
broiler chicken has been reported (Yusuf et al. 2008).
Looking to the large-scale availability of PJ pods in
Gujarat and comparatively high protein content (306–373
g kg)1) in the seeds (Shukla et al. 1990), we evaluate PJ
seed meal as a plant protein source in the supplementary

..............................................................................................

 2010 Blackwell Publishing Ltd
No claim to original US government works

feed for Labeo rohita (rohu) fingerlings by partial replacement of fish meal. Garg et al. (2002) and Hossain et al.
(2001) have reported improvement in the nutritional
quality of leguminous seeds by hydrothermical processing,
and their incorporation in the feed have improved the
growth of the carps. In the present investigation, to eliminate/reduce the levels of ANFs like tannin, trypsin

inhibitor (TI) and phytic acid as well as to improve the
nutritional quality of the PJ seed meal, we have processed
the seed meal by water soaking and by soaking + autoclaving. Test diets have been formulated by replacing the
fish meal at 20%, 35% and 50% levels by each of
unprocessed as well as processed PJ seed meal in fish
meal–based supplementary feed (D1–D9), and their effects
on the growth of carp are compared with the fish meal–
based reference diet (RD).
The present study seems to be the first attempt to investigate the use of PJ seed meal as a plant protein source in the
diet for cultivable fish species including Indian major carps
(IMCs) and is aimed at evaluating the nutritional potential of
raw and processed PJ seed meal. An attempt is also made to
determine proper processing techniques to eliminate/reduce
the levels of ANFs.

PJ seeds were obtained from Khodiyar Seed Agency,
Bhavnagar, Gujarat, India. PJ seeds were soaked in water

(1 : 3, w v)1) for 6, 12 and 24 h. Another batch of seeds
was soaked in the same way and autoclaved for 20 min at

120 C at 15 lb cm)2. Water soaked and autoclaved seeds
were oven dried at 50–60 C for 24 h and grinded
separately along with unprocessed PJ seeds into powder to
pass through 0.5-mm sieve. Because TI, tannin and phytic
acid contents of the seed meal were minimum in 24-h
water soaked (PJ24) and 24-h water soaked followed by
autoclaved samples (PJ24+) (data not shown), they have
been used in the formulation of experimental feed along
with unprocessed seed meal (PJN). The fish meal of Indian
origin was prepared from javla (small shrimps from the
cod end of dol net, from Jafarabad, Gujarat), oven dried
at 50–60 C for 24 h and grinded into powder. Before diet
formulation, the proximate composition of feed ingredients (Table 1) and amino acid analysis of unprocessed,
water soaked and autoclaved PJ seed meals along
with fish meal were performed (Table 2). Ten iso-nitrogenous (360 g kg)1 crude protein) and iso-energetic
(15 564 kJ kg)1 metabolic energy content) diets were
formulated (Table 3). Each of crude, water soaked and
autoclaved PJ seed meals was included in the experimental
diets at 200, 350 and 500 g kg)1 replacement of fish meal
and designated as D1–D9, respectively; for the control diet,

fish meal was used as a main protein source and
designated as RD (Table 3). To make all the feed
iso-nitrogenous, corn gluten (obtained from the local
market) was used to adjust total nitrogen content. Chromic
oxide was used as an external marker for the nutrient
digestibility study, and bentonite was used as a binder. The

Table 1 Proximate composition of diet ingredients and antinutritional factors (ANFs) of treated and untreated Prosopis juliflora (PJ) seed meal
(as g kg)1 dry matter unless otherwise stated)

Nutrients
Dry matter
Crude protein
Crude lipid
Ash
Crude fibre
NFE1
Gross energy (kJ kg)1)
ANFs2
Total phenols

Tannins
Phytic acids
Trypsin inhibitors
(TIU g)1)
Lignin

Fish
meal

Corn
gluten

Rice
bran

Untreated
PJ (PJN)

Soaked
PJ (PJ24)

Soaked + autoclaved
PJ (PJ24+)

965
604
101
156
30
75
19 079

914
632
79
39
22
112
19 790

911
140
103
85
70
564
14 225

902
330
119
30
38
386
15 857

906
329
118
28

37
395
15 815

907
328
118
30
37
398
15 773

1

3.7a
8.3a
3.4a
60.3a

0.03

0.14
0.17
0.11

3.6a ± 0.10
4.9b ± 0.09
3.4a ± 0.08
58.2a ± 0.96

3.5a ± 0.15
4.8b ± 0.13
3.2a ± 0.15
25.1b ± 0.36

3.6a ± 0.05

3.5a ± 0.05

3.4a ± 0.11

±
±
±
±

Nitrogen-free extract (NFE) calculated as 100 ) % (moisture ) crude protein ) crude lipid ) ash ) crude fibre).
Data are mean values ± SE. Values with the same superscript letters in the same row are not significantly different (P > 0.05) from each
other.

2

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Aquaculture Nutrition 17; e164–e173  2010 Blackwell Publishing Ltd
No claim to original US government works

Table 2 Amino acid composition of feed ingredients

Amino acid
(g per 16 g N2)

Fish meal
(javla)

PJ
normal

PJ 24 water
soaked

PJ 24+ water
soaked +
autoclaved

Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Cysteine
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Histidine
Lysine
Arginine
Tryptophan

9.43
3.32
5.33
12.0
0.27
8.0
9.62
0.47
8.01
8.60
6.73
8.02
2.03
3.24
1.28
9.32
1.13
2.09

8.73
3.49
4.98
28.39
5.02
7.77
7.39
0.31
5.02
0.35
2.70
8.60
1.93
4.68
3.43
0.74
0.93
2.48

10.49
2.98
4.32
27.03
4.41
7.33
7.26
0.42
5.05
0.65
2.90
9.09
2.25
4.92
3.28
0.75
1.31
2.55

10.75
3.22
4.56
26.51
3.81
7.38
6.93
0.29
4.87
0.38
2.65
8.22
1.83
4.84
3.22
0.95
1.77
2.45

Requirement
of carp1

1.3

1.2
0.6
0.9
1.6
2.1
1.2
0.6
1.5
0.2

PJ, Prosopis juliflora.
1
Data for essential amino acids requirement of carp from Ogino (1980).

Table 3 Ingredient and composition of the diets used in the study
Diets

Prosopis juliflora (PJ) seed meal
Reference
diet

Raw
D1

Ingredient (g kg dry weight)
Fish meal
400
294
PJ
–
200
Corn gluten1
120
120
Rice bran
394
300
25
25
Premix2
Oil premix3
50
50
01
01
Bentonite4
10
10
Cr2O35
Proximate composition (g kg)1 on dry matter basis)
Crude protein
372
361
Lipid
92
95
Ash
112
91
Crude fibre
44
41
Nitrogen-free extract
380
412
Gross energy (kJ kg)1)
15 899
15 606

Soaking
D2

D3

D4

Soaking + autoclaving
D5

D6

D7

D8

D9

)1

244
350
120
200
25
50
01
10

170
500
120
124
25
50
01
10

294
200
120
300
25
50
01
10

244
350
120
200
25
50
01
10

170
500
120
124
25
50
01
10

294
200
120
300
25
50
01
10

244
350
120
200
25
50
01
10

170
500
120
124
25
50
01
10

366
97
77
38
422
15 564

360
99
62
36
443
15 355

361
95
90
41
413
15 564

366
97
77
38
423
15 522

360
99
61
35
445
15 313

360
95
91
41
417
15 564

366
97
77
37
423
15 480

359
99
61
35
445
15 271

1

Corn gluten and Rice bran were purchased form Charotar Animal Feeds Pvt. Ltd., GIDC, V.V. Nagar, Gujarat, India.
Vitamin and mineral mixture (Vitaminetes Forte; Roche Products Ltd., Mumbai, Maharashtra, India).
3
Oil premix [2 corn oil (Tirupati active; N.K. Proteins Co., Mehsana, Gujarat, India) : 1 cod liver oil (Seacod; Universal Medicare Pvt. Ltd.,
Mumbai, Maharashtra, India)].
4
Bentonite purchased from Gujarat Minechem, Bhavnagar, Gujarat, India.
5
Chromic oxide (Cr2O3): Qualigens.
2

feed ingredients were mixed and made into moist pellets of
3 mm in diameter with hand pelletizer, oven dried at
55–60 C for 24 h and stored at 4 C.

Rohu fingerlings were obtained from Gujarat Govt.
Fish Seed Centre, Navali (Dist. Anand), acclimatized to

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Aquaculture Nutrition 17; e164–e173  2010 Blackwell Publishing Ltd
No claim to original US government works

laboratory conditions for 15 days and fed with a 1 : 1
mixture of finely powdered rice bran and groundnut oil
cake. The feeding trial was conducted in 150-L glass
aquaria (0.91 · 0.38 · 0.45 m3). Fingerlings (mean weight
3.82 ± 0.06 g) were stocked at a density of 25 fishes per
aquarium with three replicates for each treatment group.
Unchlorinated tube well water was used for the experiment.
The fishes were fed with the formulated feed twice a day at
9.00 and 15.00 h at the rate of 5% of the body weight per day
for 60 days. The fishes were weighed every week and the
feeding ration adjusted accordingly. During experimentation,
continuous aeration was provided and temperature was
maintained at 30 C. The water quality parameters like pH,
dissolved oxygen and total organic carbon were monitored
weekly following the methods of American Public Health
Association (APHA 1980) and are found to be 7.2–7.9,
6.2–7.2 and 107–115 mg L)1, respectively.
Faeces were collected once in the morning during the last
2 weeks of the experimentation by the method of Spyridakis
et al. (1989). Uneaten feed was siphoned out from the
aquaria after the last feeding in the evening. Faeces collected
from triplicate treatment groups were pooled, oven dried at
60 C and stored for digestibility study. Pooled faecal
samples for each treatment were analysed separately. At the
termination of experiment, five fishes were killed from
each aquarium and analysed for carcass composition and
intestinal digestive enzyme activity.

Feed ingredients, experimental feed, faecal samples and fish
carcass were analysed for their proximate composition following Association of Official Analytical Chemists (AOAC
1990) as follows: moisture was determined by oven drying at
105 C for 24 h, protein (N · 6.25) by Micro-Kjeldahl
digestion and distillation after acid digestion, ash by ignition
at 550 C in a Muffle furnace to constant weight, crude fibre
by Sigma kit (Catalog no. TDF-100A; Sigma, St. Louis,
Missouri, USA), lipid by Folch et al. (1957), total carbohydrate by anthrone method (Hedge & Hofreiter 1962) and
amino acids were analysed using HPLC method (Ishida et al.
1981; Central Institute of Fisheries Technology, Cochin,
Kerela, India). In processed and unprocessed test feed, total
phenol, tannin, phytic acid, TI and lignin were determined by
spectrophotometric methods (Stafford 1960; Kakade et al.
1969; Schanderi 1970; Wheeler & Ferrel 1971; Molick &
Singh 1980, respectively). The energy contents of the diets
were calculated using the average caloric conversion factors
9.45, 4.10 and 5.65 kcal g)1 for lipid, carbohydrate and

..............................................................................................

Aquaculture Nutrition 17; e164–e173  2010 Blackwell Publishing Ltd
No claim to original US government works

protein, respectively (Henken et al. 1986). Nitrogen-free
extract was computed by taking the sum of values for crude
protein, crude lipid, ash, crude fibre and moisture and subtracting this from 100 (Maynard et al. 1979). Chromic oxide
in the diets and faeces was estimated following the method of
Furukawa & Tsukahara (1966).
Chemicals used for the analysis of earlier mentioned
parameters were purchased from Qualigens; Qualigen Fine
Chemicals, Mumbai, Maharashtra, India.

The gut was rinsed with chilled distilled water, homogenized
with tissue homogenizer in cold (4 C) phosphate buffer pH 7.2
(1 : 10 w v)1), centrifuged (10 000 g · 10 min at 4 C) and
supernatant stored at )20 C for enzyme analysis. The concentration of soluble protein in pooled samples was determined
by the method of Lowry et al. (1951). In the supernatant,
a-amylase, protease and lipase activities were assayed by
using soluble starch (Qualigens), casein (Qualigens) and
olive oil (Figaro, Madrid, Spain) as substrates, respectively
(Bernfeld 1955; Kunitz 1974 and Rathelot et al. 1975).

The apparent nutrient digestibility (AD) of the diets was
calculated according to Cho et al. (1982), AD = 100 ) 100 ·
(% Cr2O3 in diet/% Cr2O3 in faeces) · (% nutrient in faeces/
% nutrient in diet).

Growth parameters were calculated according to Steffens
(1989). The data were subjected to a ANOVA, and the significance of the difference between means was determined by
TukeyÕs multiple range test (P < 0.05) using the SPSS
version 15 (Chicago, Illinois, USA). Values are expressed as
means ± SE.

The proximate composition of the feed ingredients and test
feed is shown in Table 1. There was not much variation in
nutrient content among different processed and unprocessed
PJ groups (PJN, PJ24 and PJ24+). The estimated ANF
contents of different PJ seed meal groups are presented in

0.07
0.21
6.92
0.54
0.10
0.11
0.07
3.71a ±
0.24
7.95ab ±
7.85 116.72ab ±
0.52
1.25ab ±
0.07
1.46cd ±
0.11
2.18ab ±
87.13
86.17
0.07
3.83 ±
0.28
9.32cd ±
9.75 146.36bc ±
0.63
1.47bc ±
0.08 1.15abc ±
0.14
2.62bc ±
90.02
89.55

D9

0.06
3.86 ±
0.22
8.25abc ±
6.69 116.84abc ±
0.46
1.25ab ±
0.04
1.39bcd ±
0.10
2.21ab ±
87.69
87.20
0.07 3.87 ± 0.07
3.89 ± 0.07
3.93 ±
0.25 7.49a ± 0.18
9.65d ± 0.25
9.12cd ±
7.68 96.11a ± 6.20 151.57c ± 8.64 134.09bc ±
0.56 1.09a ± 0.50 1.50c ± 0.53
1.39bc ±
d
ab
0.13 1.64 ± 0.08 1.08 ± 0.04 1.19abc ±
0.13 1.90a ± 0.10 2.77cd ± 0.13 2.44abc ±
86.65
90.92
89.82
85.40
90.13
89.30
0.04
3.83 ±
0.25 8.57abcd ±
7.13 126.48abc ±
0.53
1.32abc ±
0.23
1.28abc ±
0.12
2.43abc ±
89.28
88.60

Initial weight (g)
3.79 ± 0.07
3.74 ±
8.25abc ±
Final weight (g)
11.13e ± 0.32
Weight gain (%) 196.01d ± 8.91 121.60abc ±
SGR (% day)1)
1.77d ± 0.51
1.29abc ±
a
FCR
0.96 ± 0.04
1.33bcd ±
2.35abc ±
PER
3.24d ± 0.13
2
APD (%)
92.10
88.79
ALD (%)2
91.11
87.63

SGR: specific growth rate (% day)1) = (loge W2 ) loge W1)/t · 100.
FCR: feed conversion ratio = feed intake (g)/live weight gain (g).
PER: protein efficiency ratio = weight gain (g)/crude protein intake (g).
APD, apparent protein digestibility; ALD, apparent lipid digestibility.
1
Data are mean values ± SE. Values with the same superscript letters in the same row are not significantly different (P > 0.05) from each other.
2
Statistical analysis was not possible as determinations were performed on pooled samples.

0.07
3.80 ±
0.18 8.96bcd ±
6.58 139.66bc ±
0.52
1.40bc ±
0.08 1.25abc ±
0.10
2.54bc ±
89.92
90.04

a

D8
D7

a
a

a

D2

a

D3

a

D4

a

D5

a

D6

a

Soaking + autoclaving
Soaking
Raw

Prosopis juliflora seed meal

Reference diet D1
Parameters

Proximate composition of fish carcass and gut digestive
enzyme activity is presented in Table 5. Fishes fed RD, D4,

Diets

Rohu fingerlings readily accepted the experimental diets and
were observed to feed aggressively during experimentation.
No mortality was observed in any of the dietary groups during
the present study. The growth performance in terms of percentage weight gain, specific growth rate (SGR), feed conversion ratio (FCR) and protein efficiency ratio (PER) as well
as apparent protein digestibility (APD) and apparent lipid
digestibility (ALD) are presented in Table 4. Rohu fingerlings
fed with the RD had higher level growth performance in terms
of per cent weight gain, SGR, FCR and PER, whereas there
are not much differences in APD and ALD among different
diet groups (RD and D1–D9). Fishes exhibited higher growth
rate with processed seed meal as compared to unprocessed
seed meal at each individual inclusion level. Diet D4 (200 g
kg)1 replacement of fish meal with PJ24) produced better
growth in terms of per cent weight gain, SGR, FCR and PER
among experimental groups. As compared to RD in D4, the
values of FCR and PER are not significantly different,
whereas the values of SGR and per cent weight gain are significantly lower. Inclusion of processed and unprocessed seed
meal at the level of 500 g kg)1 replacement of fish meal (D3,
D6 and D9) depressed all the calculated parameters of growth
in comparison to inclusion at 200 and 350 g kg)1 level.

Table 4 Growth performance, feed utilization efficiencies and apparent digestibility in Labeo rohita fingerlings fed experimental diets for 60-days1

Table 1. Hydrothermical treatment of PJ seed meal resulted
in significant decrease (P < 0.05) in TI in all the autoclaved groups and in tannin (P < 0.05) in PJ24 as well as
PJ24+ groups (n = 3). Other ANFs like phytic acid, total
phenol and lignin are also decreased in processed groups
(Table 1). The amino acid content of fish meal and crude as
well as processed PJ seed meal is presented in Table 2. None
of the test feed was deficient in any of the EAAs except lysine
when compared to the requirement of carp (Ogino 1980).
However, the test feeds are found to be deficient in sulphur
amino acids (cystine + methionine) and lysine as compared
to the requirement of rohu (Singh 1987). In comparison to
test feed, methionine, lysine, valine and isoleucine levels were
observed to be higher in fish meal. Soaking of the seed meal
(PJ24) improved the level of majority of EAAs, whereas
they have been found decreased in autoclaved group (PJ24+)
as compared to unprocessed group (PJN).

..............................................................................................

Aquaculture Nutrition 17; e164–e173  2010 Blackwell Publishing Ltd
No claim to original US government works

..............................................................................................

Aquaculture Nutrition 17; e164–e173  2010 Blackwell Publishing Ltd
No claim to original US government works

2.3
1.1
0.4
0.4
0.15
0.05
0.11
±
±
±
±
±
±
±
777b
131a
42b
34bcd
1.12bc
0.32c
3.77b
±
±
±
±
±
±
±

3.8
1.1
0.2
0.4
0.15
0.05
0.19

D9

Data are mean values ± SE (n = 5). Values with the same superscript in the same row are not significantly different (P > 0.05) from each other.
a-Amylase = mg maltose liberated h)1 mg)1 protein; Protease = mg tyrosine liberated h)1 mg)1 protein; Lipase = U mg)1 protein.
2

1

761
139cd
45c
34bcd
1.12b
0.28b
3.78b
769
138bc
46c
33bcd
1.20d
0.31c
3.85c

755 ± 3.5
151e ± 6.0
49e ± 0.4
30a ± 0.2
1.53f ± 0.15
0.41e ± 0.07
4.97d ± 0.09

a

Moisture
Protein
Lipid
Ash
a-Amylase2
Protease2
Lipase2

Parameters

Reference diet

ab

±
±
±
±
±
±
±

2.6
1.5
0.5
0.8
0.13
0.01
0.04

D2

ab

±
±
±
±
±
±
±

2.3
1.2
0.5
0.6
0.09
0.01
0.10

D3

ab

774
131a
40a
35d
0.90a
0.25a
2.82a

±
±
±
±
±
±
±

2.5
1.5
0.5
0.3
0.18
0.02
0.16

ab

762
142cd
47cd
32abc
1.42e
0.34d
4.49d

±
±
±
±
±
±
±
D4
D1

6.8
1.2
0.3
0.6
0.23
0.01
0.21

D5

ab

764
140cd
46cd
32abc
1.10cd
0.30c
3.96c

±
±
±
±
±
±
±

4.3
0.9
0.3
0.5
0.10
0.02
0.15

D6

b

778
133ab
41ab
34cd
0.95a
0.30c
3.67b

±
±
±
±
±
±
±

5.6
1.5
0.4
0.3
0.11
0.03
0.13

764
143cd
48de
31ab
1.39e
0.36d
4.41d

±
±
±
±
±
±
±

8.2
0.9
0.4
0.5
0.07
0.02
0.16

763
145d
48de
30a
1.39e
0.35d
4.35d

ab

D8

ab

D7

Soaking + autoclaving
Soaking
Raw

Prosopis juliflora seed meal
Diets

The results show that the use of hydrothermically processed
PJ seed meal as a plant protein source improves the growth
of rohu fingerlings as compared to unprocessed seed meal at
their respective inclusion levels. The diet with soaked PJ seed
meal (D4) at 200 g kg)1 replacement of fish meal exhibited
higher growth of the fishes among the groups with different
test diets, though it was found to be lower as compared to
RD. Inclusion of processed seed meal at higher levels and
unprocessed seed meal at all inclusion levels failed to achieve
the growth of the fishes to a level of performance obtained
with fish meal–based RD (Table 4). It is well documented
that inclusion of plant protein sources in fish diets results in
reduced growth, and this is likely to be caused by the presence of ANFs and amino acid imbalance in plant-based feed
(Hossain et al. 2001).
Soaking and autoclaving of PJ seed meal resulted in
decrease in ANFs like tannin (P < 0.05), TI (P < 0.05)
(PJ24+), phytic acid, total phenol and lignin (Table 1).
Hossain et al. (2001), Garg et al. (2002) and several other
researchers have also reported the reduction in ANFs of
legume seeds by hydrothermical treatment. The presence of
tannin has been associated with lower nutritive value and
lower biological availability of protein, carbohydrate, amino
acids, vitamins and minerals (Makkar et al. 1987). Common
carp has been shown to tolerate addition of 2% quebracho
tannin without any adverse effect on the growth (Francis
et al. 2001). In the present study, 4.9 and 4.8 g kg)1 tannin
content of soaked (PJ24) and autoclaved (PJ24+) groups as
well as 8.3 g kg)1 tannin content of unprocessed group (PJN)
(Table 1) seem to be low to cause any adverse effect on the
growth of rohu fingerlings. TI has been reported to inhibit
growth of animals. Commercial soybean products are
reported to show 2–6 mg g)1 TI activity (Synder & Kwon
1987). Carp has not exhibited negative effect on growth when
fed with untreated and heat-treated jatropha meal with high
TI activity (Makkar & Becker 1999). Hossain et al. (2001)
have also ruled out the growth-inhibiting effect of TI
in common carp when fed with sesbania seeds with

Table 5 Proximate composition (g kg)1, wet weight) of the carcass and digestive enzyme activity of the experimental fishes at the end of the 60-day feeding experiment1

D7 and D8 had the higher carcass crude protein and crude
lipid as well as higher gut a-amylase, protease and lipase
activities. In the diet groups (D3, D6 and D9) with 500 g
kg)1 replacement of fish meal with processed and unprocessed seed meal protein and lipid deposition in the carcass
and gut digestive enzyme activities were lowest among all the
diet groups. Carcass moisture and ash content were lowest in
the fishes fed with RD.

4.7–5.2 mg g)1 of TI activity. In the present experiment,
significant reduction in TI activity (P < 0.05) has been
measured in autoclaved group (from 60.3 to 25.1 TIU g)1).
Low TI activity in all the test feed groups is unlikely to have
caused inhibition of growth in rohu (Table 1). Processing is
also found to be effective in reducing the level of phytic acid
in PJ seed meal. Phytic acid in feed is known to reduce the
bioavailability of minerals and proteins; however, the
growth in commonly cultivated fishes like carp, tilapia, trout
and salmon is reported not to be affected by phytatecontaining ingredients in the diet (Francis et al. 2001). Low
phytic acid content in processed and crude PJ seed meal
(3.2–3.4 g kg)1) (Table 1), mineral supplementation in diet
and availability of phosphorous in fish meal suggest
that presently observed phytic acid content of different
diets should not have inhibited the growth of rohu. Processing is also found to be effective in reducing the level of
total phenol and lignin (Table 1). In different PJ meals, total
phenols and lignin were found to be 3.5–3.7 and 3.4–
3.6 g kg)1, respectively (Table 1). Results suggest that
soaking and autoclaving of PJ seed meal resulted in decrease
in TI, phytic acid, tannin, total phenol and lignin; they do not
seem to be responsible for inhibiting the growth of rohu
fingerlings.
Test feeds are not found to be deficient in EAAs except
cysteine, methionine and lysine as compared to the requirement of carps (Ogino 1980; Singh 1987). In comparison to
the amino acid level of fish meal, valine, methionine, isoleucine and lysine are found to be lower in all the PJ groups
(Table 2). Soaking (PJ24) of PJ seed meal improved the level
of EAAs content as compared to unprocessed (PJN) and
autoclaved (PJ24+) groups (Table 2). In all the PJ seed
meals, EAAs are found to be higher as compared to mustard
oil cake and linseed meal (Mukhopadhyay & Ray 2001). In
the present study, autoclaving has resulted in some negative
effect on the EAA composition of PJ meal except lysine and
arginine as compared to unprocessed and soaked groups
(Table 2). During autoclaving, extended heating and over
processing may have denatured the protein and caused
reduction in amino acid availability. Hossain & Jauncey
(1990) have reported reduction in lysine in linseed and sesame seed after autoclaving. Autoclaving of sesbania meal has
not lowered its EAAs content except histidine (Hossain et al.
2001). Calculated EAAs showed that except in cystine,
methionine and lysine, none of the PJ groups were deficient
in other EAAs.
The results of the present study show that hydrothermically processed PJ seed meal in the feed of rohu found to be
effective in improving the growth in terms of per cent weight

gain, SGR, FCR and PER as compared to unprocessed seed
meal, but not to the level of growth performance achieved
with fish meal–based RD (Table 4). Fishes grew poorly when
fed with diet containing graded level of unprocessed seed
meal (D1, D2 and D3). The growth of the D4 fishes was
moderately higher as compared to fishes fed with other test
diets, whereas it was lower than those fed with RD. Inclusion
of prosopis seed meal at 20% replacement of soybean meal in
the diet of broiler chicken has been reported to have an
economic advantage (Yusuf et al. 2008). Inclusion of
processed PJ seed meal at higher replacement level (D6 and
D9) is found to depress the growth of the fishes (Table 4).
Mohanty et al. (1995) also reported poor growth rate in rohu
fry fed diets containing graded levels of oil cakes. Deficiency
of sulphur amino acids and lysine could be responsible for
presently observed lower growth rate of experimental fishes.
Another factor appears to be responsible for the reduced
growth could be PJ gum (galactomannans), a non-starch
polysaccharide. PJ seeds are reported to be rich in PJ seed
gum (galactomannans), the main storage carbohydrate lying
between the seed coat and cotyledons (Azero & Andrade
2006; Gallao et al. 2007). Guar gum galactomannan is
reported to be responsible for reducing the availability of
nutrients in the diets of salmonid and is found to be one of
the factors responsible for hampering the growth of carp
(Storebakken 1985; Garg et al. 2002). Thus, galactomannan
content of PJ seed could be responsible for reduced growth of
rohu. Among the test diets, higher growth of fishes with
soaked seed meal at 20% replacement level (D4) may be
attributed to the availability of EAAs because of the partial
replacement of fish meal and presence of ANFs in permissible limit in the formulated feed. The results suggest that
hydrothermically processed PJ seed meal appear to be a
better plant protein source for increasing the growth of IMCs
as compared to raw and processed soybean, moong, cowpea
and guar reported by Garg et al. (2002) in the feed for rohu.
Our results are in agreement with the findings of Garg et al.
(2002) and Hossain et al. (2001) who have reported
improvement of nutritional quality of leguminous seeds by
hydrothermical treatment. Fishes fed with RD exhibited
highest per cent weight gain (P < 0.05), SGR (P < 0.05),
FCR and PER. Not much difference is observed in the values
of FCR among the groups with different test diets and it was
found to be lower in RD. The value of PER in D4 is higher
among the groups with different test diets, whereas its value
in RD is greater. This can be attributed to the availability of
good quality protein to the fishes and presence of ANFs in
permissible limit in the test diets. The FCR and PER values
obtained in the present studies were better than those

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Aquaculture Nutrition 17; e164–e173  2010 Blackwell Publishing Ltd
No claim to original US government works

obtained by Mukhopadhyay & Ray (2001) and Garg et al.
(2002) for plant protein–based diet.
Carcass composition and intestinal enzyme activities of
fishes were influenced by the processing of seed meal. Fishes
fed with soaked and autoclaved seed meal at lower inclusion
level (D4, D5, D7 and D8) had higher crude protein and
lipid content as compared to their higher inclusion levels
(D3, D6, D9) (Table 5). Intestinal a-amylase, protease and
lipase activities were found to be higher in the fishes fed
with soaked (D4) and autoclaved groups (D7, D8) as
compared to crude seed meal (D1–D3). Moisture and ash
content of carcass are found to be similar with different test
diets, though they have increased with higher levels of
inclusion of test feed (D3, D6 and D9) and are also found
to be higher as compared to RD. Highest crude protein and
lipid content as well as lowest moisture and ash content of
carcass with highest intestinal enzyme activity have been
observed in fishes fed with the RD (Table 5). Similar trend
in muscle protein and fat levels were reported in Oreochromis niloticus (Keembiyehetty & De Silva 1993) and rainbow trout (Gomes et al. 1993) fed with higher amounts of
cowpea and black gram meal as well as pea and rapeseed
meals, respectively. This observation is in agreement with
Hossain et al. (2001) who reported significantly higher
moisture and lowest lipid content of whole body with higher
level of sesbania meal in the feed of common carp. Saha &
Ray (1998) also observed decrease in muscle protein and fat
content as well as significantly low digestive enzyme activities with increasing level of Chuni in the diet of rohu.
Deficiency of some of the EAAs and probably presence of
non-starch polysaccharides (NSPs) could be responsible for
reduced deposition of protein and lipid as well as increased
deposition of moisture and ash with increasing concentration of test feed. The muscle protein and lipid content and
digestive enzyme activities correlate with the growth pattern
of fish fed on different experimental diets.
The APD and ALD of processed PJ seed meal at lower
inclusion level (20% and 35%) were found to be quite similar
to fish meal–based RD, whereas they were lower with higher
inclusion (50%) of seed meal (Table 4). Mukhopadhyay &
Ray (2001) observed decreased APD with increased level of
incorporated linseed meal protein in rohu fingerlings. Gomes
et al. (1993) reported that inclusion of coextruded pea and
rapeseed meal in the diet of rainbow trout at 16–24%
inclusion level improved digestibility; whereas at higher
inclusion level, digestibility was found to be hampered. The
pattern of presently observed nutrient digestibility of the
diets corresponds to the growth trend of fishes and nutritional quality of test diet.

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Aquaculture Nutrition 17; e164–e173  2010 Blackwell Publishing Ltd
No claim to original US government works

The results of the present study show that processing of PJ
seed meal improved the nutritional quality of test diet and
growth performance of fishes as compared to unprocessed
seed meal, although not to the level of performance obtained
with fish meal–based RD. However, processed seed meal can
be used in the diet at a lower level replacement of fish meal
looking to its easy availability and nutrient utilization in fish.
Reduced growth performance of fishes at higher inclusion
level of PJ seed meal might be related to amino acid imbalance and presence of non-starch polysaccharides (galactomannans) of PJ seed meal.

The authors thank the University Grant Commission (UGC)
for the research grant [UGC Major Research Project #
No.F.31-228/2005 (SR)] provided for this investigation. The
authors are very much grateful to the reviewers for very
many constructive suggestions that led to thorough revision
substantially improving the manuscript.

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