Aquaculture Reports 14 (2019) 100202

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Aquaculture Reports
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Fish performance, nutrient digestibilities, and hepatic and intestinal T

morphologies in grouper Epinephelus fuscoguttatus fed fermented copra meal
⁎
Roger Edward P. Mamauaga, , Janice A. Ragazab, Trisha Nacionalesa
a
Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo, 5021, Philippines
b
Department of Biology, Ateneo de Manila University, Katipunan Avenue, Loyola Heights, Quezon City, NCR, 1108, Philippines

A R T I C LE I N FO A B S T R A C T

Keywords: Protein enhanced copra meal (PECM®) is an alternative, cheap, and sustainable source of plant protein for the
Protein enhanced copra meal aquafeed industry, albeit its use on carnivorous fish species has been very limited. A 70-day feeding trial using
Grouper grouper Epinephelus fuscoguttatus (initial mean body weight of 1.86 ± 0.19 g) tested fermented copra meal as
Digestibility feed ingredient. Six isonitrogenous (crude protein of 45%) and iso-lipidic (crude fat of 11%) diets consisted of
Hepatic and intestinal morphology
PECM®: a control diet at 0% soybean meal replacement (C); four diets replacing soybean meal at 25% (FC25),
Alternative plant protein source
Fermented copra meal
50% (FC50), 75% (FC75), 100% (FC100) – all with methionine and lysine supplementation; and 100% soybean
replacement without methionine and lysine supplementation (FCW100). Growth and feed performance were not
significantly (P > 0.05) affected by PECM® replacement of soybean meal up to 100%, even without methionine
and lysine supplementation. Chemical body composition was likewise not significantly (P > 0.05) altered.
PECM® when used as a grouper feed ingredient has protein, lipid, carbohydrate and dry matter digestibilities of
89.28%, 78.63%, 82.57%, and 48%, respectively. Hepatic and intestinal morphologies displayed no apparent
pathological changes. PECM® can be efficiently utilized by grouper and can replace soybean meal up to 100%
(16% in diet) for normal fish performance and digestive organ functions.

1. Introduction
Soybean meal is a staple aquafeed ingredient and proven beneficial
(Ayadi et al., 2012). However, its price has increased significantly for
the past years and supply has created a demand conflict between
aquaculture feed and human food. Candidate plant protein sources
when compared to fish meal protein are found insufficient in protein
and essential amino acids. Increased efficiencies are often achieved
through processing and addition of amino acid/s to the feed (Rust,
2003).
Coconut production is an important contributor to the Philippine
economy. The Philippines is the world’s largest producer of coconut –
with 19,500,000 tons produced in 2009 (FAO, 2014). Copra meal
comes from oil-extracted dried coconut kernels produced from coconut
product facilities. Copra meal, albeit a by-product of coconut produc-
tion contains 23% protein, 12.91% lipid, 6.6% fiber, and 39.29% car-
bohydrates (Mukhopadhyay and Ray, 1999). It is a cheap source of
protein for poultry, livestock, and fish (Guarte et al., 1996; Knudsen and
Jensen, 1997). However, raw copra meal is characterized by inferior
amino acid profile, poor digestibility (Thorne et al.,1990; Sundu
et al.,2009), ubiquitous anti-nutrients (Mukhopadhyay and Ray, 1999;
Sundu et al., 2009) and aflatoxin (Head et al., 1999), high fiber content
(Dairo and Fasuyi, 2008; Diarra and Malouin, 2014), and low bulk
density (Sundu et al., 2014). The nutrient profile of raw copra meal can
be improved through several physico-chemical processing. Several
studies have indicated that raw and processed or soaked copra meal can
be incorporated in rohu (Labeo rohita) diets (Mukhopadhyay and Ray,
1999); tilapia Sarotherodon mosambicus (Jackson and Capper, 1982);
and carp Cyprinus carpio (Hasan et al., 1997) at 25 to 30% inclusion
levels without causing a significant decrease in performance para-
meters.
Recent studies have also shown that processing plant-based in-
gredients has significantly improved nutrient profile and digestibility
while eliminating anti-nutrients (Lee et al.,2016; Mamauag et al.,
2011). Enzymatic hydrolysis is a process wherein commercially avail-
able enzyme is introduced into a bio-processing system using a solid-
state fermentation process converting under-utilized or poor nutrient
profile ingredient to functional protein (Kristinsson and Rasco, 2000).
Products of enzymatic hydrolysis have proteins of low molecular
weight, with good amino acid profile, and high digestibility compared
to unprocessed protein with its high molecular weight (Mamauag et al.,
2011).

⁎
Corresponding author.
E-mail address: ermamauag@seafdec.org.ph (R.E.P. Mamauag).

https://doi.org/10.1016/j.aqrep.2019.100202
Received 28 February 2019; Received in revised form 19 April 2019; Accepted 2 June 2019
Available online 13 June 2019
2352-5134/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/BY/4.0/).
R.E.P. Mamauag, et al. Aquaculture Reports 14 (2019) 100202

Table 1 replacement without methionine and lysine supplementation

Feed formulation and proximate composition of six diets using protein en- (FCW100). Amino acid supplementation levels in the treatment diets
hanced copra meal PECM® as soybean meal replacement (g 100g−1). were derived from the difference in methionine and lysine amino acid
Ingredients PECM® C FC25 FC50 FC75 FC100 FCW100 levels from the replacement of soybean meal with PECM®. All diets
formulated were isonitrogenous and iso-lipidic, containing crude pro-
Peruvian fish meal1 40 40 40 40 40 40 tein and crude fat of 45% and 11%, respectively, to meet nutrient re-
Defatted soybean 16 12 8 4 0 0
quirements of juvenile grouper (Millamena and Golez, 2001). The main
meal2
Shrimp meal 10 10 10 10 10 10 dietary protein sources are derived from fish meal (crude protein,
Squid meal 5 5 5 5 5 5 68.3% and crude lipid, 5.9%) and defatted soybean meal (crude pro-
Rice bran3 5 4.95 4.91 4.87 4.83 5 tein, 43.6% and crude lipid, 1.5%). The PECM® contains 41% crude
PECM®4 0 4 8 12 16 16
protein and 5% crude fat. After milling (Restch ZM 200, Haan, Ger-
Bread flour5 8 8 8 8 8 8
Cod liver oil6 4 4 4 4 4 4
many) and sieving, dry ingredients were mixed, followed by liquid in-
Soybean oil7 6 6 6 6 6 6 gredients (blended oils and water), and finally by cooked bread flour in
Vitaminmix8a 3 3 3 3 3 3 a food processor. The dough produced 1.2–2.2 mm diameter pellets
Mineral mix8b 3 3 3 3 3 3 using a pelletizer. Pellets retained 15% moisture by drying using a
Methionine 0 0.03 0.05 0.07 0.09 0
convection oven for 4 h at 60 C.
Lysine 0 0.02 0.04 0.06 0.08 0
Proximate
Composition (in
2.2. Feeding setup and trial
%DM)
Crude protein 41.32 45.17 45.31 45.24 45.50 45.96 45.72
Crude fat 5.47 11.99 11.53 11.04 11.18 11.69 11.90 Juvenile groupers obtained from a marine fisheries hatchery
Ash 8.19 11.98 12.13 11.93 11.80 11.89 12.26 (Southeast Asian Fisheries Development Center, Aquaculture
NFE9 43.18 23.82 19.85 21.50 18.72 24.23 24.12 Department, Tigabauan, Iloilo) were acclimated for 2 weeks and fed
Crude fiber 5.82 3.04 3.18 3.38 2.79 3.22 4.00
control diet at Fish Nutrition Wet Laboratory (SEAFDEC/AQD,
1
Marcela Farm, Bohol, Phil., crude protein, 68.3%; lipid, 5.9. Tigbauan, Iloilo). After acclimation, triplicate groups of 15 juvenile
2
La Filipina Uy Gonco Inc., Iloilo, Phil., crude protein, 43.6%; lipid, 1.5. groupers (1.86 ± 0.19 g) were kept in 250-L tanks. Twice daily at 0800
3
MCR Agri Venture, Phil. and 1600 h, groupers were hand-fed experimental diets to apparent
4
Potein enhanced copra meal, National Institute of Molecular Biology and satiation. To calculate for actual feed intake, uneaten feeds were re-
Biotechnology, University of the Philippines Los Baños, Phil. moved. Water quality was monitored daily with temperatures 27 to 29
5
Roger’s Trading Corp., Phil. C, salinity 31 to 33 g kg−1, and pH levels 8.2 to 8.4 throughout the 70-
6

7
Alyson Chemical Enterprises, Phil. day feeding period. Seawater flow of 2.4 L min−1 to the tanks and 12 h:
Trans Asia Philippines Manufacturing Industrial Corp., Phil. 8Feed Source
12 h, light: dark conditions were sustained.
Trading, CKCIT, Manila, Phil.
8a
Vitamin mix (kg−1): Vitamin A (1,200,000 IU), D3 (200,000 IU), E
(20,000 IU), B1 (8000 mg), B2 (8000 mg), B6 (5000 mg), B12 (2000 mg), niacin 2.3. Chemical composition of feed and carcass
(40,000 mg), calcium pantothenate (20,000 mg), biotin (40 mg), folic acid
(1800 mg), and ethoxyquin (500 mg). Chemical composition of both feed and grouper carcass were ana-
8b
Mineral mix (kg−1): iron (40,000 mg), manganese (10,000 mg), zinc lyzed. Twenty groupers from the stock tank served as initial samples for
(40,000 mg), copper (4000 mg), iodine (1800 mg), cobalt (20 mg), and sele-
whole body chemical composition. After the trial, three groupers per
nium (200 mg).
9 tank were randomly selected and freeze-dried. Crude protein, crude
Nitrogen free extract, calculated by difference.
lipid, moisture and ash were quantified with AOAC methods (AOAC,
1995). Profile of PECM® (Table 2) and diet (Table 3) total amino acids
Fermented copra meal has been previously utilized as black tiger
were analyzed in triplicate by the Pacific Lab Services, Singapore
shrimp (Penaeus monodon) aquafeed to replace dietary protein from fish
meal for up to 40% without adversely affecting growth, feed efficiency,
Table 2
and survival (Apines-Amar et al., 2016). Furthermore, the same in-
PECM® total amino acid profile (g 100g−1 PECM®).
gredient can be used to replace soybean meal in milkfish diet for up to
Each value is expressed as mean ± S.E.M. of data from
20% without affecting performance parameters and when used in triplicate groups.
longer duration in commercial cage culture systems (Apines-Amar
Amino acid %
et al., 2015). With the increasing by-products derived from coconut oil
production, copra meal has a huge potential to be an alternative, cheap, Alanine 1.00 ± 0.04
and sustainable source of protein for fish feed industry. There are Arginine 2.05 ± 0.11
limited research works on inclusion of dietary fermented copra meal for Aspartic acid 1.73 ± 0.21
carnivorous fish species. Hence, this research tested fermented copra as Cystine 0.48 ± 0.20
Glutamic acid 4.24 ± 0.14
plant protein source to replace soybean meal in grouper Epinephelus Glycine 1.12 ± 0.30
fuscoguttatus diets. Histidine 0.71 ± 0.21
Isoleucine 0.61 ± 0.10
Leucine 1.38 ± 0.25
2. Materials and methods Lysine 0.71 ± 0.03
Methionine 0.35 ± 0.02
Phenylalanine 1.01 ± 0.11
2.1. Grouper diets Proline 0.80 ± 0.24
Serine 0.90 ± 0.07
Six diets (Table 1) were prepared with varying protein enhanced Taurine ND1
copra meal PECM® levels (National Institute of Molecular Biology and Threonine 0.70 ± 0.14
Tryptophan 0.21 ± 0.06
Biotechnology, University of the Philippines-Los Baños): a control diet
Tyrosine 0.71 ± 0.17
at 0% soybean meal replacement (C); four diets replacing soybean meal Valine 1.17 ± 0.11
at 25% (FC25), 50% (FC50), 75% (FC75), 100% (FC100) – all with
1
methionine and lysine supplementation; and 100% soybean Not detected.

2
R.E.P. Mamauag, et al. Aquaculture Reports 14 (2019) 100202

Table 3
Analyzed total amino acid profile of the diets (g 100 g−1 diet). Each value is expressed as mean ± S.E.M. of data from triplicate groups.
Amino acid C FC25 FC50 FC75 FC100 FCW100

Alanine 2.10 ± 0.22 2.18 ± 0.18 2.10 ± 0.19 2.21 ± 0.18 2.09 ± 0.16 2.10 ± 0.15
Arginine 3.01 ± 0.14 2.87 ± 0.14 3.09 ± 0.15 3.07 ± 0.18 2.97 ± 0.15 2.94 ± 0.17
Aspartic acid 1.43 ± 0.10 1.38 ± 0.09 1.44 ± 0.08 1.48 ± 0.14 1.45 ± 0.07 1.42 ± 0.08
Cystine 0.99 ± 0.09 1.09 ± 0.12 0.97 ± 0.11 0.99 ± 0.11 1.01 ± 0.14 0.92 ± 0.12
Glutamic acid 5.69 ± 0.20 5.47 ± 0.22 5.98 ± 0.21 5.78 ± 0.24 5.91 ± 0.26 5.97 ± 0.28
Glycine 1.95 ± 0.18 2.08 ± 0.13 1.92 ± 0.15 1.97 ± 0.14 2.02 ± 0.10 1.94 ± 0.11
Histidine 1.08 ± 0.10 1.09 ± 0.10 1.10 ± 0.12 1.09 ± 0.13 1.09 ± 0.11 1.06 ± 0.14
Isoleucine 0.98 ± 0.14 1.03 ± 0.15 0.99 ± 0.14 1,00 ± 0.09 0.99 ± 0.10 1.00 ± 0.08
Leucine 1.72 ± 0.12 1.75 ± 0.12 1.83 ± 0.99 1.70 ± 0.12 1.72 ± 0.13 1.85 ± 0.14
Lysine 2.73 ± 0.10 2.89 ± 0.18 2.75 ± 0.13 2.81 ± 0.12 2.79 ± 0.12 2.85 ± 0.13
Methionine 1.24 ± 0.14 1.33 ± 0.13 1.28 ± 0.13 1.25 ± 0.11 1.27 ± 0.10 1.31 ± 0.08
Phenylalanine 1.15 ± 0.09 1.14 ± 0.12 1.17 ± 0.11 1.14 ± 0.14 1.14 ± 0.15 1.13 ± 0.14
Proline 1.08 ± 0.14 1.10 ± 0.16 1.07 ± 0.13 1.10 ± 0.12 1.09 ± 0.16 1.20 ± 0.17
Serine 0.95 ± 0.15 1.00 ± 0.17 0.96 ± 0.20 0.99 ± 0.23 1.00 ± 0.21 1.00 ± 0.25
Taurine ND1 ND1 ND1 ND1 ND1 ND1
Threonine 1.07 ± 0.21 1.13 ± 0.26 1.07 ± 0.25 1.11 ± 0.19 1.11 ± 0.20 1.08 ± 0.21
Tryptophan 0.47 ± 0.18 0.52 ± 0.16 0.50 ± 0.14 0.49 ± 0.12 0.50 ± 0.11 0.48 ± 0.10
Tyrosine 0.91 ± 0.11 0.98 ± 0.11 0.95 ± 0.12 0.92 ± 0.11 0.95 ± 0.10 0.92 ± 0.11
Valine 1.38 ± 0.20 1.37 ± 0.18 1.38 ± 0.17 1.40 ± 0.15 1.34 ± 0.16 1.40 ± 0.14

1
Not detected.

(AOAC 985.28, 994.12). 2.6. Statistical analysis

Data and differences among dietary treatments were tested using

2.4. Digestibility experiment
As an ingredient, PECM® was tested for apparent nutrient digest-
ibilities. Two diets: reference diet and test diet were formulated, with
the former similar to the control diet (Table 4). The test diet consisted
of 30% PECM® and the rest from each ingredient of the reference diet
(Cho et al., 1982). Chromic oxide was used as indicator for both diets.
For 4 days, randomly stocked groupers (˜20 g, 10 groupers per tank in
triplicate) were fed control diet (without indicator). Twice daily at
0700 and 1500 h, groupers were fed reference and test diets. After 3
days, fecal matter was collected immediately after excretion. Feces
were collected for 21 days. Uneaten feeds were also removed. The
nutrient digestibility coefficients were then calculated (Cho et al.,
1982).
2.5. Hepatic and intestinal histology
Haematoxylin and eosin stained distal intestine samples of 5 μm
were examined with a light microscope (Olympus CX41-RF, Tokyo,
Japan) with images recorded using a digital camera (Olympus DP22,
Tokyo, Japan). Hepatic and intestinal morphologies were evaluated
based on the methods of Hu et al (2013), Kroghdahl et al. (2003), and
Caballero et al. (2004). Hepatosomatic indices were also calculated by
measuring hepatic weight against whole body weight in triplicate.
Table 4
Diets used for the in vivo digestibility testing of PECM® (g kg−1).
Ingredient Reference
one-way analysis of variance and Tukey’s test as post-hoc at a sig-
nificant level of P < 0.05 (IBM SPSS ver. 20, IBM Corporation,
Armonk, NY, USA).
3. Results
3.1. Growth performance, feed efficiency, proximate composition, and
nutrient digestibilities
PECM® did not significantly (P > 0.05) affect both feed conversion
efficiency (FCE) and feed intake (FI) (Table 5). High (> 80%) survival
rates were evident and similar among treatments (P > 0.05). In all
treatments, percent weight gain was similar (P > 0.05). However,
groupers fed diets with 30% PECM® showed a higher numerical value.
Whole-body composition showed insignificant (P > 0.05) difference
among dietary treatments. PECM® was calculated to have apparent
nutrient digestibility for protein of 89.28%; 78.63% for fat; 82.57% for
carbohydrates; and 48% for dry matter. All diets showed similar amino
acid profiles.
3.2. Histology
There were no anomalies found in the structure of the digestive tract
(Fig. 1) in all treatments (P > 0.05). The mucosal epithelium consisted
of normal enterocytes with prominent villi and microvilli. Average-
sized enterocytes with vacuoles were present in the lamina propria.
Goblet cells were also well-distributed in the mucosal epithelium. He-
patic cells showed homogenous hepatocyte nuclei and vacuolation
(Fig. 2). Moreover, hepatosomatic indices were similar (P > 0.05).
PECM®

Danish fish meal 40 28 4. Discussion and conclusion

Squid meal 5 3.5
Shrimp meal 10 7
The current research shows partial replacement of soybean meal
Soybean, defatted 16 11.2
Wheat flour 8 5.6 with fermented copra meal up to 16% (6% of the dietary total protein)
Rice bran 9 6 even without methionine and lysine supplementation in diets of
Cod liver oil 2.5 1.75 grouper, a carnivorous species. Fish growth, feed utilization, proximate
Soybean oil 2.5 1.75 composition, and hepatic and intestinal morphologies were not sig-
V/M mix 6 4.2
nificantly affected by the addition of PECM®. Previous studies reported
PECM® 0 30
Chromic oxide 1 1 that even at low levels, diets with unprocessed copra meal cause sig-
nificantly depressed growth and feed efficiencies (Mukhopadhyay and

3
R.E.P. Mamauag, et al. Aquaculture Reports 14 (2019) 100202

Table 5
Performance parameters and whole-body proximate composition (% DM) of groupers.
Initial fish C FC25 FC50 FC75 FC100 FCW100
Performance parameters

Initial body weight (g) 1.82 ± 0.10 1.89 ± 0.06 1.88 ± 0.02 1.88 ± 0.05 1.85 ± 0.07 1.92 ± 0.02
Final body weight (g) 18.78 ± 1.33 19.13 ± 2.20 19.06 ± 1.69 21.05 ± 0.87 19.83 ± 1.39 20.84 ± 1.23
Percent weight gain (%) 931 ± 8.2 914 ± 10.8 916 ± 11.72 1011 ± 9.4 974 ± 15.01 985 ± 12.39
Survival (%) 73.3 ± 3.84 68.9 ± 5.87 66.7 ± 7.69 75.6 ± 5.87 71.1 ± 9.68 73.3 ± 3.84
Feed intake1 338.3 ± 18.79 363.6 ± 8.16 341.6 ± 2.75 367.2 ± 13.63 324.9 ± 4.23 344 ± 11.05
FCE2 0.53 ± 0.02 0.47 ± 0.08 0.46 ± 0.01 0.59 ± 0.05 0.57 ± 0.11 0.59 ± 0.06
Proximate Composition
Crude protein 63.16 55.71 54.39 53.70 55.03 52.22 54.23
Crude fat 13.72 24.21 27.14 26.58 24.47 27.04 26.13
Ash 17.66 18.91 16.85 17.69 18.25 17.46 16.25
NFE 5.40 1.07 1.56 1.92 2.22 3.27 3.37
Fiber 0.16 0.09 0.05 0.09 0.02 ND3 0.01

Each value is the mean ± S.E.M. of data from triplicate groups. Within a row, absence of letters indicates no significant difference between treatments.
1
Feed intake (g dry diet per fish per 70 days).
2
Feed conversion efficiency = wet weight gain (g) /dry feed intake (g).
3
Not detected.

Ray, 1999). Poor growth, feed utilization, and feed palatability are
mainly attributed to the high tannin content in copra meal which ne-
gatively affects feed palatability, feed efficiency, and nutrient absorp-
tion. Due to its high fiber content and anti-nutrients, inclusion levels of
unprocessed copra to aquafeed are limited to 15% for omnivorous fish
and 10% for carnivorous fish species (Obirikorang et al., 2016). Al-
though several studies have suggested soaking copra meal prior to feed
inclusion for rohu (Mukhopadhyay and Ray, 1999) and 68% inclusion
level for tilapia (Obirikorang et al., 2016), the two fish species are
mainly herbivorous species or require minimum amount of protein in
their diets.
The PECM® was produced through a bio-processing system using
solid state fermentation employing the fungus Aspergillus niger in di-
gesting the protein of copra meal (Pham, 2017). Agro-industrial car-
bohydrate wastes are characterized by the presence of cellulose, lignin,
and hemicelluloses which are not fully utilized as energy due to its poor
digestibility (Hertrampf and Piedad-Pascual (2012)). A. niger has been a
standard ingredient to produce citric acid and extracellular food en-
zymes to digest the cellulose component of plant feeds (Thongsook and
Chaijamarus, 2014). The PECM® produced from A. niger fermentation
was initially tested on poultry and tilapia feeds replacing soybean meal,
results of which showed no significant difference in both feed efficiency
and growth (Pham, 2017). Processed copra meal was used in diets of
milkfish Chanos chanos and resulted in significant improvement of
weight gain at 5% inclusion level. Moreover, higher copra meal level up
to 20% did not show any noticeable difference with the control treat-
ment (Amar et al., 2015). It was likewise tested on black tiger shrimp
Penaeus monodon and showed that fermented copra meal can be in-
corporated in the diet by as much as 40% without affecting feed effi-
ciency, survival, and growth (Amar et al., 2016).
Result of the current research indicates that PECM® can be utilized
as dietary plant protein for carnivorous grouper. Copra meal is rich in
mannooligosaccharides – β-1,4-mannooligosaccharides (MOS), which
have been reported to modulate innate immune response in macro-
phage model in mouse, rendering MOS as a prebiotic and im-
munostimulant (Mulimani and Naganagaouda, 2010; Ng et al., 2010).
Through enzymatic processing, the MOS-containing β-mannooligosa-
caccharide from copra meal can be extracted. Rungrassamee et al.,

Fig. 1. Intestinal histology of grouper fed C (A), FC100 (B) and FCW100 (C), showing normal structures such as goblet cells (g), lamina propria (lp), villi (v),
epithelium (e), and lymphocyte nuclei (n).

4
R.E.P. Mamauag, et al.
Fig. 2. Hepatic histology of grouper fed C (A), FC100 (B) and FCW100 (C), showing normal, large and spherical nucleus which is centrally located and homogenous
in size.
2013 reported that Bacillus mannanase mannan endo-1,4-β mannosi-
dase effectively degrades heteromannan and mannan into mannooli-
gosaccharide, mannose, galactose and glucose. Short- or long-chain
oligomannosides are released, contingent on the types of enzymes
present, source of mannanase, and hydrolytic conditions (Puls, 1997).
The biological mechanism of MOS is poorly understood as it is a rare
sugar. However, MOS have been observed to act as a prebiotic and
stimulate local intestinal and systemic immune responses. The use of
MOS as an additive has been reported in separate studies to enhance
growth and immune system of freshwater crayfish Astacus leptodactylus
(Mazlum et al., 2011) and spiny lobster Panulirus ornatus (Sang and
Fotedar, 2010).
Intestinal morphological changes as affected by the inclusion of
PECM® have not been investigated in the study of Apines-Amar et al.
(2016, 2017). Intestinal pathohistological changes were not evident in
the current research. The mucosal epithelium consisted of normal en-
terocytes with goblet cells and prominent villi and microvilli (Berillis
et al., 2017). The normal intestinal morphology is expected as PECM®
neither contains alcohol-soluble substances nor other anti-nutrients.
Many intestinal morphological abnormalities that decrease digestive
function are attributed to alcohol-soluble components which naturally
occur in processed leguminous seeds (Berilis et al., 2017). The normal
intestinal morphological structure of grouper implies an efficient nu-
trient absorption that led to improved fish growth. Moreover, PECM®
abnormal alterations in the hepatic morphology of groups fed PECM®
were absent. Hansen et al. (2006) reported normal hepatic morphology
in cod fed a combination of plant protein (i.e. wheat gluten and soybean
protein concentrate) at levels up to a maximum of 44%. In addition,
sunflower meal replacing 30% of protein from fish meal showed no
histological alteration in sharpsnout sea bream Diplodus puntazzo
(Mérida et al (2010)). The study of Martinez-Llorens et al. (2012) ob-
served insignificant hepatic pathological changes in gilthead sea bream
fed carob seed germ meal at 52% inclusion level.
Limiting amino acids in proteins such as methionine and lysine are
commonly sourced from plants (Gaitlin et al., 2007). Supplementation
of dietary methionine and lysine can improve weight gain (Khan and
Abidi, 2011; Alam et al., 2011) and promote intestinal growth and
enzyme activities in the digestive system (Tang et al., 2009). The sup-
plementation of methionine and lysine aimed to exclude the factor of
5
Aquaculture Reports 14 (2019) 100202
amino acid deficiency. The result suggested that even without the
supplementation of crystalline amino acids, PECM® can still replace
soybean meal at a 16% level. Analyzed amino acid composition of the
diets showed similar lysine and methionine levels – all of which are
within the required levels for juvenile grouper. Dietary methionine and
lysine requirements for juvenile grouper were reported at 1.31% of diet
(Luo et al., 2005) and 2.83% of diet (Luo et al., 2006), respectively.
Raw copra meal has digestibilities of 65.35%, 100%, and 18.74%
for protein, lipid, and carbohydrate, respectively when fed to grass carp
Ctenopharyngodon idella (Law, 1986). It has a digestibility of 79.90%
protein when fed to tropical catfish Mystus nemurus (Khan, 1994). Most
copra meal digestibility values were evaluated using fish species with
minimal protein requirement.
The present study indicates that fermentation of copra can sig-
nificantly improve nutrient digestibility of the ingredient when used in
fish species requiring high amount of dietary protein. Its use as dietary
ingredient did not negatively alter hepatic and intestinal morphologies.
Moreover, it can replace soybean meal up to 100% (16% in diet) for
normal fish performance and digestive organ functions of juvenile
grouper. Additional studies on the use of fermentation process to fur-
ther improve nutrient quality of fish meal alternative ingredients are
recommended.
Acknowledgements
We would like to thank the National Institute of Molecular Biology
and Biotechnology, University of the Philippines-Los Baños for pro-
viding the PECM®. We would also like to thank the Ateneo de Manila
University for the publication financial assistance. This study is funded
by SEAFDEC/AQD under 5210-TRD-FD0217.
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