ABSTRACT

AYOOLA, AYUUB AYODELE. Replacement of Fishmeal with Alternative Protein Source

in Aquaculture Diets. (Under the direction of Professor Peter Ferket).

Fisheries have played a very vital role in improving the food security status of the
people; it contributes about 15-16% to the total animal protein consumed by 2.9 billion
people in low-income and food-deficient countries. An estimated 520 million people, nearly
8% of the world population rely on income from fisheries for sustenance. With the continued
increase in the awareness of health benefits, the global demand for aquatic foods, even in
developed countries is expected to continue to rise. Aquaculture production is responsible for
50% of the global fishery production. Fishmeal is the preferred protein ingredient in
aquaculture feed and contributes significantly to the variable production cost in the
aquaculture industry. However, decreasing fishmeal supply relative to demand and increasing
costs threaten the sustainability and growth of the aquaculture industry. The way to sustain
the industry is to remove or reduce the use of fishmeal. Past studies have demonstrated that
complete or partial substitution of fishmeal with alternative proteins does not adversely affect
fish performance.
Two experiments were conducted to evaluate the effect of replacing fishmeal
with different animal proteins in aquaculture feed using omnivorous and carnivorous fish
models. In experiment 1, the effect of complete replacement of fishmeal with fermented
lactic acid-stabilized mechanically deboned poultry meat residue (MDM), poultry by-product

meal (PBM) and yeast extract (YE), was tested on the growth performance and protein
efficiency of the omnivorous Nile tilapia (Oreochromis niloticus). There were no significant
treatment effects on specific growth rate. However, the body weight of the fish fed with FM
was significantly greater than fish fed with diets containing PBM, YE and MDM. Apparently
due to feed form differences, MDM resulted in significantly higher FCR than FM, but neither
of these treatments were significantly different from YE or PBM. In contrast, PBM resulted
in significantly lower PER than FM but neither of these treatments was significantly different
from YE or fermented MDM. There were no significant treatment effects on body
composition. Although plasma IGF-1 concentrations increased during the experiment, there
were no significant treatment effects on the plasma IGF-1 concentration among treatments at
the end of the experiment. The results of this experiment demonstrate that YE, MDM and
PBM are feasible fishmeal alternatives in Nile tilapia diets. However, PBM is the most
economical alternative to FM in Nile tilapia feed, although it has a slightly reduced protein
quality.
Experiment 2 was conducted to evaluate the effects of dietary replacement of FM
with fermented lactic acid-stabilized MDM on growth performance of HSB (Morone
saxatilis X M. chrysops). Replacement with MDM affects feeding behaviors and the amount
of feed intake by HSB. Apparently, due to different level of feed intake, the SGR, BW, BL
and plasma IGF-1 concentration were also affected. However, the replacement of FM with
different levels of fermented MDM in HSB diet does not have significant effect on the

growth parameters; FCR, PER, ASI, HSI, hepatic IGF-1 gene expression and carcass
proximate composition. Generally, the fish fed with the 50% and 75% performed better than
the control diets that contain the 0% replacement whereas fish fed with 100% replacement
did not perform comparatively well. Results demonstrate that fermented MDM can
satisfactorily provide between 50% to 75% protein in commercial HSB diet when substituted
with FM. The results from both experiments agreed with the conclusion of previous studies
affirming that partial and total replacement of FM with animal proteins does not affect the
growth performance of fish, however level of replacement depends on fish species.

 Copyright 2010 by Ayoola Ayuub Ayodele

All Rights Reserved

Replacement of Fishmeal with Alternative Protein Sources in Aquaculture Diets

by
Ayuub Ayodele Ayoola

A thesis submitted to the Graduate Faculty of

North Carolina State University
in partial fulfillment of the
requirements for the Degree of
Master of Science

Nutrition and Food Science

Raleigh, North Carolina

2010

APPROVED BY:

_______________________________
Peter Ferket, Ph.D.
Committee Chair
________________________________

Russell Borski, Ph.D.

______________________________
Jonathan Allen, Ph.D.
Committee Co-Chair

DEDICATION

To:
God, for his mercies and faithfulness.
Three special women in my life my Mama, Peju and Teni.

ii

BIOGRAPHY

Ayuub Ayodele Ayoola was born on July 30, 1980 in Lagos, Nigeria. He is the fifth born in a
family of six children. He had a bachelor degree in Food Science and Technology from the
Federal University of Agriculture Abeokuta, Nigeria. During his undergraduate study, he
benefited from Oyo State of Nigeria Scholar award and graduated among top 5% in his
graduating set. He worked briefly in a food manufacturing company after his graduation to
raise funds for his graduate school education. He started his graduate program at NC State
University, Raleigh, United States in August 2009. Ayuub hopes to continue with his PhD
program in Spring, 2011.

iii

ACKNOWLEDGMENTS

My appreciation goes to Almighty God for his mercy over me and my family throughout the
challenging period of my program. Many people contributed to the success of this work, all
of whom am grateful. To Dr. Ferket, my major advisor, I say a big Thank You for
accepting me into your research group, also providing funds and support for the completion
of my MS degree. I appreciate your going beyond the call of duty to ensure that this work is
completed. A special thanks goes to Dr. Allen, for believing in ability and supporting me in
all forms throughout this program. I am deeply grateful to Dr. Borski for all his advice and
support. I owe special gratitude to all my friends at NC State; Margaret Jackson, for proof
reading the sections in the thesis, Jose Santa-Cruz, Wilmer Pacheco, David Roseneri,
Marcelo, Ilana, Indira, and Dr. Megan Swartz. The present document would not have been
possible without the advice and technical contribution of Dr. Ramon, Eugene Won, William
Johnstone, Annett Israel and all the people in Dr. Ferkets laboratory and Dr. Borskis
laboratory. My family deserves special appreciation. To my wife, thank you for taking very
good care of our daughter and for all her sacrifices. I say thank you to my parents and
siblings for their moral and financial support. My parents in-law for their prayers and
supports. Lastly, the encouraging smiles of my daughter Oluwateniola is a treasure I cant
quantify.

iv

TABLE OF CONTENTS

LIST OF TABLES....ix
LIST OF FIGURES.....xii
Chapter 1: LITERATURE REVIEW.....1
1.0 FOOD SECURITY........2
1.1 IMPORTANCE OF FISH AND AQUACULTURE TO ALEVIATE
POVERTY AND MALNUTRITION......2
1.2 AQUACULTURE..........5
1.3 FISHMEAL.......7
1.3.1 Fishmeal Alternatives ......12
1.4. HYPOTHESIS AND RESEARCH OBJECTIVES ...20
1.4.1 Hypothesis 1..........21
1.4.2 Objective 1........22
1.4.3 Hypothesis 2......22
1.5.4 Objective 2....22
References........23
Chapter 2: Replacement of fishmeal by alternative protein ingredients in feeds for Nile tilapia
(Oreochromis niloticus).........31

2.0 ABSTRACT.32
2.1 INTRODUCTION.......34
2.3. MATERIALS AND METHODS........36
2.3.1 Ingredient preparation, feed formulation and feed manufacturing...36
2.3.2 Water quality management...... 42
2.3.3 Growth Study .......42
2.3.4 Carcass Analysis ......44
2.3.5 Radioimmunoassay-Plasma IGF-1 Concentration....45
2.3.6 Statistical Analysis ...........45
2.3.7 Animal ethics ...........46
2.4. RESULTS ..46
2.4.1 Water Quality ...........46
2.4.2 Growth Performance Characteristics ...........47
2.4.3 Radioimmunoassy- Plasma IGF-1 concentration ....53
2.4.4 Carcass Composition ...........55
2.4.5 Protein efficiency ratio .....57
2.5. DISCUSSION ....59
References..68

vi

CHAPTER 3: Replacement of fishmeal by fermented mechanically deboned poultry meat in

feeds for Hybrid striped bass (Morone chrysops X M. saxatlis)..............73
3.1 ABSTRACT...........74
3.2 INTRODUCTION.........77
3.3. MATERIALS AND METHODS..........82
3.3.1 Ingredient preparation, feed formulation, and feed manufacturing....82
3.3.2 Pellet durability test87
3.3.3 Water quality management 90
3.3.4 Growth Study .........90
3.3.5 Carcass Analysis ............92
3.3.6 Conditional indices 93
3.3.7 Radioimmunoassay-Plasma IGF-1 Concentration .93
3.3.8 RNA isolation and Quantitative real-time PCR .........94
3.3.9 Statistical Analysis .........95
3.3.10 Animal ethics ...........96
3.4. RESULTS 96
3.4.1 Water Quality .........96
3.4.2 Growth Performance Characteristics .....97

vii

3.4.3 Radioimmunoassay-Plasma IGF-1, Hepatic IGF-1 mRNA expression

and Compositional Indices............102
3.4.4 Carcass Composition....104
3.4.5 Protein Efficiency Ratio .......106
3.5 DISCUSSION .....108
References..........115
Chapter 4: SUMMARY AND CONCLUSION ....123
4.1 Research summary.....124
4.2 Conclusion ....127
4.3 Recommendation.......128
References........129

viii

LIST OF TABLES

Chapter 1
Table 1. Classification of fish feeds based upon their energy levels and processing
technology.............10

Table 2. Estimated use of fishmeal in feed for various fish species groups in 2000 and
2010 .........11

Table 3. Percentage of essential amino acids (EAA) in fishmeal (FM), rendered meat
meal (MM), poultry by-product meal (PBM), blood meal (BM), soybean
meal (SBM) Percentage of crude protein in the meal (in parenthesis).13

Chapter 2
Table1. Nutrient composition of Dietary protein sources......38

Table 2. Feed formulations and proximate analyses of diets for the evaluation of
fishmeal replacement with yeast extract (NuPro, Alltech Inc.), fermented
mechanically deboned meat (MDM) and poultry by-product meal (PM) in
Nile tilapia diet .....41

Table 3. Effect of dietary replacement of fishmeal with yeast extract, fermented

mechanically deboned meat and poultry by-product meal on the change in
the mean body weight of juvenile Nile tilapia......49

Table 4. Effect of dietary replacement of fishmeal with yeast extract, fermented

mechanically deboned meat and poultry by-product meal on specific growth
rate of juvenile Nile tilapia. ........ 50

ix

Table 5. Effect of dietary replacement of fishmeal with yeast extract, fermented

mechanically deboned meat and poultry by-product meal on mean body
length of juvenile Nile tilapia .......51

Table 6. Effect of dietary replacement of fishmeal with yeast extract, fermented

mechanically deboned meat and poultry by-product meal on the dry
matter, crude protein, ether extract, ash content and gross energy
composition of juvenile Nile tilapia ......................................................56

Chapter 3
Table 1. Nutrient composition of dietary protein sources.....84

Table 2. Feed formulations and proximate analyses of diets for the evaluation of
fishmeal replacement with fermented mechanically deboned meat (MDM)
in HSB diet ...86

Table 3. Effect of dietary replacement of fishmeal with fermented mechanically

deboned meat on the pellet durability of the feeds....89

Table 4. Effect of dietary replacement of fishmeal with fermented mechanically

deboned meat on the growth response parameters of Hybrid striped bass
over a 77-day growth study in tanks .......101

Table 5. Effect of dietary replacement of fishmeal with fermented mechanically

deboned meat on the plasma and hepatic IGF-1 and compositional indices
of Hybrid striped bass measured on day 77 of a 77-day growth study in
tanks ....103

Table 6. Effect of dietary replacement of fishmeal with fermented mechanically

deboned meat on the dry matter, crude protein, ether extract, ash content
and gross energy composition of Hybrid striped bass measured at day 11
after a 77-day growth study in tanks ...105

xi

LIST OF FIGURES

Chapter 1
Figure 1. 5 year commodity price chart for fishmeal from IMF Commodity Price
Data......9
Chapter 2
Figure 1. Effect of dietary replacement of fishmeal with yeast extract, fermented
mechanically deboned meat and poultry by-product meal on the feed
conversion ratio of juvenile Nile tilapia ....52

Figure 2. Effect of dietary replacement of fishmeal with yeast extract, fermented

mechanically deboned meat and poultry by-product meal on the
Radioimmunoassay-Plasma IGF-I concentration of juvenile Nile tilapia
............54

Figure 3. Effect of dietary replacement of fishmeal with yeast extract, fermented

mechanically deboned meat and poultry by-product meal on the Protein
efficiency ratio of juvenile Nile tilapia .....58
Chapter 3
Figure 1: Effect of dietary replacement of fishmeal with fermented mechanically
deboned meat on the pellet durability of the feed......88

Figure 2. Effect of dietary replacement of fishmeal with fermented mechanically

deboned meat on the feeding response time of juvenile hybrid striped bass
.......99
Figure 3. Effect of dietary replacement of fishmeal with fermented mechanically
deboned meat on the Protein efficiency ratio of juvenile hybrid striped
bass
..107

xii

CHAPTER 1

LITERATURE REVIEW

1.0 FOOD SECURITY

Food security is the term that is used to express the accessibility of people to
sufficient, safe and nutritious food to maintain a healthy and active life (WHO, 2010). Food
security is achieved when all people, at all times, have physical, social and economic access
to sufficient, safe and nutritious food that meets their dietary needs and food preferences for
an active and healthy life (FAO, 2002; Seligman et al., 2010). Therefore, improved food
security is a necessary precursor for global initiative towards reduction of hunger and
poverty, and for economic development. One aim of the UN Millennium Development Goals
is to reduce by half the proportion of people suffering from hunger by 2015 (UN, 2010).
However, the continuing population and consumption growth will mean that the global
demand for food will continue to increase. Presently, over 820 million people are affected by
hunger in developing countries and these numbers are not decreasing rapidly enough,
particularly in Africa and Southern Asia (FAO, 2006). Lack of quality food is still the main
cause of under-nutrition among children, which is very common in developing areas (UN,
2010).

1.1 IMPORTANCE OF FISH AND AQUACULTURE TO ALEVIATE POVERTY

AND MALNUTRITION
Fisheries have always played a very significant socio-economic role in many
countries and communities. As a subsistence produce, fish is a vital resource towards poverty

reduction and food security for most poor households (Cinner et al., 2009). In the
Philippines, 84 % of people involved in aquaculture are locals (Bergquist, 2007). Income
generated from the fisheries sector or through fish trade is the most important indirect
contribution to food security. In sub-Sahara Africa, fishing and fish related employment
provides both part-time and full-time jobs to 6 and 9 million people , respectively. There are
about 43.5 million fishers in the world, and there are at least four other people associated
with each fisher in fish-related jobs, including processors, traders and small-scale operators
(FAO, 2002). Thus, the fishing industry supports over 170 million people with income.
Taking into account their families and dependents, one can therefore estimate a total of 520
million people or nearly 8 percent of the total world population directly relies on income
from fisheries (FAO, 2009). This contributes greatly to their purchasing power to achieve
greater food security, providing people with an important source of income with which to
buy food. (Simon et al. ,2009).
Fish has always been a source of rich food for poor people and played an important
role in improving a developing countrys food security and nutrition for its people. Fish is a
major source of high-quality dietary protein, essential vitamins, minerals, and other
micronutrients for about 1 billion people, many isolated in rural communities of developing
and low income countries (FAO, 2003; Stephen et al., 2010). For example, more than 200
million Africans eat fish regularly (Stephen et al., 2010). Globally, fish contributes about 1516% of the total animal protein consumed by 2.9 billion people, including the low-income

and food-deficient countries (LIFDCs) (FAO, 2009; Heck et al., 2007). The proportion fish
can exceed 50% of animal protein in the poorest countries, especially where other sources of
animal protein are scarce or expensive (John, 2009). A few hundred grams of fish, consumed
at a subsistence level, can make the difference between adequate and inadequate nutrition,
between recovered health and prolonged illness, or between food security and starvation.
Furthermore, fish is often the most accessible and affordable source of protein in many poor
neighborhoods of urban communities in developed countries. Fish-based food products also
contribute to local and national economic sustainability through trade and exports.
Fish contributes more to the diet of people in developing countries than their
counterparts in the developed world (Normile, 2002). Fish contribute to direct nutritional
food security in countries where the staple crop is particularly low in protein (Smith et al.,
2010). Most especially in the developing countries, fish is an indispensable source of high
quality protein and other nutrients, especially vitamins A and D, phosphorus, magnesium,
iron, iodine, zinc, calcium, and selenium (Lunven, 1982; Dahl et al., 2006). Marine fish is
also an important source of iodine (Fenton et al., 1997; Bonham et al., 2009; AlvarezPedrerol et al., 2010). Like other meat protein, fish protein is easily digestible and
complements dietary protein (amino acids) provided by cereals and legumes consumed in
many developing countries. In combination with a vegetable based diet, fish provides a
complementary effect to the essential amino acids that are present in low quantities in
vegetarian diets. The beneficial effect of improved protein balance on health is obvious even

when a small quantity of fish is consumed (Claire, 2000). Fish can also provide up to 180
calories per capita per day (John, 2009). In addition to being a good source of protein, fish oil
is a vital source of the essential fatty acids, such as Eicosapentaenoic acid (EPA), and
Docosahexaenoic acid (DHA) which are important for brain development in unborn and
infant children (Bonham et al., 2009). Fish oil has also been shown to prevent cardiovascular
diseases (Mozaffarian, 1991; Joensen et al., 2009; Heidarsdottir et al., 2010; Baik et al.,
2010; Eschen et al., 2010; Heine-Brring et al., 2010; Lemieux et al., 2010).

1.2 AQUACULTURE
The nutritional benefits of fish and fish oil consumption on human health, including
the prevention of cancer, diabetes and heart diseases, have been well established (Lunven,
1982; Fenton et al., 1997; Mozaffarian, 1991; Dahl et al., 2006; Joensen et al., 2009;
Alvarez-Pedrerol et al., 2010; Heidarsdottir et al., 2010; Baik et al., 2010; Eschen et al.,
2010; Heine-Brring et al., 2010; Lemieux et al., 2010). As public awareness about the
health benefits of fish consumption continues to increase, the global demand for aquatic
foods is also expected to continue to rise (Gina, 2009). Furthermore, the world's population
is expected to grow by more than 30 % by 2050, resulting in an estimated 2.3 billion more
mouths to feed, with the major growth expected in the developing countries where fish is the
primary source of protein (UN, 2010). Aquaculture is recognized as the only way to meet
these increasing demands for aquatic foods (Allan, 2004). Half of the sea foods consumed

worldwide are from commercial fishing (i.e. fish caught in the wild open waters) (FAO,
2009). The other half is farm-raised fish grown under controlled conditions known as
aquaculture (Allan, 2004). The amount of fish produced globally from aquaculture rose from
6 % in the 1970s to about 50 % of the total fish consumed in the world in 2006.
Furthermore, aquaculture is also an important income-generating sector of many economies
with considerable prospects for job creation, poverty alleviation, community development,
and food security. It provides fish for domestic markets and for international markets. The
domestic market improves national food security, and production for international markets
creates employment, provides income, and brings in foreign exchange; thereby indirectly
contributing to national food security.
The exponential growth of the aquaculture sector during the past two decades is a
result of the progressive intensification of production systems. A major contributor to this
intensive production system is the use of manufactured feeds formulated to meet the
nutritional requirements of the targeted fish species (FAO, 2009). Feeds account for up to 70
% of the variable cost of a commercial aquaculture operation (Webster et al., 1999) for many
fish species. Feed production costs are driven by the cost of fishmeal, an important protein
source in fish feeds. The price of fishmeal has increased more than two fold in recent years
(Figure 1). It rose from about US$600 per metric ton in 2005 to about US$2000 per metric
ton in the first quarter of 2010 (IMF, 2010). Furthermore, the provisions of fishmeal are not

adequate to sustain the current rate of growth of aquaculture in addition to the demand from
other animal feed industries.

1.3 FISHMEAL
Fishmeal is generally added to animal diets to increase feed efficiency and growth
through better feed palatability; it enhances nutrient uptake, digestion, and absorption (Mile
and Chapman, 2010). Fishmeal is valuable because of the quality of its protein; the amino
acids that make up the protein are present in just the right balance for animal nutrition (FAO,
2001; Balios, 2003). The balanced amino acid composition of fishmeal complements other
animal and vegetable nutrients in the agricultural feed to provide synergistic effects that
promote fast animal growth (Mile and Chapman, 2010). The availability of the essential
amino acids, phospholipids, and fatty acids (Docosahexaenoic acid and Eicosapentaenoic
acid) are high in fishmeal and promote optimum development, growth, and reproduction.
Fishmeal has high biological value as a feedstuff, as it has a high level of digestible essential
amino acids, such as lysine, methionine and leucine, which are often deficient in exclusive
grain feeds, the typical base for most animal feeds (Keller, 1990; Hall, 1992; FAO, 2001;
Balios, 2003). Furthermore, fishmeal has low fiber content and is also a valuable source of
vitamins B1, B2, B6 and B12 in addition to calcium, phosphorous, magnesium, potassium,
and trace elements including zinc, iodine, iron, copper, manganese, cobalt, selenium, and
fluorine (FAO, 2001; Hall, 1992). The lack of nutritional inhibitors in fishmeal also makes

this meal more attractive than plant proteins for use in aquaculture diets. Aquaculture feeds
typically have a higher percentage of fishmeal than feeds for other animal species. The level
of fishmeal in the feed formulation varies depending on whether the fish species is a
carnivore or omnivore because of the variations in their biochemical utilization of nutrients
(Table 1; Martin, 1999).

In a typical commercial feed for tilapia, 30-40 % is protein

ingredient, while, the protein ingredient level is about 42-50 % in a typical salmon diet
(Table 1; NRC, 1993). The aquaculture feed industry consumes a considerable amount of the
global fishmeal production. About 46 % of the total annual fishmeal produced is used to
generate aquaculture feeds. This figure is expected to rise because of the public attention that
has been shifted to the consumption of the seafood products (FAO, 2009). Table 2, shows
the percentage use of fishmeal in some typical feeds for different fish species. However, the
inconsistency of supply, the growing demand and price, among other problems, are limiting
the use of fishmeal and putting greater pressure on the feed industry to find an economical
alternative source of protein (Hardy, 2007).

Fishmeal - Monthly Price - Commodity Prices US $ per metric ton

US$ per metric ton

2500
2000
Fishmeal Monthly
Price Commodity
Prices US $
per metric
ton

1500
1000
500

Ju

l- 0
5
ov
-0
M 5
ar
-0
Ju 6
l- 0
N 6
ov
M 06
ar
-0
Ju 7
l- 0
N 7
ov
M 07
ar
-0
Ju 8
l- 0
N 8
ov
M 08
ar
-0
Ju 9
l- 0
N 9
ov
M 09
ar
-1
0

Month

Figure 1. 5-year commodity price chart for fishmeal from IMF Commodity Price Data
Source: IMF Data base, 2010

Table 1. Classification of fish feeds based upon their energy levels and processing
technology

Digestible energy (MJ)

Protein (%)
Fat (%)

High energy
(Carnivorous)
15- 22
42-47
22-35

Processing technology

Cooker Extruder

Species

Salmon, Eel,
Trout

Source: NRC (1993)

10

Medium energy
Low energy
(Carnivorous)
(Omnivorous)
14-18
13-15
45-47
30-40
12-23
8-12
Extruder expenderPellet mills
pellet mills
Sea bass, Sea
Tilapia, Carp,
bream, Turbot
Mullet

Table 2. Estimated use of fishmeal in feed for various fish species groups in 2000 and 2010

Species group
Salmon
Trout
Marine fish
Flat fish
Shrimp
Cat fish
Carp
Other*
Total

2000
(%)
35
30
45
55
25
2
4

2010
(%)
25
20
40
45
20
0
3

2000
(000mt)
455
180
377
40
487
0
337
629
2117

*Includes eels, milk fish, tilapia and other carnivorous fresh water species
Courtesy: Pike and Barlow, 2003

11

2010
(000mt)
406
139
628
145
576
0
602
489
2854

1.3.1 Fishmeal Alternatives

There are several protein sources that have the potential of replacing fishmeal in
aquaculture feeds without affecting the growth performance of fish (Tacon et al., 2009).
Research outcomes on the nutritional requirements of aquatic organisms and the
technological advances in feed production suggest that there is the possibility of partial or
total replacement of fishmeal with protein from other sources. These alternative protein
sources include animal proteins from rendering or slaughter, plant protein concentrates and
novel proteins such as algae, yeast, dried distillers grains with solubles (DDGS) and insect
meal. Furthermore, research findings have identified the essential nutrients in fishmeal and a
way of incorporating them into the alternatives. Table 3 shows the essential amino acids and
crude protein in fishmeal and other potential protein sources for aquaculture feed. Plant
based aquaculture feeds containing soybean meal protein, canola meal, extruded pea seed
meal, wheat and corn meal supplemented with lysine and methionine has been used in the
formulation of aquaculture feed for catfish, tilapia and carp without affecting the growth
performance of the fish (Tacon et al., 2009).

12

Table 3. Percentage of essential amino acids (EAA) 1 in fishmeal (FM), rendered meat meal
(MM), poultry by-product meal (PBM), blood meal (BM), soybean meal (SBM) Percentage
of crude protein in the meal (in parenthesis).
Essential Amino Acid

Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine + Cystine3
Phenylalanine + Tryosine4
Threonine
Tryptophan
Valine

MM
(55.6%)2

FM
(64.5%)2

PBM
(59.7%)2

3.82
1.45
2.66
4.48
4.72
2.31
4.35
2.31
0.57
2.77

3.60
0.89
1.64
2.85
2.93
1.25
2.99
1.64
0.34
2.52

4.06
1.09
2.30
4.11
3.06
1.94
3.97
0.94
0.46
2.86

BM
(89.2%
)2
3.75
5.14
0.97
10.82
7.45
2.32
8.47
3.76
1.04
7.48

SBM
(50.0%)2
3.67
1.22
2.14
3.63
3.08
1.43
4.20
1.89
0.69
2.55

The percentage values for the EAA composition of each feedstuff were taken from the 1993 NRC (National
Research Council, Nutrient Requirements of Fish, National Academy of Sciences, Washington, DC).
2
Percentage of total crude protein in feedstuff.
3
Cystine can be synthesized from methionine.
4
Tyrosine can be synthesized from phenylalanine
Source: (Mile and Chapman, 2010)

13

The complete or partial replacement of fishmeal with plant based protein is possible
without loss of growth performance (Webster et al., 1992; Webster et al., 1995a; Gur 1997;
Middleton et al., 2000; Kaushik et al., 2004). Plant proteins are similar to fishmeal in terms
of the protein content and apparent protein and amino acid digestibility. However, the amino
acid profile of the protein from plant sources does not match the amino acid requirement of
some fish species as well as fishmeal does (Hardy, 2007). Methionine is the limiting amino
acid in soybean meal (SBM) and soy protein concentrates, while corn gluten meal is deficient
in lysine; and lysine and arginine is limiting in wheat gluten meal. By blending SBM with
grain protein concentrates, the amino acid profile can be adjusted to partially overcome
limitations of individual plant proteins. Replacement of fishmeal with a combination of
SBM and DDGS in feed of channel catfish, Ictalurus punctatus, and blue catfish, I. furcatus,
does not affect the growth performance of the fish (Webster et al. 1992; Webster et al.,
1995a). Middleton et al. (2000) evaluated the possibility of using culled jewel sweet potatoes
and co-extruded poultry silage as a feed ingredient for hybrid tilapia (Oreochromis niloticus
X O. massambicus). The outcome of their studies show that the formulation formed an
acceptable feed, and this co-extruded meal could be used up to 33% in the hybrid tilapia diet
without any adverse effect on the fish observable economic variables which included a
consumer panel sensory analysis. The European seabass, Dicentrarchus labrax, maintained
growth performance when 98 % of the fish meal was replaced by plant protein sources
(Kaushik et al., 2004). In carnivorous fish, total replacement rather than partial replacement

14

of fishmeal with plant based protein presents a more challenging situation. The growth
performance of some carnivorous fish is not affected when the fishmeal was partially
substituted (Dabrowski et al., 1989; Kaushik et al., 1995; McGoogan and Gatlin, 1997;
Carter and Hauler, 2000; Zhou et al., 2005). McGoogan and Gatlin (1997) observed no
effect on the growth performance of Red Drum, Sciaenops ocellatus, when fishmeal was
substituted with SBM in all but 10 % of the total protein content in the diet when
supplemented with sulfur amino acids.

Up to 40 % and 33 % of fishmeal in Cobia,

(Rachycentron canadum), and Atlantic Salmon diets can be replaced with SBM, respectively,
without significant differences in growth performance (Carter and Hauler, 2000; Zhou et al.,
2005).
However, Kaushik et al, (1995) observed that replacement of fishmeal with casein
and soy flour meals in rainbow trout feed had a negative effect on growth performance.
Plant protein tends to lower feed intake by reducing diet palatability when replacement levels
are high, or by affecting the health of the fish in other ways, such as the condition describe as
distal enteritis in Atlantic salmon and rainbow trout fed with high soybean meal (Refstie et
al., 2000). Furthermore, some plant proteins contain phosphorus as phosphorus phytate. The
phytate makes phosphorus in seeds unavailable to monogastric animals, including fish. Also,
phytate interferes with the bioavailability of divalent trace elements, especially zinc, making
it necessary to over fortify feeds to ensure adequate dietary zinc intake in fish feed containing

15

high level of phytate especially in the presence of high dietary calcium levels (Richardson et
al., 1985; Gatlin and Philips, 1989).
Replacement of fishmeal with animal rendering by-product meals may be a more
suitable alternative for some species of fish, especially the carnivorous species. Animal
proteins have long been used in feed. Generally, they have inferior protein digestibility, the
ash content is very high in the case of meat, bone and poultry by-product meal; and the
amino acid profile of blood and feather meal is not close to the optimal growth requirement
of the fish. However, animal proteins are free of anti nutritional factors, are palatable and
cheaper than fishmeal. The possibility of replacing fishmeal with fishery by-products, such
as shrimp head meal, fermented fish silage and squid meal, have been tested in various
aquaculture feeds. Toledo et al. (1986 and 1987) partially replaced 15 % of fishmeal with
shrimp head meal in Blue tilapia, Oreochromis aureus, feed without any adverse effects on
feed and growth efficiency.

Fagbenro and Jauncey have studied on fish silage as an

alternative to fishmeal in aquaculture feed. They found that lactic acid bacteria-fermented
fish silage can be used to replace fishmeal in Nile tilapia, O. niloticus (Fagbenro and
Jauncey, 1993, 1994a, 1995) and Mozambique Tilapia Oreochromis mossambicus (Hossain
et al. 1992). Furthermore, they found that the fermented silage could be preserved for up to 6
months at 30oC without any significant effect on the nutritional quality. Lapie and BiguerasBenitez (1992) used formic acid instead of lactic acid to ferment the fish silage and found
that there was no effect on the growth and feed efficiency in Nile Tilapia fed with such feed.

16

Different land animal protein products have been made into meals and evaluated as
fishmeal alternatives in various feed. These animal products include poultry by-product meal,
mechanically deboned meat, hydrolyzed feather meal, blood meal, and meat and bone meal.
They are high in crude protein and fat content, depending on the animal. However this crude
protein is usually lacking in one or more of the essential amino acids. Lysine, methionine
and iso-leucine are generally the limiting amino acids in animal sources of fishmeal
alternatives (Tacon et al., 1985; NRC, 1993). Poultry by-product meal is limited most in
lysine (Tacon and Jackson, 1985; Nengas et al., 1999; Sevgili, 2002), while methionine was
reported as the most limiting amino acid in meat and bone meal and blood meal.
Furthermore, hydrolyzed feather meal is limiting in both lysine and methionine, while blood
meal is also deficient in iso-leucine. However, some suggested way of overcoming these
deficiencies is by supplementing the deficient amino acids or by mixing complementary
alternatives to obtain the desired essential amino acid profile (Davies et al., 1989; Tacon et
al., 1983). Hydrolyzed feather meal could replace up to 66 % of the fishmeal in feed for O.
niloticus fingerlings and fry without any adverse effect on the growth performance (Falaye
1982; Bishop et al., 1995). Past research has established precedence for the partial and total
replacement of fishmeal as a protein source with poultry by-product meal, the amount of
fishmeal replacement possible is species specific (Fowler, 1991; Gouveia, 1992; Hasan et al.,
1993, Hasan and Das, 1993; Steffens, 1994; Sadiku and Jauncey, 1995; Appelbaum et al.,
1996; El-Sayed, 1998, Nengas et al., 1999; Webster et al., 2000; Davis and Arnold, 2000;

17

Sevgili, 2002). Menghong et al., (2008) studied the effect of partial replacement of fishmeal
with rendered animal protein ingredients: poultry by product meal and meat and bone meal
alone and in combination with lysine and methionine supplements in practical feed for Gibel
carp Carassius auratus gibelius. Their results show that the optimal performance is observed
in Gibel carp when the rendered animal proteins supplemented with lysine and methionine is
used at 66.7 % replacement of the fishmeal. Furthermore, they reported that inclusion at this
level lowered the feed cost. Rodriquez-Serna et al. (1996) evaluated the effect of substituting
commercially defatted animal by-product meal on the performance of O. niloticus fry. The
animal by-product meal is a formulation of blood meal, meat and bone meal, hydrolyzed
feather meal and fish meal in ratio 1:1 with soybean oil. Their results show this alternative
could replace up to 75 % of the fishmeal and if the formulation is supplemented with soybean
oil, it could be used up to 100 % without any adverse effect on the growth performance of the
fish. Grouper, Epinephelus coioides, and Gilthead seabream, Sparus aurata, maintained
growth, feed efficiency, and protein efficiency when fishmeal was replaced up to 80 % and
44 % with meat and bone meal in their diets, respectively (Robaina et al., 1997; Millamena,
2002). Based on outcomes of several scientific evaluations, animal proteins represent good
alternatives that can be used as fishmeal substitutes in aquaculture feed.
Finally, novel proteins is another area of active research in the aquaculture feed
industry. Novel proteins are proteins obtained from single cell organisms and invertebrates,
but they are often too costly to be considered as an alternative protein source to fishmeal in

18

aquaculture feed. However, because of the recent increasing cost of fishmeal, researchers
have started evaluating the economic feasibility and optimum usage of these novel proteins
as fishmeal substitutes. Partial replacement of fishmeal with algae is possible (Broun, 1980;
Appler, 1982; Zeinhom, 2004; El-Hindawy et al., 2006), especially in tropical areas where
they are found in abundant amounts. Tartiel et al (2008) observed that partial substitution of
fishmeal with dried microalgae, Chlorella spp and Scenedesmus spp, in Nile tilapia
fingerling diets does not affect fish growth performance, feed efficiency and body
composition. Their 90 day growth trial indicated that growth performance, feed conversion
ratio and protein productive value were significantly better in fish fed diets containing 50 %
of both Chlorella spp and Scenedesmus spp as compared to the standard diet. However,
there was a decrease in growth performance when dietary inclusion of algae meal is over
20% of the feed formulation (Tartiel et al., 2008). Only about 10-15 % of dietary protein
requirement can be met by algae in fish diets without compromising growth and feed
utilization. Poor growth was attributed to the high carbohydrate content, which contained a
little fraction of both mono and disaccharides; and this is believed to have affected
digestibility (FAO, 2010).
Yeast is another novel protein that has been studied as a fishmeal alternative. Ebrahim
et al. (2008) studied the effect of partial and complete replacement of fishmeal with yeast
protein, Saccharomyces cerevisiae. The feed was supplemented with biogenic L-carintine
(methionine plus lysine mixture) at a rate of 100 mg/100 g of diet on growth performance,

19

carcass composition and feed utilization of Nile tilapia fingerlings. The best growth
performance, feed and protein efficiency ratios, without any adverse effects was observed
with 50 and 75 % replacement supplemented with biogenic L-carintine. The feasibility of the
commercial application of any of these novel proteins in substituting fishmeal depends on
economic feasibility and costs (FAO, 2010c). In the meantime, their use may be limited to
high valued fish and as supplements in aquaculture feed because of their omega 3 content
that has health benefits.

1.4. HYPOTHESIS AND RESEARCH OBJECTIVES

From ancient times, fishing has been a key source of food for the humans. It provides
a means of livelihood, employment and economic benefits to those engaged in this and
associated activities. Generally, income from fishing activities contributes 16-29 % of the
total typical household income in developing countries (World Fish Center, 2010). Many of
the world's poorest peoples nutrition depend on fish; about 1 billion people rely on fish for
at least 30 % of their animal protein (FAO, 2009). Furthermore, fishing is an integral part of
the process of ensuring food security and poverty alleviation in developing countries (DFID,
2005). FAO (2004) reported that aquaculture provided 43 % of the total food supply of fish.
The demand for seafood has been on the increase worldwide; the total consumption of fish
hit 95.5 million metric tons in 1990 from 50 million metric tons in 1976 and with 6.6 %
annual incremental contribution from aquaculture. It is estimated that by 2020 the figure will

20

rise to about 50 % (World Fish Center, 2010). This development has put a lot of pressure on
aquaculture production resources; fishmeal is a major resource in aquaculture feed
composition. The increasing global demand and decreasing availability of fishmeal has led
to the skyrocketing price of fishmeal and hence, the cost of aquaculture production has
increased. The prices went from the US$ 600/metric ton in 2005 to US$ 2000/metric ton in
June, 2010, and the trend is likely to continue (IMF, 2010). The world aquaculture industry
can no longer completely rely on fishmeal to meet production demands. Consequently, there
is great economic and environmentally sustainability incentives to find less expensive protein
sources to replace fishmeal in feeds for aquaculture applications
Various research outcomes have shown that alternative protein sources derived from
grains such as corn, wheat, barley, SBM, and animal rendering products such mechanically
deboned meal, poultry by-product meal, meat and bone meal, etc. can be used to formulate
nutritious aquaculture feed at various levels of inclusions in different feeds. The objective of
these studies is to evaluate potential fishmeal alternatives under the following hypothesis and
objectives:
1.4.1 Hypothesis 1:
Alternative protein source, such as yeast extract, mechanically deboned poultry meat,
and poultry by-product can replace fishmeal in Nile tilapia feed without compromising
growth performance.

21

1.4.2 Objective 1:
Objective of this study are to replace the fishmeal (FM) protein portion of total crude
protein requirement for Nile tilapia with mechanically deboned meat, yeast extract, and
poultry by-product meal, at 6 % of total FM and the determination of the effects on the
following important economic and growth performance indicators and fish proximate
composition: body weight (gained market weight), specific growth rate, RadioimmunoassyPlasma IGF-1 concentration, feed conversion ratio, protein efficiency ratio, fish proximate
composition.

1.4.3 Hypothesis 2:
Fermented mechanically deboned meat can be used as an alternative protein source in
replacing fishmeal in hybrid striped bass feed.
1.5.4 Objective 2:
The objectives of this trial is to replace FM in a hybrid striped bass feed at 25, 50, 75,
and 100 % with fermented mechanically deboned meat with the determination of the effects
on the following important economic and growth performance indicators and fish proximate
composition: body weight (gained market weight), specific growth rate, RadioimmunoassyPlasma IGF-1 concentration, feed conversion ratio, protein efficiency ratio, fish proximate
composition and hepatic IGF-1 mRNA expression.

22

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CHAPTER 2

Replacement of fishmeal by alternative protein ingredients in feeds for Nile tilapia

(Oreochromis niloticus).

A. Ayoola1, R. Malheiros1, P. Ferket1, R. Borski2, C.Stark1

1

Departments of Poultry Science1 and Biology2, College of Agriculture and Life

Sciences, North Carolina State University, Raleigh.
February 4, 2010

31

2.0 ABSTRACT
Because of its declining global availability and increasing demand, fishmeal is a
major contributor to the rising cost of fish feeds and fish production. Therefore, there is a
great economic and environmental sustainability incentive to find less expensive protein
sources to replace fishmeal in feeds for aquaculture application. Nile tilapia, an omnivorous
species, is a feasible species to feed diets that contain proteins from alternative sources other
than fishmeal. An experiment was conducted to evaluate the replacement of fishmeal at a
dietary inclusion level of 6% with poultry by-product meal (PBM), fermented lactic acidstabilized deboned poultry meat residue (MDM), and yeast extract (Nupro, Alltech Inc.), on
growth performance and protein efficiency of Nile Tilapia (Oreochromis niloticus). Each of
the 4 experimental diets were manufactured into 2mm crumbled pellets and fed twice daily at
a rate of about 3% of average body weight per day. Each treatment was replicated in 3
1000L recirculation tanks containing 35 fish per tank. The trial continued for 105 days,
beginning at an average Body weight (BW) of 91 g. BW were measured at 34, 72, and 105
days, and specific growth rate was calculated. Blood samples for IGF-1 measurements were
collected at day 0 and 105. At 106 days, 3 fish per tank were randomly sampled, frozen, and
then the carcasses were analyzed for crude protein, ether extracted fat, and energy (kcal)
content. Feed conversion ratio (FCR, grams of feed/gram weight gain) and protein efficiency
ratio (PER) from 1-105 days were calculated. There were significant treatment effects on
specific growth rate. The final average weight of the fish fed with FM was significantly

32

greater than fish fed diets containing MDM but neither is different from yeast extract and
PBM (448.35g vs versus 411.18g, 421.62g and 437.02g respectively, P< 0.05). Apparently
due to feed form differences, fermented MDM resulted in significantly higher FCR than FM
(1.82 vs. 1.62, p<.05), but neither of these treatments were significantly different from yeast
extract or PBM (1.75 and 1.68, respectively). In contrast, PBM resulted in significantly
lower PER than FM (1.95 vs. 2.13, p<.05), but neither of these treatments were significantly
different from yeast extract or fermented MDM (2.02 and 2.04, respectively). There were no
significant treatment effects on 105 d body composition. Although plasma IGF-1
concentration increase during the experiment, (65 ng/ml for 0d versus 125 mg/ml for 105d,
p<0.05), there were no significant treatment effects observed on day 105 IGF-1. The results
of this experiment demonstrate that yeast extract, mechanically deboned meat and poultry
by-product meal proteins are feasible alternatives for FM in diets for Nile tilapia. PBM is the
most economical alternative to FM in Nile tilapia feed, although it has an inferior protein
efficiency ratio.

Key words: Nile tilapia, Fishmeal, yeast extract, poultry by-product protein.

33

2.1 INTRODUCTION
Tilapia is the third most important fish cultivar in term of production after salmon and
carp (FAO, 2009). The global annual production of tilapia in 2007 was about 2.2 million
tonnes and is projected to reach about 2.5 million tonnes in 2010 (FAO, 2009). During the
end of the 20th century, tilapia has the highest rate of annual growth among all finfish,
exceeding that of salmonids and cyprinids (De Silva, 2001). Tilapia plays an important role
in food security and poverty alleviation in the developing countries such as Indonesia, the
Philippines, Thailand and Taiwan Province of China (De Silva, 2004). In particular, the
Oreochromis niloticus species has impacted on aquaculture development since the 1970s,
and has become the preferred tilapia species for aquaculture, especially in the developing
countries in Asia and Africa (Smith and Pullin, 1984). In low income and protein deficient
countries, O. niloticus is commonly cultured in backyard and/or home garden ponds of
relatively poor water quality, such as sewage-fed ponds, to supplement the income of poor
households as well as to make available a fresh source of animal proteins to the family
(Edwards. 1990; Edwards et al., 1990; Khalil and Hussein, 1997). There have been no
reported health effects after consumption of such tilapia (Nandeesha, 2002).

Presently, tilapia production from aquaculture accounts for about 2.5 times more than
production from wild capture fisheries, although the reverse was the case before the 1980s
(De Silva, 2004). In tilapia aquaculture farming like most finfish aquaculture systems, the
production of diets in commercial quantities is usually been based on the use of fishmeal

34

(FM) as the main source of dietary protein (Yilmaz et. al, 2003), although the dependency is
of a lesser extent than that for carnivorous fishes and shrimps. The biological value and the
nutritional qualities of fishmeal, such as the amino acid profile, essential fatty acids and
vitamins to cultured fishes has been widely reported (Tacon, 1993; ADCP, 1983; FDS,
1994).

The price of FM has risen at an increasing rate because of its inconsistent low supply
and increasing demand. As a result, future availability and cost are uncertain (Muzinic et al.,
2006; Yilmaz et. al., 2003). Consequently, the aquaculture feed manufacturing industry is at
a critical juncture as the supply-demand price constraint situation for fishmeal threatens the
profitability and sustainability of tilapia aquaculture. Thus, there is an urgent need to finding
alternatives to fishmeal in aquaculture feed. Alternative economical protein sources from
rendered animal protein, such as poultry by product meal (PBM) and mechanically deboned
meat (MDM), could be used at various percentages of inclusion in an aqua feed to improve
cost effectiveness (Allan et al., 2000; Bureau et al., 2000; Kureshy et al., 2000; Stone et al.,
2000). These FM replacers have been tested successfully in feeds for fish species such as
Chinook salmon (Fowler, 1990;1991), silver Sea bream (El-Sayed, 1994), rainbow trout
(Steffens, 1994; Bureau et al., 2000), red drum (Moon and Gatlin, 1994; Kureshy et al.,
2000), gilthead Sea bream (Robaina et al., 1997; Nengas et al., 1999), Indian major carp
(Hasan et al., 1997), Australian snapper (Quartararo et al., 1998), Australian silver perch

35

(Allan et al., 2000; Stone et al., 2000), Nile tilapia (El-Sayed, 1998), sunshine bass (Webster
et al., 2000), grouper (Milliamena, 2002), and mirror carp (Yilmaz et, al, 2003).
The purpose of this study was to test if mechanically deboned meat, yeast extract
(NuPro, Alltech Inc.), and poultry by-product meal could serve as a replacement of
fishmeal (FM) protein while meeting the total crude protein requirement for Nile tilapia
(Oreochromis niloticus). Dependent variables in this study include growth rate, feed
conversion ratio, feed consumption and digestibility, and protein efficiency ratio.

We

hypothesize that these alternative protein sources can effectively replace dietary inclusion of
FM for tilapia; thereby reduce production costs while maintaining high quality fish for
consumer market. Additionally, we measured circulating insulin-like growth factor-1 (IGF1) as evidence suggests it may serve as a proxy for growth rate in fishes (Picha et al., 2008).
To our knowledge, a comparison of circulating IGF-1 to growth rates in response to
alternative dietary proteins has not been assessed in Nile tilapia

2.3. MATERIALS AND METHODS

2.3.1 Ingredient preparation, feed formulation and feed manufacturing
This study was designed to compare nutritional value of same inclusion levels of
dietary protein sources for Nile Tilapia similar in protein content to fishmeal. The protein
sources used in the experimental diets included high protein soybean meal (48% CP) (Cargill

36

Inc., Raleigh, NC), poultry by-product meal (Valley Protein Inc., Fayetteville, NC),
mechanically deboned poultry meat (MDM) (Townsends Inc., Siler City, NC), the yeast
extract, NUPRO (Alltech, Nichoasville, KY), and menhaden fishmeal (Special SelectTM,
Omega Protein, Inc., Houston, TX). The nutrient profiles of the protein sources used in this
study are illustrated in Table 1. Except for the MDM, all protein source meals were included
in the experimental diets as purchased from commercial sources. The MDM used in this
study was a by-product of poultry processing. After the removal of breast meat filet, the
residual soft tissue (meat and some fat) was separated from the bone using a Beehive Model
RSTD06 separator/mechanical deboner (Provisur Technologies, Sandy, UT) equipped with
0.5-0.8 mm holes in the cylinder. The MDM was then stabilized to pH<4.7 by lactic acid
fermentation using a procedure as described by Middleton et al. (2001). The MDM was
mixed with 12% dextrose (wt/wt) and inoculated with 4 billion cfu of lactobacillus
acidophilus per g of fermentation mixtures and maintained at ambient temperature in a closed
but vented container for a period of 14 days. This MDM silage was then dried at 60 C for 48
hours (Blue M Drying Oven, General Signal, Atlanta, GA) to a moisture content of about
12% and then ground to a meal.

37

Table 1. Nutrient composition of Dietary protein sources

Nutrient
Dry matter, %
Crude protein,%
Crude fat, %
Crude fiber
Ash, %
Neutral detergent
Acid detergent fiber, %
Calcium, %
Potassium, %
Sodium,%
Chloride, %
Total phosphorus,%

FM1
90.2
62.3
9.4
0.90
18.4
5.2
0.70
0.40
0.55
3.04

SBM2
90.4
48.5
1.3
3.65
6.0
7.0
6.3
0.4
2.58
0.02
0.03
0.64

MDM3
88.0
61.0
22.5
0.04
2.3
1.6
0.08
0.07
0.07
0.35

PBM4
96.7
65.0
12.7
0.32
12.7
23.8
5.5
2.9
0.70
0.38
0.50
2.15

YE5
94.5
51.5
0.9
8.2
0.3
1.80
0.15
0.15
1.50

Magnesium, %
Zinc, Mg/ Kg
Iron, Mg/ KG
Manganese, M
Copper, Mg/kg
Lysine, %
Methionine, %
Cysteine, %
Leucine, %
Isoleucine, %
Threonine
Tryptophan
Phenylalanine
Tyrosine, %
Valine, %

0.14
148
480
34
11.0
4.81
1.77
0.57
4.66
2.57
2.64
0.66
2.56
2.03
3.37

0.36
68
238
52
19.0
3.04
0.70
0.72
3.70
2.20
1.93
0.63
2.49
1.75
2.57

0.03
106
389
10
12.
3.06
0.85
0.55
3.51
1.77
2.13
0.46
1.62
0.83
2.52

0.12
119
207
10
9.0
3.42
0.95
0.67
3.99
2.01
2.37
0.50
1.84
0.94
2.86

2.92
0.81
0.58
3.93
2.24
2.04
1.58
2.17
1.58
2.64

FM is menhaden fishmeal (Special SelectTM, Omega Protein, Inc., Houston, TX)

SBM is high protein soybean meal (Cargil Inc., Raleigh, NC)
3
MDM is mechanically deboned poultry meat (MDM) (Townsends Inc., Siler City, NC)
4
PM is poultry by-product meal (Valley Protein Inc., Fayetteville, NC)
5
YE is yeast Extract was supplied as NUPRO (Alltech Inc., Nicholasville, KY).
1
2

38

Table 2 describes the ingredient formulation and nutrient analysis of the experimental
diets. Two basal diets were mixed prior to the addition of the test protein source ingredients.
One basal diet was prepared to include 6% fishmeal, poultry by-product meal, or yeast
extract meal. Because the MDM silage meal contained more fat and less ash (calcium and
phosphorus from bone) than the other protein source ingredients, the MDM diet was
formulated to contain less added fat and more limestone and dicalcium phosphate than the
other test diets.
All of the feed was manufactured at the North Carolina State University Feed Mill
Educational Unit in accordance with current Good Manufacturing Processes (cGMPs).
Ingredients were ground with a hammer mill (Model 06036EP3E364TS, 8 ton/hr capacity,
California Pellet Mill Co., Crawfordsville, IN) equipped with 1.5 mm (4/64 inch) screens.
The basal diets were produced in a 2 ton capacity double ribbon mixer (Model TRDB 1260604, Hayes & Stolz Ind. Mfg. Co., Fort Worth, TX) and the test diets were mixed in a 500
lb. capacity double ribbon mixer (Model SRM304, Scott Equipment Co., New Prague, MN).
All diets were pellet processed at 185-195 F (85-91 C) with a 30 s retention time in the
conditioner (Master Model HD, Size 46 cm X 122 cm (18 X 48 ), 1 ton/hr capacity,
California Pellet Mill Co., Crawfordsville, IN). Pellets were manufactured with a pellet mill
(Model PM 1112-2, 1 ton/hr capacity, California Pellet Mill Co., Crawfordsville, IN) with a
4.4 mm X 35 mm die (11/64 X 1 3/8 ). Ambient air was used to cool pellets in a counterflow cooler (Model VK09x09KL, 1 ton/hr capacity, Geelen counter-flow USA, Inc.,

39

Orlando, Florida). The cooled pellets were passed through a crumble roller to produce 2 mm
pellet crumbles and then sieved to remove all fines.

40

Table 2. Feed formulations and proximate analyses of diets for the evaluation of fishmeal
replacement with yeast extract (NuPro, Alltech Inc.), fermented mechanically deboned meat
(MDM) and poultry by-product meal (PM) in Nile tilapia diet
Experimental Diet
Ingredients

FM

YE

PBM3

MDM4

-------------------------- (% of Diet as is basis) ------------------Soybean Meal (48% CP)

Corn
Wheat
Fishmeal
Yeast Extract1
Poultry by-product meal
MDM
Poultry fat
Limestone
Dicalcium Phoshate (18.5% P)
Salt
Se Premix2
Ascorbic Acid
Trace Mineral Premix3
Vitamin Premix4
Choline Chloride, (60%)
Lysine-HCl
Total
Determined Nutrient Analysis
Dry matter (%)
Crude Protein (%)
Crude Fat (%)
Acid Det. Fiber (%)
Neutral Det. Fiber (%)
Non-fiber CHO (%)
Ash (%)
Calcium (%)
Phosphorus (%)
Sulfur (%)
Magnesium (%)
Sodium (%)
Potassium (%)
Cooper, ppm
Iron, ppm
Manganese, ppm
Zinc, ppm

50.80
12.20
25.00
6.00
---4.43
0.44
0.33
0.22
0.15
0.13
0.10
0.10
0.10
0
100.00

50.80
12.20
25.00
-6.00
--4.43
0.44
0.33
0.22
0.15
0.13
0.10
0.10
0.10
0
100.00

50.80
12.20
25.00
--6.00
-4.43
0.44
0.33
0.22
0.15
0.13
0.10
0.10
0.10
0
100.00

50.60
11.50
25.00
---6.00
3.94
0.68
1.29
0.27
0.15
0.13
0.10
0.10
0.10
0.14
100.00

86.92
28.9
6.18
6.57
8.72
37.41
5.72
0.68
0.73
0.27
0.21
0.13
1.11
14
276
98
116

87.73
28.29
6.31
5.04
9.33
38.65
5.15
0.66
0.69
0.31
0.24
0.14
1.28
18
271
120
142

87.37
30.67
6.05
6.77
9.89
35.22
5.55
0.66
0.73
0.28
0.21
0.10
1.12
14
236
105
121

86.72
27.03
7.28
5.32
9.52
38.84
5.54
0.57
0.65
0.26
0.21
0.10
1.08
14
250
103
116

Yeast Extract was supplied as NUPRO (Alltech Inc., Nicholasville, KY).

NaSeO3 premix provided 0.3 mg Se/kg of complete feed.
Each kilogram of mineral premix (.1% inclusion) supplied the following per kg of complete feed: 60 mg Zn as ZnSO4.H2O; 60 mg Mn as MnSO4.H2O; 40 mg Fe as FeSO4.H2O; 5 mg Cu as
CuSO4; 1.25 mg I as Ca(IO3)2; 1 mg Co as CoSO4.
4
Each kilogram of vitamin premix (.1% inclusion) supplied the following per kg of complete feed: vitamin A, 13,200 IU; cholecalciferol, 4,000 IU; alpha-tocopherol, 66 IU; niacin, 110 mg;
pantothenic acid, 22 mg; riboflavin, 13.2 mg; pyridoxine, 8 mg; menadione, 4 mg; folic acid, 2.2 mg; thiamin, 4 mg; biotin, 0.253 mg; vitamin B12, 0.04 mg; ethoxyquin, 100 mg.
2

41

2.3.2 Water quality management

Water temperature and dissolved oxygen (DO) were measured in the pond twice daily
(08:30 and 15:30 h) at a depth of 0.75 m using an oxygen meter (Model 85, YSI Industries,
Yellow Springs, OH, USA). This was done to ensure that DO levels in the tanks were
optimal for fish growth and well-being. Total ammonia, nitrite, pH and alkalinity were
measured once weekly (13:00 h) using a spectrophotometer (Model DREL/2000, HACH
Inc., Loveland, CO). Total alkalinity was measured once weekly (13:00 h) by titration using
a digital titrator (Model AL-36DT, HACH Inc., Loveland, CO). The salinity was also
measured once weekly (13:00 h) using a refractometer (YSI 85, YSI Industries, Yellow
Springs, OH, USA)

2.3.3 Growth Study

In order to demonstrate the effect of each treatment on growth parameters, a 105-day
feeding trial was carried out at the NCSU Tidewater Research Station (Plymouth, NC). Sex
reversed juvenile male Nile tilapia fish (Company, City, FL. USA) were transferred from
Lake Wheeler Fish Barn to Tidewater. Fish with average weight of 91 grams were randomly
allocated at a stocking density of 35 fish per tank among three replicate tanks per
experimental diet. Each 1000 L recirculation tank was equipped with external standpipes
controlling the depth of the water. Mechanical filtration on the system was provided by a

42

hydro-tech filter with 60 micron sized screens and a BF10 bubble bead filter (bio-filtration as
well). This system was also equipped with an ultraviolet filter (Aquatic Ecosystems Catalog
# ES26). All the tanks were maintained to the same water quality parameters throughout the
experiment; however there was fluctuation in salinity levels, due to occasional system filter
flushed and addition of rock salt.
The fish were fed the experimental 2 mm-crumbled feeds in the morning and
afternoon of each day. Because the crumbled pellet feeds sank to the bottom of the tanks,
they were placed in a B-net (32 mm mesh size) feeding bag and suspended in tanks at the
water surface. Fish obtain feeds placed in pick wire bags, and they were fed at a rate of about
3% of average body weight per day. The amount of feed consumed was measured daily by
determining the pick wire feeding bag weight difference before and after feeding. All fish
were anesthetized, weighed and measured for total length calculated individually at 0, 34, 72,
105 days of the experiment.

The mortality weight was determined and recorded as it

occurred and it was used to adjust feed conversion ratio (FCR). Specific growth rate (SGR)
was calculated as [(ln W2 ln W1)/ (T2 T1) X 100], where W2 is the weight at the end of
the growth interval and W1 is the weight at the beginning of the growth interval, while T2T1 represents the duration (days) of the growing interval. Specific growth rates were
calculated during the following time intervals: 0-34 d, 34-72 d, 72-105 d, and 0-105 d.
Protein efficiency ratio (PER) was calculated as the total amount of the protein consumed
divided by the total amount of body protein gained during the 105 d period. Five fish per

43

treatment were collected from each treatment tank at 15, 34, 72 and 105 days. The samples
were euthanized, measured, weighed and were frozen in liquid nitrogen, then stored at -20oC
in polythene bags until analyses.

2.3.4 Carcass Analysis

The frozen sampled fish were ground into paste in a grinder (Model No: RU 05,
Manual Grinding Machines, Crocean Industry Co., Ltd. Taiwan) and then dried at 60 C for
48 hours in a drying oven (Blue M Drying oven, General Signal Atlanta, GA) prior to the
chemical analysis. The samples were then ground to a fine powder with a laboratory grinder
(Mortar grinder Mill Model 2, Fritsch Pulverisette, Mount Holly, NJ, USA). Chemical
analyses were done on a dry matter basis. Protein (N6.25) was determined by the Dumas
method using a LECO nitrogen automatic analyzer (Tru Spec N, LECO Corporation, St.
Joseph, Michigan 49085, US), gross energy content was determined using a bomb
calorimetery (C5000, IKA Calorimeterm, IKA Werke Labortechnik, Staufen, Germany).
Percentage fat was determined by the ether extraction method (AOAC, 2010).

Percentage

ash was determined by placing diets in a muffle furnace (600oC) for 24 h, and moisture was
determined by drying 24 hours at 65 oC in a drying oven (Blue M Drying oven, General
Signal Atlanta, GA) (AOAC 1990).

44

2.3.5 Radioimmunoassy- Plasma IGF-1 concentration

All fish were sampled individually for plasma IGF-I analysis at the beginning (day 0)
and the end of the trail (day 105). Blood was taken via heparinized syringe from the caudal
vein of the anesthetized fish. Circulating levels of total IGF-I were measured in duplicate
from acid/ethanol extracted plasma by radioimmunoassay (RIA) using recombinant
barramundi IGF-I as tracer and standard, rabbit anti-barramundi IGF-I primary antibody
(Novozymes GroPep; Adelaide, Australia) and goat anti-rabbit secondary antibody (Sigma;
St. Louis, Missouri) according to method described by Shimizu et al., 2000 and Picha et al.,
2008. Barramundi IGF-I was iodinated using the chloramine-T method and purified by
column chromatography. Tracer (125I-barramundi IGF-I) was diluted to 20,000 cpm for each
assay tube.

2.3.6 Statistical Analysis

The experiment was designed and statistically analyzed using a completely
randomized block design consisting four dietary treatments. Tank mean values were
considered experimental observation units for statistical analysis. Statistical significance of
the main treatment effects on each measurement was performed using the JMP procedure of
SAS (SAS Institute, 2009). Treatment variable effects were considered significant at p< 0.05.

45

The LS Means procedure of JMP was used to determine significant differences among the
individual treatments after ANOVA.

2.3.7 Animal ethics

The experiment herein reported was conducted in accordance to the guidelines of the
Institutional Animal Care and Use Committee at North Carolina State University.

2.4. RESULTS
2.4.1 Water Quality
Water quality was for each experimental unit tank was controlled to be within the
critical limits for optimum growth of tilapia. Water temperature and dissolved oxygen were
measured daily and pH, nitrate, alkalinity, salinity and ammonia concentration were
measured weekly and adjusted to be within the critical minimum and maximum limit for
each parameter throughout the duration of the experiment. The meanSEM for total
alkalinity, salinity and pH (125 8.09 ppm, 2.140.25 ppm and 8.00 respectively) were
recorded, along with the means for total ammonia-nitrogen and nitrite-nitrogen (0.04.008
ppm, 0.480.09 ppm respectively). Daily meanSEM temperatures and dissolved oxygen
were 25.430.0797 oC and 6.960.726, respectively (Table 3). Water quality measurements
were not significantly different among treatments.

46

2.4.2 Growth Performance Characteristics

No mortality <1% was observed among the treatment groups throughout the
experimental period. These general performance characteristics indicate the husbandry
practices (water quality, feeding rates, and basal diet formulations, fish handling, etc.) were
adequate. Regardless of dietary treatment, the fish were fed with same amount of feed
during each measured growth phase. Fish feeding was maintained at around 3% body weight
and was adjusted during each sampling period throughout the experiment.
The mean body weight (BW), specific growth rate (SGR), body length (BL) during
the sampling periods did not differ significantly among groups (Table 3,4 and 5). However, a
statistically significant treatment effect was observed on final mean BW (Table 3) and the
overall SGR (Table 4). In comparison to those fish fed the diet containing FM, fish fed the
diets containing MDM had lower final mean BW (411.2g vs 448.3g, p<0.05) and lower SGR
(3.03% vs 3.39%, p<.05), but neither of these parameters were significantly different from
those fed with diets containing PBM or YE. Because the same amount of feed was offered to
all experimental treatment groups, treatment differences in feed conversion ratio (FCR, g
feed intake/g BW gain) was apparently associated with the observed differences in BW gain
and SGR. In comparison to those fish fed the diet containing FM, fish fed the diets
containing MDM had reduced higher FCR (1.62 vs 1.82, p<0.05); whereas the FCR of the
fish consuming diets containing YE or PBM were not significantly different from the other

47

two treatment groups. Evidently, fish fed the diets containing the fermented MDM in place
of FM had reduced growth performance characteristics.

48

Table 3. Effect of dietary replacement of fishmeal with yeast extract, fermented mechanically
deboned meat and poultry by-product meal on the mean body weight of juvenile Nile tilapia.1

Dietary
Treatment

FM2
MDM3
YE4
PM5
P-Value
SEM(5)

--------------------------- Days of Experimental Treatment ------------------------0

15
34
72
105
------------------------------------- (g/fish) ---------------------------------------------92.250.84 133.158.34 194.546.78 305.283.67 448.351.32 a
92.822.64 136.1811.5 181.767.79 282.697.58 411.1811.3 b
91.092.43 128.443.44 175.143.57 286.302.73 421.621.43 ab
91.750.49 137.661.79 184.054.19 299.340.03 437.021.27 ab
0.9206
0.8215
0.2714
0.061
0.0355
1.8564
7.3644
5.8489
4.4244
5.7627

a,b

Mean values with different letter superscript within a column are significantly different (p<0.05)
Values are means SD, N= 2 tanks/treatment; 37 fish/tank.
2
FM= Fishmeal
3
MDM= Fermented mechanically deboned meat
4
YE= Yeast extract (NuPro, Alltech Inc.)
5
PBM= Poultry by-product meal
1

49

Table 4. Effect of dietary replacement of fishmeal with yeast extract, fermented mechanically
deboned meat and poultry by-product meal on specific growth rate of juvenile Nile tilapia.1

Dietary
Treatment

FM2
MDM3
YE4
PM5
P-Value
SEM(5)

-------------------------- Days of Experimental Treatment -------------0-15

15-34
34-72
72-105
0-105
---------------------------- (% BW change/fish) --------------------------2.730.62 3.230.79 2.910.28
4.340.15
3.390.01 a
2.890.59 2.400.19 2.660.08
3.890.11
3.030.08b
2.490.07 2.460.01 2.930.03
4.100.13
3.150.04ab
3.060.15 2.440.31 3.030.16
4.170.04
3.290.01ab
0.8159
0.5545
0.4281
0.1954
0.0179
0.4337
0.4401
0.1487
0.1149
0.0453

a,b

Mean values with different letter superscript within a column are significantly different (p<0.05)
Values are means SD; N= 2 tanks/treatment; 37 fish/tank.
2
FM= Fishmeal
3
MDM= Fermented mechanically deboned meat
4
YE= Yeast extract (NuPro, Alltech Inc.)
5
PBM= Poultry by-product meal
1

50

Table 5. Effect of dietary replacement of fishmeal with yeast extract, fermented mechanically
deboned meat and poultry by-product meal on mean body length of juvenile Nile tilapia.1

Dietary
Treatment

FM2
MDM3
YE4
PBM5
P-Value
SEM(5)

------------------------ Days of Experimental Treatment -------------------0

15
34
72
105
------------------------------------ (mm/fish) -----------------------------------179.930.1 190.001.7 223.440.4 252.780.4 265.470.1
175.238.6 170.7714
221.773.2 243.637.9 260.603.8
174.721.8 181.900.6 218.501.4 247.020.3 261.010.7
176.230.9 189.150.9 222.380.5 251.090.6 265.290.5
0.836
0.3316
0.3593
0.4557
0.2799
4.4256
7.1381
1.7827
3.9803
1.9507

Values are means SD; N= 2 tanks/treatment; 37 fish/tank.

FM= Fishmeal
3
MDM= Fermented mechanically deboned meat
4
YE= Yeast extract (NuPro, Alltech Inc.)
5
PBM= Poultry by-product meal
2

51

Effect of dietary replacement of fishmeal with yeast extract,

fermented mechanically deboned meat and poultry by product on
the feed conversion ratio of juvenile Nile tilapia

Feed conversion ratio (total feed

consumed by fish/body weight gained
by fish

1.9
1.85
1.8
1.75
1.7
FCR
1.65
1.6
1.55
1.5

ab

ab

YE

PM

1.45
FM

MDM

Dietary treatments

Figure 1. Feed conversion ratio (mean SD; N= 2 tanks/ treatment; 37 fish/tank) of Nile
tilapia over a 105-day growth study in tanks. Fish were fed with diets containing 6% fishmeal
(FM) or diets in which fishmeal was replaced with 6% yeast extract (YE), poultry by-product
meal (PBM), and fermented mechanically deboned meat (MDM). Columns depicting FCR
for treatments with different alphabetical script are significantly different (p<0.05).

52

2.4.3 Radioimmunoassy- Plasma IGF-1 concentration

Relative to levels at the start of the experiment (day 0), plasma IGF-I level was
increased among all groups at day 105, but there were no significant dietary treatment effects
observed (Figure 1, p>0.05).

53

Effect of dietary replacement of fishmeal with yeast extract,

fermented mechanically deboned meat and poultry by-product meal
on Radioimmunoassay- plasma IGF-I concentration of juvenile Nile
tilapia over a 105-day growth study in tanks.

Radioim m unoassayP lasm a IG F-I (ng/m l)

160
140
120
100
80

Baseline- Plasma IGF-I

concentration at day 0

60
40
20
0
FM

MDM

YE

PM

Dietary treatments

Figure 2. Plasma IGF-I levels (mean SD, N= 2 tanks/ treatment; 37 fish/tank) of Nile
tilapia over a 105-day growth study in tanks. Fish were fed with diets containing 6% fishmeal
(FM) or diets in which fishmeal was replaced with 6% yeast extract (YE), poultry by-product
meal (PBM), and fermented mechanically deboned meat (MDM). There were no significant
dietary treatment effects on plasma IGF-I (p> 0.05).

54

2.4.4 Carcass Composition

At the end of the experimental period, carcass composition as determined by

proximate analysis was similar for all sampled fish, regardless of dietary treatment. Table 8
shows that there was no significant difference (p>0.05) among experimental diets on carcass
dry matter, crude protein, ash, crude fat, and gross energy content.

55

Table 6.

Effect of dietary replacement of fishmeal with yeast extract, fermented

mechanically deboned meat and poultry by-product meal on the dry matter, crude protein,
ether extract, ash content and gross energy composition of juvenile Nile tilapia.1
Dietary
Treatment
2

FM
YE3
MDM4
PBM5
P-value
SEM(5)6

Dry Matter

Crude
Crude Fat
ASH
Protein
----------------------------- (%) ------------------------------32.250.79
23.141.49
9.951.40 10.011.01
31.820.82
22.972.06
10.411.69 7.400.99
32.250.49
23.090.99
10.071.58 8.250.84
32.580.44
22.821.06
9.921.19 7.501.20
0.9017
0.4607
0.8968
0.2144
1.1999
1.9409
0.0257
2.4675

Values are means SD; N= 2 tanks/ treatment; 37 fish/tank.

FM= Fishmeal
3
YE= Yeast extract (NuPro, Alltech Inc.)
4
MDM= Fermented mechanically deboned meat
5
PBM= Poultry by-product meal
6
SEM = Pooled standard error of the mean with 5 degrees of freedom.
2

56

Gross Energy
(Kcal/g)
5710167
5803160
5617432
5697196
0.3795
181.7403

2.4.5 Protein efficiency ratio

Protein efficiency ratio (PER) is an indicator of the quality of the dietary protein in
the diet, relative to the amino acid balance, digestibility, and bioavailability. Significant
dietary treatment effects were observed on PER (Figure 3, p<0.05). In comparison to those
fish fed the diets containing FM, those fish fed the PM had significantly lower PER (2.13 Vs
1.95, p<.05), whereas the PER of those fish fed the diets containing YE and MDM were not
significantly different from the other two treatment groups.

57

Effect of dietary replacement of fishmeal with yeast extract,

fermented mechanically deboned meat and poultry by product on
the feed conversion ratio of juvenile Nile tilapia

Protein efficiency ratio (Body weight

gain over the period observed/ total
protein intake by fish

2.15
2.1
2.05
2
PER
1.95
1.9
1.85

ab

FM

MDM

ab

YE

PM

1.8
Dietary treatments

Figure 3. Protein efficiency ratio (mean SD; N= 2 tanks/ treatment; 37 fish/tank) of Nile
tilapia over a 105-day growth study in tanks. Fish were fed with diets containing 6% fishmeal
(FM) or diets in which fishmeal was replaced with 6% yeast extract (YE), poultry by-product
meal (PBM), and fermented mechanically deboned meat (MDM). There was significant
differences in the protein efficiency ratio among the fish fed with different diets, columns
with different alphabetical script are significantly different (p<0.05).

58

2.5. DISCUSSION
Traditionally, FM is the main and preferred source of protein used in agricultural feed
industry because of the excellent nutritional composition. However, the rising demand and
limited supply make FM an expensive protein source (FAO, 2006). The FM price has risen
from about US$ 600 per metric ton in 2005 to about US$ 2000 per metric ton in first quarter
of 2010 (IMF, 2010). The inconsistency of supply, the growing demand and price among
other problems are limiting the use of FM, putting a great pressure on the feed resource
industry to find economical alternative source of protein. Alternative protein sources such as
PBM (Fowler, 1991; Gouveia, 1992; Hasan et al., 1993, Hasan and Das, 1993; Steffens,
1994; Sadiku and Jauncey, 1995; Appelbaum et al., 1996; El-Sayed, 1998, Nengas et al.,
1999; Webster et al., 2000; Davis and Arnold, 2000; Sevgili, 2002), MDM ( Robaina et al.,
1997; Bureau et al., 2000; Webster et al., 2000; Grouper, Milliamena, 2002), feather meal
(Rodriquez-Serna et al., 1996; Falaye 1982, Bishop et al., 1995), soybean meal (Richardson
et al., 1985; Gatlin and Philips, 1989), yeast ((Mahnken et al., 1980; Tacon and Jackson,
1985; Tacon, 1994; Aires Oliva-Teles et al., 2001; Ebrahim et al., 2008), oil seed (Refstie et
al., 2000), and algae (Broun, 1980; Appler H.N, 1982; Zeinhom, 2004; El-Hindawy et al.,
2006; Tartiel et al., 2008) have been tested using different fish models.
In this study, we evaluated PBM, MDM and YE protein sources as alternatives to FM
in the Nile tilapia (Oreochromis niloticus) diet. Treatments were either fed a balanced tilapia
diet (28% crude protein, 7% crude fat) containing 6% FM or diets in which the FM has been

59

replaced with other alternative protein sources: PBM, MDM and YE. These alternative
protein sources have varying levels of digestibility and bioavailability that has profound
effects on their use.
The suitability of each of the alternative evaluated in this study is determined using
treatment performance indicators that include final body weight, specific growth rate (SGR),
final body length, feed conversion ratio (FCR), protein efficiency ratio (PER) and plasma
IGF-1 concentration. SGR measures the rate of body weight change within a specific time
frame and a high positive SGR indicates consumed dietary feed nutrients are partitioned
towards optimum growth. In contrast, FCR, expressed as the amount of feed consumed per
unit of body weight gained, is an important indicator of feed utilization efficiency, balance of
bioavailable nutrients, and partitioning dietary nutrients towards growth (Luzzana et al.,
2003; Angelidis et al., 2005). Therefore, SGR and FCR together can be used to assess the
palatability and acceptability of feed and its dietary constituents. Apart from favorable
economic attributes, minimizing FCR has significant environmental benefits, as a greater
proportion of feed nutrients are converted to animal biomass and less nutrients are emitted
into the environment where it may have adverse ecological consequences. Moreover, energy
costs and environmental emissions associated with the manufacture and transport of feed
decreases as FCR decreases for animal production. PER, the amount of body protein gained
per gram of protein consumed, is a biological assay of the quality of protein in the diet. A
high PER value is indicative of good protein digestibility and bioavailability, and the

60

constituent amino acid profile satisfy the requirement for optimum body protein accretion
and growth. Plasma IGF-1 concentration is another performance indicator that is regulated by
the quantity and nutritional quality of dietary proteins (Phillips et al., 1978; Prewitt et al.,
1982; Isley et al., 1983; Takahashi et al., 1990; Miura et al., 1991). Plasma IGF-1 is a
peptide hormone that indicates the growth potential; the higher the concentration, the better
the growth potential of the fish.
PBM is a rendered protein commodity made from the ground, rendered, clean parts of
the carcass of slaughtered poultry, such as necks, feet, undeveloped eggs, intestines,
exclusive of feathers, except in such amounts as might occur unavoidably in good processing
practices (AAFCO, 2010). PBM has been widely studied as an alternate protein source to FM
in various fish species diets (Gallagher and LaDouceur, 1995; Negre et al., 1999; Emre et
al., 2003; Wang et al., 2005). As compared to FM, PBM contains a marginally lower
concentration of one or more amino acids essential for fish, including methionnine+cystine,
lysine, and phenylalanine (Tacon and Jackson, 1985; Davies et al., 1991; Nengas et al., 1999;
Sevgili, 2002). Wang et al., (2005) observed that PBM could replace 30% to 50% of FM in
Cuneate drum diet without affecting the SGR, final BW and FCR. Replacement of FM with
PBM at 80% and 100% levels in the pacific white shrimp and sunshine bass diet respectively
does not have negative effects on the FCR and BW (Emre et al., 2003). Gallagher and
LaDouceur (1995) observed that juvenile Palmetto bass fed with a diet containing 12% FM
and 36% low-ash PBM had weight gains similar to fish fed with a diet containing 47% FM.

61

Negre et al. (1999) reported that 100% replacement of FM with PBM does not adversely
affect the growth performance of Sea breams. PBM seems to be a good dietary protein
alternative to FM in fish diet.
The observed performance indicators of Nile tilapia in this study suggested that PBM
can comprise up to 6% of a practical diet and a complete replacement of FM without
compromising growth performance. Final body weight (FBW), final body length (FBL),
SGR, Plasma IGF-1 concentration and FCR of the Nile tilapia fed with PBM as total
replacement of FM were similar to those fed with FM diets (Control). These results are in
agreement with Webster et al., (1999); who reported that Sunshine bass fed with a diet
containing PBM and SBM as a complete replacement of FM had similar growth performance
as fish fed diets containing FM. The FCR and PER values observed in this study were within
the range reported by Soltan (2009) who replaced dietary FM with PBM plus different grain
sources and enzyme supplements in Nile tilapia diets. However, we observed that fish
consuming the diet containing PBM had a significantly lower PER than those fed the diet
containing FM (1.95 vs 2.13, P<.05). The lower PER value resulting from the PBM diet
could be due to the: i) limiting amino acid (histidine, methionnine+cystine, lysine and
phenylalanine) content (Tacon and Jackson, 1985; Nengas et al., 1999; Sevgili, 2002), ii) the

presence of high percentage of bone, feather, connective tissue and skin contents, which
are difficult for fish to digest (Davies et al., 1989; Fowler, 1990; Hasan et al., 1997; Hardy,
2000; Sevgili, 2002), iii) subjection of the protein to excessive heat during the rendering

62

process of PBM may compromise the bioavailability of amino acids (Nengas et al., 1999;

Webster et al. 2000; Sevgili, 2002). High processing temperature of PBM may reduce
digestibility of protein and amino acids due to the lysine and cystine+cystein losses
(Opstvedt et al., 1984; McCallum and Higgs, 1989). Nontheless, the results of this study
confirm the conclusions from previous studies that PBM could be used as alternative to FM
at various levels without adversely affecting the growth performance of fish: 20% in feeds
for Chinook Salmon (Fowler, 1991), 25% for silver Sea bream (El-Sayed, 1994), 21% for
Australian Snapper (Quartararo et al., 1998), 71% for gilthead Sea bream (Nengas et al.,
1999), and 14% for red drum (Kureshy et al., 2000) and sunshine bass (Webster et al., 2000).
Based on previous studies and our findings, PBM is a good alternative to FM in Nile tilapia
diets and PBM could be used to replace FM in aquaculture feed without affecting growth
performance.
Poultry MDM is another potential animal protein alternative evaluated in this study.
Like PBM, MDM is also a valuable and locally available co-product of poultry processing,
but it contains much less indigestible components as it is free of hard bone tissues and
feathers. It has an excellent nutritional composition containing high quality protein and fat
and is low ash content (Webb et al., 1976; Trindale et al., 2004). The incorporation of MBM
at up to 24% in aquaculture feeds does not affect fish growth performance for several
species: Sea bream, (Robaina et al., 1997); Rainbow trout, (Bureau et al., 2000); Sunshine
bass, (Webster et al., 2000) and Grouper, (Milliamena, 2002). However, Wang et al. (2005)

63

demonstrated that replacing FM with poultry MDM at 10.5% (to replace 30% of the fish
meal) in feed formulation for Cuneate drum resulted in reduced SGR, FBW and FCR, which
corroborates with the FAO report that claimed MDM cannot be used beyond 10% in
aquaculture feeds without having detrimental effects on growth (FAO, 2009). In this study,
fermented MBM was incorporated into Nile tilapia feed at 6% to replace 100% of the FM.
We observed no large difference in final BW, SGR, final BL, plasma IGF-1 concentration
and PER when compared with the fish fed with diet containing FM (Control diet). Our study
results agree with the conclusion of some previous studies that MBM could be used at 10%
(to replace 100% FM) in feeds for Red drum without affecting the growth (Kureshy et al.,
2000). However, in our study, the replacement of FM with fermented MDM had a marginally
adverse affected on FCR of Nile tilapia. This effect on the FCR was probably due to feed
wastage rather than compromised nutrient utilization or digestibility. The fermented MDM
was not heat processed prior to incorporation into the experimental diet, so when this diet
was subjected to the heat of pellet processing, MDM functioned as a very effective pellet
binder, resulting in more durable, larger pellets. The heat application coagulates the
solubilized protein between the muscle chunks of the MDM, binding them tightly together
with other ingredients (Schnell et al., 1970). After the crumbling process the MDM diet had
larger particle sizes compared to the other diets tested (2000 microns vs 1200 micron). This
large particle size may have caused a physical limitation to feed consumption rather than a
chemical or nutritional limitation. The crumbled test diets were not sieved to a uniform size

64

before feeding them to fish. This can be easily corrected by properly adjusting the feed
manufacturing process. Consequently, we conclude that fermented MDM is a good protein
source for Nile tilapia diets.
YE is a novel protein that has been widely used as feed supplement in the agricultural
feed industry. It is by obtained removing the cell wall of yeast. The production of YE has low
effect on the ecosystem compared to FM production. YE have higher digestible concentrated
protein content than either whole or hydrolyzed yeast. It contains about 50% crude protein
and 1% crude fat. Furthermore, YE is an excellent source of nucleotides, with concentrations
as high as 5% of the dry weight. Whole yeast has been incorporated in aquaculture feed at
levels of 1530%, to replace 25-50% of the FM (Tacon and Jackson, 1985; Tacon, 1994)
without adverse effects on the growth performance of Tilapia. Mahnken et al., (1980) used
Candida spp. as a substitute for FM in Salmon diets and found that the fish accepted
substitution levels from 25%-50% without adversely affecting the growth performance of
Salmon.

Aires Oliva-Teles et al., (2001) reported that brewers yeast (Saccaromyces

cereisae) can replace 50% of FM protein without adversely affecting the growth performance
of juvenile Sea bass (Dicentrarchus labrax). They also reported no beneficial effect after
supplementing the brewers yeast diets with methionine. There is limited information on the
use of YE as a FM replacement in fish diet. Our observation when we used 6% YE to replace
6% FM (a complete FM replacement) in Nile tilapia diet show no effect on the growth
performance when compare to fish fed the FM diet. It is important to note that the market

65

cost FM and YE is similar, so there is little economic advantage at the present time of
replacing FM with YE in tilapia diets unless it is for the sake of environmental sustainability.
Overall, our findings in Nile tilapia using PBM, fermented MDM and YE as FM
alternatives agree with the previous reports on that alternative protein sources can be used in
tilapia feed without affecting the growth performance. In this study, adequate amino acid and
other nutrient requirements were provided in the experimental diets. The difference in the
value of some growth parameters in the experimental diets could not be attributed to a lack of
nutrients but rather to processing conditions, digestibility, palatability, or a combination of
these factors (Nengas et al., 1999; Webster et al., 2000). The FCR values observed in our
study were higher than those reported by Wu et al. (2002) for Nile tilapia fed diets containing
corn gluten meal, full-fat soy, and synthetic amino acids (FCR 1.67-1.79), but our results
were within the 1.7-2.3 FCR values reported by Siddiqui et al., (1991) for tilapia with an
initial weight of 19 g and fed a 34% protein diet for 98 days in outdoor concrete tanks. In
contrast, Stickney and McGeachin (1984) fed a 32% protein diet to tilapia with an initial
weight of 4.4 g for 84 days in aquaria and obtained FCR values of 1.9-2.8. The FCR values
of 1.62-1.75 observed in our study were similar to the 1.67-1.79 FCR values reported by Wu
et al., (2002), Siddiqui et al., (1991) and Stickney and McGeachin (1984). The PER value are
within the 0.72-2.15 range reported by Siddiqui et al. (1988) but were higher than the PER
range of 1.42-1.56 reported by Wu et al. (2002) for Nile tilapia fed diets containing corn
gluten meal, full-fat soy, and synthetic amino acids. The plasma IGF-1 concentration and

66

proximate composition of the carcass was not affected by the replacement of FM with the
alternative proteins. Chemical analysis of the carcass showed no significant differences in
dry matter, crude protein, crude fat, ash or gross energy content among the juvenile Nile
tilapia fed with the test diets. Other scientists reported similar results (Davis et al., 1978;
Mazid et al., 1979; Winfree et al., 1981; and Jauncey, 1982).
The observed effects of the replacement of FM with PBM, fermented MDM and YE
in feed on growth performance and body compositions of Nile tilapia (Oreochromis
niloticus) corroborates with the previous studies, implying that YE, PBM and fermented
MDM are feasible alternative sources of protein for juvenile Nile tilapia. However, the
current price/ton of FM, MDM (similar to high quality pet-food grade PM) and PM are
$1375, $700 and $317.50 respectively (Feedstuff, 2010). YE is a commercial product that
has similar price with FM. The replacement of FM with PBM and fermented MDM are the
most economically feasible of all the alternative protein sources tested. Although, PBM has a
relatively inferior PER, more quantity can be fed to the fish, even though this may come at
the expense of increased nutrient emissions and reduced water quality. The FCR of the
fermented MDM can be improved by adjusting the feed processing condition to ensure a diet
of good particle size is produced. Also, considering the cost and availability of these
alternative protein sources, a certain degree of compromise in quality in favor of economy
might be acceptable. Therefore, all future protein sources studied should be considered for
protein replacement in feeds for Nile tilapia and perhaps other species of fish.

67

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31. Stone, D.A.J., Allan, G.L., Parkinson, S., Rowland, S.J., 2000. Replacement of fish
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growth using meat meal products. Aquaculture 186, 311 326.

32. Spring, P. and D.F. Fegan. 2005. Mycotoxins a rising threat to aquaculture? In:
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33. Tacon, A.G.J., 1994. Feed ingredients for carnivorous fish species: alternatives to
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38. Wu Y. Victor, Ronald Rosati, Kathleen Warne (2000). Growth, feed conversion,
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72

CHAPTER 3

Replacement of fishmeal with fermented mechanically deboned poultry meat in feeds

for Hybrid striped bass (Morone chrysops X M. saxatlis).

A. Ayoola1, R. Malheiros1, P. Ferket1, E. Won2, R. Borski2, C. Stark1

1

Departments of Poultry Science1 and Biology2, College of Agriculture and Life

Sciences, North Carolina State University, Raleigh.
August, 2010

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3.1 ABSTRACT
Lactic acid preserved mechanically deboned poultry meat (MDM) is a high-quality
protein source that may be a lower cost alternative to fishmeal (FM) in diets for hybrid
striped bass (HSB). A trial was conducted to evaluate of the effects of replacement of FM
with fermented lactic acid-stabilized MDM in the diet of HSB on growth performance and
protein efficiency of palmetto HSB (Morone saxatilis X M. chrysops). Five iso-caloric and
iso-nitrogenous diets were formulated with different level of FM replacements (0%, 25%,
50%, 75%, and 100%) and manufactured into 2mm sinking pellets. Juvenile (41.935.95g)
fish were randomly distributed among 15, 76 L recirculating culture tanks containing 5 fish
each; 3 replicate tanks were assigned per treatment. Throughout the 77-d trial, the fish were
fed twice daily by dropping some pellets in each tank until feeding activity of fish ceased.
The time lag for fish in each tank to initiate feeding behavior was recorded at each feeding.
Feed intake (FI) was recorded daily, and body length (BL) and body weight (BW) were
determined on each fish at 77 d, and 1-77 d feed/gain (FCR) and specific growth rate (SGR)
was calculated. At 77 d, caudal vein blood samples were collected from each fish for plasma
IGF-1 assay.

Adipose fat and liver tissue sample were collected to determine the adipose

somatic index (ASI) and hepatosomatic Index (HSI) respectively. Hepatic IGF-1 mRNA
levels were also quantified by quantitative PCR (qPCR). Three fish per tank were also
sampled at 77 d for proximate analysis of carcasses, and protein efficiency ratio (PER)
determinations.

74

Fish fed the diet that replaced all of the FM with MDM (100% treatment) exhibited
significantly longer response time to the initiation of feeding behavior than all other dietary
treatments that included FM (17 vs 12 s, P<.05), and this difference was more pronounced
during the first 30 d of the trial. FI of the 25% treatment was significantly greater than the
100% treatment (689.5 g vs 534 g, p<.05) but not different from the 0%, 50% and 75%
treatments (583.4 g, 667.7 g and 644 g, respectively). SGR was positively correlated with FI
and was greater among fish in 25% treatment group than the 0% and 100% treatment groups
(0.015 vs 0.012 and 0.012, p<.05). Neither of these groups was different from 50% and 75%
treatments (0.013 and 0.014, respectively). The 75% treatment resulted in significantly
greater 77 d BW than the 100% treatment (157.1 g vs 125.75 g, p<.05), but was not different
from the 0%, 25% and 50% treatments (138.20 g, 146.5 g and 146.06 g respectively).
Similarly, the 75% treatment resulted in greater 77 d BL than the 100% treatment (235.85
mm vs 215.83 mm, p<.05), but did not differ from the 0%, 25% and 50% treatments (222.6
mm, 220.22 mm and 224.89 mm, respectively). Plasma IGF-1 was higher in 50% treatment
group than the 100% group (41.81 vs 32.22, p<.05), but neither of these groups were
different from 0%, 25% and 75% treatments (37.51, 40.44 and 41.11 respectively). Dietary
replacement of FM with fermented MDM had no (effect p>0.05) on FCR, PER, ASI, HSI,
hepatic IGF-1 gene expression and carcass proximate composition among treatments.
In conclusion, the fish fed with diets replacing 50% and 75% of the FM with MDM
performed better than those fed diets that replaced 0% or 100% of the FM with MDM.

75

Evidently, it is feasible to replace up to 75% of the FM in HSB diets with fermented

mechanically deboned meat and achieve equal or better growth performance.

76

3.2 INTRODUCTION
Hybrid Striped Bass (HSB) are a cross between striped bass, Morone saxatilis, and
white bass, M.chrysops. The crossing of a female striped bass with a male white bass (the
original cross) is called the Palmetto Bass, and the crossing of a male striped bass with a
female white bass is called the Sunshine Bass (the reciprocal cross) (Pine et. al, 2005;
Muzinic et al., 2006). Hybrid striped bass have traits that are favorable for intensive
aquaculture production, including rapid growth rates, broad physiological tolerances, and a
high market value (Carlberg et al. 1984). The HSB aquacultural industry is a rapidly growing
industry in the United States (Gerald, 2004) due to the vacuum created by cessation of the
commercial natural striped bass stock. The annual production in the U.S. has increased from
about 400,000 pounds in 1987 to about 11.5 million pounds in 2003 (Margaret and Bill 2003,
2004; Carl et. al, 1999). Hybrid striped bass is now the fifth largest aquaculture industry in
the US in terms of volume and the fourth largest in terms of value (Carlberg and Massingill,
2005; Margaret and Bill, 2003; Dunning and Daniels 2001) due to the increasing demand for
the high market value of the flesh and the improved fish culture technology.
Although, the revenue from HSB is increasing, the cost of production is increasing at
a greater rate (Pine et al., 2005). Like other culture fish and crustaceans, feed cost is a
considerable portion of operational expense for HSB aquaculture: it accounts for about 40%
to 50% of the total variable production cost (FAO, 2006; Dunning 1998, D.Abramo et al.,
2000) and reaches as high as 70% (Webster et al., 1999). The high cost of these feeds is

77

attributed to protein content (Webster et al., 1999). HSB is a carnivorous finfish requiring
high quality protein in their diet. Currently, most of this protein comes from fishmeal, with
dietary inclusion levels of about 35%-45% (Tacon and Metian, 2008; Brown et al., 1992;
Nematipour et al.,1992). In 2006, the aquaculture sector consumed over 3.7 million tonnes of
fishmeal, which accounts for about 68.2% of the total world fishmeal production (Tacon and
Metian, 2008).
Substitution of fish meal with an alternative protein source could sustain and stabilize
the continued growth of the hybrid striped bass industry. Driven by the increasing global
demand and the inconsistency in the price and supply of fishmeal, considerable research has
been done to evaluate the total or partial replacement of fishmeal with less expensive plant
and animal protein meals that may help to reduce feed costs. However, the inclusion level of
these alternative proteins in HSB diets depends on the source of the protein. Soybean meal
can replace up to 85% of fishmeal as a protein source in HSB feeds (Webster, 1995).
Juvenile palmetto HSB (Morone saxatlis X M. chrysops) can be fed with 16% fishmeal and
44% soybean meal (48% CP) without adverse effect on the growth performance (Gallagher,
1994). Similarly, the diet of palmetto HSB (Morone saxatlis X M. chrysops) could contain
23% and 40% soybean meal without any effect on the growth performances (Brown et al.,
1997). Webster et al., (1999, 2000) observed that diets in which the fishmeal is completely
replaced with a combination of meat and bone meal (MBM), poultry by-product (PBM),
soybean and distiller dried grains with soluble (DDGS) and plant source proteins can be fed

78

to sunshine bass (Morone chrysops X M. saxatlis) without any adverse effects on the weight
gain, growth rate and body composition (Webster et al., 1999). Similarly, turkey meal and
poultry by-product meal could be used in sunshine HSB (Morone chrysops X M. saxatlis)
diets to completely replace the fishmeal without adverse effects on body weight gain, specific
growth rate, and feed conversion ratio (Pine et al., 2008; Muzinic et al., 2006).
Another alternative protein source to fishmeal may be mechanically deboned meat
(MDM). MDM is a potentially valuable and locally available by-product of poultry
processing in the USA. MDM may be a high-value candidate as a fishmeal replacement in
fish feed because of its excellent nutritional composition, low ash content, and quality.
Poultry meat is an important diet for many countries due to its relatively inexpensive cost
(Chiarini et al., 2009). MDM from poultry is predominantly obtained from poultry carcasses
with the valuable parts such as wings, legs and breast removed. The soft and hard tissues of
carcass parts after the meat is removed during poultry processing is mechanically separated
using a high pressure chamber device with small holes in it (Baker and Bruce, 1995;
Froning, 1981). Through the application of high pressure, carcass or bone-attached meat, fat
and skin separates from the bones and those materials pass through the barrel sieve (around
0.5-0.8 mm in diameter), whilst the bone part remains inside the barrel and is discharged
separately (Baker and Bruce, 1995). Ordinarily, MDM should not contain bone particles
within the ground meat fraction obtained, and the amount of small fragments should not
exceed 0.3% (Trindale et al., 2004).

MDM is frequently used in the formation of

79

comminuted meat products due to its fine consistency and relatively low cost (Melten et al,
2005). MDM contains a significant amount of sarcoplasmic proteins (Webb et al., 1976) and
as a result of the infusion of bone marrow in MDM there is a great variation in fatty acid
content and a high percentage of cholesterol and phospholipids in MDM (Al-Najdawi
Abdullad, 2002). The protein content is MDM is usually around 12-17% protein and this
figure depends heavily on the amount of fat present and at the same time. Some fine bone
particles (<0.5 mm) remain in the meat mass, so MDM has a higher calcium content
(varying from 60 to 280 mg Ca/100 g MDM) than ground poultry meat (Trindale et al.,
2004). MDM has a neutral pH value of around 6.3-6.5 (Murphy and Silbert, 1992), which has
a strong negative impact on the development of the curing color.
The microbiological status of MDM is of great importance and care has to be taken
that bones and carcasses, processed for the production of mechanically deboned meat, exhibit
a low bacteria count. MDM has a large surface area and is therefore susceptible to high levels
of bacterial growth, which is influenced also by the processing technology and hygiene
(Miettinen et al., 2001; Berrang et al., 2002; Chasseignaux et al., 2002; Vitas et al., 2004;
Barbalho et al., 2005; Reiter et al., 2005). The bacteria count in MDM should not be higher
than in ordinary minced muscle meat and should not exceed 105-106 colony-forming units
(cfu)/g (Chasseignaux et al., 2002; Vitas et al., 2004; Barbalho et al., 2005; Reiter et al.,
2005). Also, the presence of bone marrow in mechanically deboned meat can speed up
oxidation of fat, given that bone marrow contains a fair amount of metals such as iron,

80

magnesium and copper, which act in a pro-oxidative manner. Finally, the fat content of
MDM can vary dramatically and accelerate the oxidation of the products (Ordez et al.,
1999).
Lactic acid fermentation of ground poultry carcasses is a viable method to stabilize
whole poultry carcasses at ambient temperatures and allow the nutrients in the carcasses to be
utilized more efficiently as feed ingredients (Blake and Donald, 1992 and 1995; Murphy and
Silbert, 1992; Cai et al., 1994a; Cai et al., 1994b; Cai and Sander, 1995; Middleton et al.,
1999). Fermented silage has a pH of 4-4.5 compared to neutral pH (6.3- 6.5) of fresh tissue
(Murphy and Silbert, 1992). The low pH of fermented grounded meat with lactic acidproducing bacteria is very effective in inactivating pathogenic viruses, bacteria and prevents
undesirable degradation processes (Wooley et al., 1981; Cai et al., 1994a; Erikson et al.,
2004) and bacteria (Talkington et al., 1981a, Talkington et al., 1981b). Murphy and Silbert
(1992) reported that acid silage (pH=4.2) was produced when broiler carcasses were
fermented with 17.5% glucose or 10-20% of other fermentable carbohydrates. Fermentation
of MDM helps with decontamination of the product, increases its shelf life, improves the
palatability through improved flavour characteristics and the possibility of using in a feeds
production (Erickson et al., 2004). The objective of the present study was to evaluate the
feasibility of either partially or totally replacing fishmeal with fermented mechanically
deboned meal in diets for Palmetto bass (Morone saxatlis X M. chrysops).

81

3.3. MATERIALS AND METHODS

3.3.1 Ingredient preparation, feed formulation, and feed manufacturing
This study was designed to evaluate nutritional value of the replacement of fish meal
(FM) with fermented mechanically deboned meat (MDM) in hybrid striped bass (HSB) diets.
Five iso-amino acids and iso-caloric (3200 kcal metabolizeable energy/kg of diet) diets
manufactured as slow sinking pellets were formulated with commercially available
ingredients (Table 2). The protein sources used in the experimental diets included high
protein soybean meal (48% CP) (Cargill Inc., Raleigh, NC), mechanically deboned meat
(MDM) from poultry carcasses (NC State University, Poultry Farm, NC State Raleigh, NC),
menhaden fishmeal (Special SelectTM, Omega Protein, Inc., Houston, TX), and poultry byproduct meal (PBM) (Valley Protein Inc., Fayetteville, NC). Their nutrient profiles are
illustrated in Table 2. The MDM was then stabilized to pH<4.7 by lactic acid fermentation
using a procedure as described by Middleton et al., (2001). It was mixed with 12% dextrose
(wt/wt) and inoculated with 4 billion cfu of lactobacillus acidophilus per g of fermentation
mixtures and maintained at ambient temperature in a closed but vented container for a period
for 14 days.
A standard commercial diet (D1) for HSB was formulated to contain about 41%
protein, 10% lipid and 3,200 kcal/kg estimated metabolizeable energy (Table 2). Protein in
the D1 diet, which contained no MDM, was supplied by a mix of animal and plant proteins
that included about 37.19% soybean meal (SBM), 25% Menhaden fishmeal (FM) and 5%

82

PM and that are considered typical of commercial formulations. MDM was tested at four
dietary inclusion levels: 25% (D2), 50% (D3), 75% (D4), and 100% (D5), respectively, at the
expense of FM (Table 2). All diets contained minimum requirement of all essential nutrients
to satisfy the needs of HSB (NRC, 1993).

83

Table 1. Nutrient composition of dietary protein sources

Nutrient
Dry matter, %
Crude protein,%
Crude fat, %
Crude fiber
Ash, %
Neutral detergent
Acid detergent fiber, %
Calcium, %
Potassium, %
Sodium,%
Chloride, %
Total phosphorus,%
Magnesium, %
Zinc, Mg/ Kg
Iron, Mg/ KG
Manganese, M
Copper, Mg/kg
Lysine, %
Methionine, %
Cysteine, %
Leucine, %
Isoleucine, %
Threonine
Tryptophan
Phenylalanine
Tyrosine, %
Valine, %

FM1
90.2
62.3
9.4
0.90
18.4
5.2
0.70
0.40
0.55
3.04
0.14
148
480
34
11.0
4.81
1.77
0.57
4.66
2.57
2.64
0.66
2.56
2.03
3.37

SBM2
90.4
48.5
1.3
3.65
6.0
7.0
6.3
0.4
2.58
0.02
0.03
0.64
0.36
68
238
52
19.0
3.04
0.70
0.72
3.70
2.20
1.93
0.63
2.49
1.75
2.57

MDM3
88.0
61.0
22.5
0.04
2.3
1.6
0.08
0.07
0.07
0.35
0.03
106
389
10
12.
3.06
0.85
0.55
3.51
1.77
2.13
0.46
1.62
0.83
2.52

FM is menhaden fishmeal (Special SelectTM, Omega Protein, Inc., Houston, TX)

SBM is high protein soybean meal (Cargil Inc., Raleigh, NC)
3
MDM is mechanically deboned poultry meat (MDM) (Townsends Inc., Siler City, NC)
4
PM is poultry by-product meal (Valley Protein Inc., Fayetteville, NC)
1
2

84

PM4
96.7
65.0
12.7
0.32
12.7
23.8
5.5
2.9
0.70
0.38
0.50
2.15
0.12
119
207
10
9.0
3.42
0.95
0.67
3.99
2.01
2.37
0.50
1.84
0.94
2.86

Table 2 describes the ingredient formulation and nutrient analysis of the experimental
diets. All of the feed was manufactured at the North Carolina State University Feed Mill
Educational Unit in accordance with current Good Manufacturing Processes (cGMPs). The
diets were prepared by mixing lactic acid fermented MDM with other ingredients according
to their respective formulations (Table 2) in a mixer (Model N1-20, Triumph manufacturing
company, Cincinnatus, Ohio, USA) for about 20 minutes, followed by the addition of 600
ml water until a stiff dough was obtained. The moist diet was extruded through a meat
mincer with a die (A-200, The Hobart MFG Co, Troy, Ohio, USA). The resulting pellets
were then dried at 60 C for 72 hours (Blue M Drying Oven, General Signal, Atlanta, GA).
The cooled pellets were crumbled and kept in air tight polyethylene bags and stored at
ambient temperature in the laboratory over the period of the experiment. The durability of
each dietary treatment pellet was determined before through pellet durability analysis before
the start of the experiment.

85

Table 2. Feed formulations and proximate analyses of diets for the evaluation of fishmeal
replacement with fermented mechanically deboned meat (MDM) in HSB diet
Experimental diet number (% FM : % MDM)

Ingredients
CARGIL SBM

D11
D22
D33
D44
D55
(100 : 0)
(75 : 25)
(50 : 50)
(25 : 75)
(0 : 100)
-------------------------- (% of Diet as is basis) ------------------37.19
22.84
24.04
25.42
27.93

FISH MEAL, MENHADEN

25.00

18.75

12.50

6.25

SOFT WHEAT

24.38

30.90

30.24

29.48

28.03

POULTRY FAT

6.14

3.84

4.12

4.42

4.88

POULTRY MEAL

5.00

5.00

5.00

5.00

5.00

LYSINE

1.15

1.39

1.55

1.71

1.86

DL METHIONINE

0.55

0.59

0.64

0.70

0.75

DICAL P 18.5

0.21

0.72

1.47

2.22

2.98

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.10

0.08

0.11

0.15

0.20

0.25

Fermented Poultry MDM

15.15

19.11

22.98

26.29

LIMESTONE

0.43

0.87

1.31

1.74

Determined Nutrient Analysis

ME POULTRY, KCAL/KG

3200

3200

3200

3200

3200

CRUDE PROTEIN, %

40.75

43.00

43.00

43.00

43.00

CRUDE FAT, %

10.00

7.37

7.14

6.93

6.87

CRUDE FIBRE, %

2.33

1.96

1.93

1.90

1.89

CALCIUM, %

1.65

1.65

1.65

1.65

1.65

TOTAL PHOSPHORUS, %

1.160

1.195

1.180

1.166

1.157

AVAIL. PHOS. POULTRY

0.950

0.950

0.950

0.950

0.950

SODIUM, %

0.170

0.170

0.170

0.170

0.170

POTASSIUM, %

1.253

1.043

1.075

1.110

1.166

CHLORIDE, %

0.457

0.499

0.529

0.558

0.584

Na+K-Cl, MEQ/KG

265.77

200.00

200.00

200.91

207.69

NCSU TRACE MINERAL PREMIX

NCSU VITAMIN PREMIX

SODIUM SELENITE PREMIX

MICRO SALT

D = Diet contains only fishmeal as a major source of protein

D = Diet contains 75% fishmeal and 25% fermented mechanical deboned meat as a major source of protein
D = Diet contains 50% fishmeal and 50% fermented mechanical deboned meat as a major source of protein
4
D = Diet contains 25% fishmeal and 75% fermented mechanical deboned meat as a major source of protein
5
D = Diet contains only fermented mechanical deboned meat as a major source of protein
8
NaSeO3 premix provided 0.3 mg Se/kg of complete feed. 6Each kilogram of mineral premix (.1% inclusion) supplied the following per kg of complete feed: 60 mg Zn as ZnSO4.H2O; 60
mg Mn as MnSO4.H2O; 40 mg Fe as FeSO4.H2O; 5 mg Cu as CuSO4; 1.25 mg I as Ca(IO3)2; 1 mg Co as CoSO4. 7Each kilogram of vitamin premix (.1% inclusion) supplied the following
per kg of complete feed: vitamin A, 13,200 IU; cholecalciferol, 4,000 IU; alpha-tocopherol, 66 IU; niacin, 110 mg; pantothenic acid, 22 mg; riboflavin, 13.2 mg; pyridoxine, 8 mg;
menadione, 4 mg; folic acid, 2.2 mg; thiamin, 4 mg; biotin, 0.253 mg; vitamin B12, 0.04 mg; ethoxyquin, 100 mg.
2

86

3.3.2 Pellet durability test

The pellet durability index is an indicator of the hardness of the pellet. It was determined at
two different tumbling periods, 90 and 210 seconds. The durability of the pellet increases
with the increase in the level of replacement of fishmeal with fermented mechanically
deboned poultry meat in both tumbling periods (Figure 1). There is statistically difference in
the durability indices of the diets (Table 3).

87

PELLET DURABILITY INDEX

100

Percentage

95

90

90 Seconds
210 Seconds

85

80

75
0%

25%

50%

75%

100%

DIETARY TREATMENTS

Figure 1: Effect of dietary replacement of fishmeal with fermented mechanically deboned

meat on the pellet durability of the feed.

88

Table 3. Effect of dietary replacement of fishmeal with fermented mechanically deboned

meat on the pellet durability of the feeds.1

% Dietary
Replacement
of FM by
MDM
0
25
50
75
100
Source of
Variation
MDM Level
Linear

Pellet Durability Index

90

210

Seconds
92.50.5c
80.510.5d
bc
93.50.5
85.01.0c
95.00.0ab
89.50.5b
a
96.00.0
91.50.5ab
96.50.5a
93.00.0a
----------------P-Value-------------0.0032
0.0001

0.0001
0.0001

Values are means SD

SEM = Pooled standard error of the mean with 40 degrees of freedom
3
Pellet durability = 91.55+1.05X, where X= % FM replacement
2

89

3.3.3 Water quality management

The recirculation water system was well aerated and backwashed weekly to ensure
good water quality was maintained. Water temperature and dissolved oxygen (DO) were
measured in the tanks twice daily (08:30 and 15:30 h) using an oxygen meter (Model 85, YSI
Industries, Yellow Springs, OH, USA). This was done to ensure that DO levels in the tanks
were optimal for fish growth and well-being. Total ammonia, nitrite, pH and alkalinity were
measured once weekly (13:00 h) using a spectrophotometer (Model DREL/2000, HACH
Inc., Loveland, CO). Total alkalinity was measured once weekly (13:00 h) by titration using
a digital titrator (Model AL-36DT, HACH Inc., Loveland, CO). The salinity was also
measured once weekly (13:00 h) using a refractometer (YSI 85, YSI Industries, Yellow
Springs, OH, USA)

3.3.4 Growth Study

In order to demonstrate the effect of each treatment on growth parameters, a 77-day
feeding trial was carried out at the NCSU fish research facilities (Varsity Drive, Raleigh,
NC). Fish with average weight of 32 grams were randomly allocated at a stocking density of
5 fish per tank among three replicate tanks per experimental diet. Fish were allowed to
acclimatize to the new environment and standard diet for one week before experimental data
were collected. Each 80 L recirculation tank was equipped with central standpipes for
controlling the depth of the water. Biofiltration and UV sterilization were provided for the

90

recirculating culture systems. All the tanks were maintained to the same water quality
parameters throughout the experiment; however there was fluctuation in salinity levels, due
to occasional system back flush and addition of salt to maintain high water calcium
requirements of HSB.
The fish were fed the crumbled experimental feeds in the morning and afternoon of
each day (08:00 and 17:30 h). At each feeding, some pellets were dropped in each tank until
no feeding activity of fish was observed. Fish were fed at a rate of about 3% of average body
weight per day, and the amount of feed consumed was measured weekly by determining the
feeding bag weight difference before and after feeding. All fish were anesthetized, weighed
individually at 0, 35 and 77 days of the experiment. At days 35 and 77, 2 and 3 fish were
randomly sampled, respectively, from each tank euthanized and the length of sampled fish
was measured. Blood samples were collected for plasma IGF I analysis, and the liver was
weighed and sampled for RNA quantification. At day 77, the adipose fat of each fish samples
were collected and weighed.
The mortality weight was determined and recorded as it occurred. Specific growth
rate (SGR) was calculated as [(ln W2 ln W1)/ (T2 T1) X 100], where W2 is the weight at
the end of the growth interval and W1 is the weight at the beginning of the growth interval,
while T2-T1 represents the duration (days) of the growing interval. Specific growth rates
were calculated during the following time intervals: 0-35 d, 35-75 d and 0 -77 d. The feed
conversion ratios (FCR) were calculated as the total weight of feed consumed divided by the

91

total weight gained. Protein efficiency ratios (PER) were calculated as the total amount of the
protein consumed divided by the total amount of body protein gained during the 77 d period.
A total of 5 fish were collected from each treatment tank at 35 and 77 days. The samples
were measured, weighed and were frozen in liquid nitrogen, then stored at -20oC in
polyethylene bags until analyses.

3.3.5 Carcass Analysis

The frozen sampled fish were ground into paste in a grinder (Model No: RU 05,
Manual Grinding Machines, Crocean Industry Co., Ltd. Taiwan) and then dried at 60 C for
48 hours in a drying oven (Blue M Drying oven, General Signal Atlanta, GA) prior to the
chemical analysis. The samples were then ground to a fine powder with a laboratory grinder
(Mortar grinder Mill Model 2, Fritsch Pulverisette, Mount Holly, NJ, USA). Chemical
analysis was done on dry matter basis. Protein (N6.25) was determined by the Dumas
method using a LECO nitrogen automatic analyzer (Tru Spec N, LECO Corporation, St.
Joseph, Michigan 49085, US), gross energy content was determined using a bomb
calorimeter (C5000, IKA Calorimeterm, IKA Werke Labortechnik, Staufen, Germany).
Percentage fat was determined by the ether extraction method.

Percentage ash was

determined by placing diets in a muffle furnace (600 C) for 24 h, and moisture was
determined by drying 24 hours at 65 C in a drying oven (Blue M Drying oven, General
Signal Atlanta, GA).

92

3.3.6 Conditional indices

At the termination of the growth trial, three fish per tank were euthanized and
dissected to determine body condition indices. Conditional indices were calculated according
to the following:
Hepatosomatic index (HSI) =(Liver weight / fish mass) X 100
Adiposomatic index (ASI) = Adipose fat weight/fish mass) X 100

3.3.7 Radioimmunoassy- Plasma IGF-1 concentration

All fish were sampled individually for plasma IGF-I analysis at the beginning (day
35) and the end of the trial (day 77). Blood was taken via a heparinized syringe from the
caudal vein of the anesthetized fish. Circulating levels of total IGF-I were measured from
acid/ethanol extracted plasma by radioimmunoassay (RIA) using recombinant barramundi
IGF-I as tracer and standard, rabbit anti-barramundi IGF-I primary antibody (Novozymes
GroPep; Adelaide, Australia) and goat anti-rabbit secondary antibody (Sigma; St. Louis,
Missouri) according to method described by Shimizu et al. 2000 and Picha et al. 2006.
Barramundi IGF-I was iodinated using the chloramine-T method and purified by column
chromatography. Tracer (125I-barramundi IGF-I) was diluted to 20,000 cpm for each assay
tube.

93

3.3.8 RNA isolation and Quantitative real-time PCR

Total RNA was extracted from liver samples using TRI Reagent (Molecular Research
Center, Cincinnati, OH), precipitation by high salt and LiCl, and treatment with Plant RNA
Isolation Aid (Ambion) to remove the excess glycogen commonly present in liver tissue.
The absorbance ratio at 260:280 nm (A260/280) ranged from 1.8 to 2, as determined by
spectrophotometry using a Nanodrop ND-1000 (Nanodrop Technologies; Wilmington DE).
RNA quality was also confirmed by gel electrophoresis (1% agarose, 0.6 g/ml ethidium
bromide) before and after DNase treatment (DNA-free; Ambion; Austin, TX).
Complementary DNA (cDNA) was synthesized from 1 g of DNase-treated total RNA using
a High Capacity cDNA Reverse Transcription (Applied Biosystems, Foster City, CA) kit
according to the manufacturers protocol. Enzymatic reactions for all samples were
performed at the same time with identical reagents, as to reduce the variability of DNase and
RT efficiency between samples.
Hepatic IGF-1 mRNA expression was determined by quantitative real-time PCR
(qRT-PCR) according to previously published methods (Picha et al. 2008) using genespecific primers and Taq Man probe (forward: 5'-TTGTGTGTGGAG AGAGAGGCTTT-3';
probe:

5'-TTTCAGTAAACCT

ACAGGCTATGGCC-3';

reverse:

5'-TGACCGCC

GTGCATTG-3'). Analysis was performed on an ABI 7900 HT Sequence Detection System

using Brilliant II qPCR Master Mix (Stratagene; La Jolla, CA), 900 nM primers, 250 nM
probe, and 50ng of cDNA in a total reaction volume of 10 L. The qRT-PCR cycling

94

parameters were: 50 C for 2 minutes, 95 C for 10 minutes and then 40 cycles of 95 C for
15 seconds and 60 C for 1 minute. The absence of genomic DNA contamination was
assessed using two negative control templates in place of cDNA: sterile water (No-Template
Control; NTC) and DNase-treated RNA (No-Amplification Control, NAC).
Cycle threshold (Ct) values for experimental samples were transformed using a
standard curve of serially diluted cDNA versus Ct values (R2 = 0.99) and normalized to
reflect the amount of cDNA template per ng total RNA. Since liver mass and total hepatic
RNA values fluctuate depending on the nutritional state of the fish, data was further
normalized to total liver RNA production as a function of body weight. Total hepatic IGF-1
gene expression, relative to body mass, was calculated by the following equation: [(ng total
liver RNA) (ng cDNA per ng RNA)] / (g body weight)] (Picha et al. 2008).

3.3.9 Statistical Analysis

The experiment was designed and statistically analyzed as a completely randomized
designed consisting of four dietary treatments. Tank mean values were considered
experimental observation units for statistical analysis. For IGF-I it is the mean of individuals
measured within a treatment. Regression analysis and statistical significance of the main
treatment effects on each measurement was performed using the JMP procedure of SAS
(SAS Institute, 2009). Treatment variable effects were considered significant at p< 0.05. The

95

Tukey LS Means procedure of JMP was used to determine significant difference among the
individual treatments.

3.3.10 Animal ethics

The experiment herein reported was conducted in accordance to the guidelines of the
Institutional Animal Care and Use Committee at North Carolina State University.

3.4. RESULTS
3.4.1 Water Quality
The HSB used in this experiment adapted well to the environment and began normal
feeding within 24 hours. Throughout the study, water quality for each experimental unit tank
was controlled to be within the critical limits acceptable for optimum growth and health of
HSB (Boyd, 1979; Mazik et al., 1991). The meanSEM for total alkalinity, salinity and pH
(195 5.0904 ppm, 5.140.26 ppm and 8.020.01, respectively) were recorded, along with
the means for total ammonia-nitrogen and nitrite-nitrogen (0.0560.008 ppm, 0.480.056
ppm respectively).

Daily meanSEM temperatures and dissolved oxygen were

23.430.079oC and 7.060.52mg/L respectively.

96

3.4.2 Growth Performance Characteristics

The overall growth performance of the HSB observed in this experiment is shown in
Table 3.

No mortality was observed among the treatment groups throughout the

experimental period. These general performance characteristics indicate that the experimental
cultivation condition (water quality, feeding rates, and basal diet formulations, fish handling,
etc.) were adequate. Initial weight of fish stocked in the growth trial did not differ
statistically among the dietary treatments and averaged 41.935.95g. A statistical difference
was observed among treatments behavioral response to feed during periods of the study
(figure 1, table 3). Generally, at the onset of the experiment, it took the fish a longer time to
respond in initiating feeding behavior when experimental diets were offered than during the
latter periods of the experiment, likely because of behavior conditioning (figure 1). There
was a strong statistical difference between the feeding response time of the fish fed with diet
containing no FM and those fed with other diets; however the statistical difference between
treatments decreased towards the latter period of the experiment, with no statistically
significant differences observed among treatments for feeding response time during the last 7
days of the experiment. Regardless of the observational period effects, the replacement of
FM with fermented MDM had a cubic effect on the feeding response time. Fish fed diets that
replaced 0%, 25%, 50% and 75% of the FM with fermented MDM responded significantly
faster to the presence of feed dropped into their tanks than those fed diet containing 100% of
the FM replaced with fermented MDM (table 3). The diet containing 50% of the FM replaced

97

by MDM had the shortest response time to feeding behavior, while the fish fed the 100%
replacement diet had the longest response time over the experiment.

98

Effect of dietary replacment of fishmeal with fermented mechanical deboned meat on the feed response
time of juvenile Hybrid striped bass over a 77-days growth study in tanks
45
40
0% Replacement

Time (seconds)

35

25% Replacement
P<0.005

30

50% Replacement
75% Replacement

25

100% Replacement
20

P>0.05

15

P<0.02

10

P>0.05

5
0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77

Days of experiment

Figure 2. Feeding response time (mean SEM; N= 3 tanks/ treatment; 5 fish/tank) of Hybrid
striped bass to dietary replacement of fishmeal (FM) with fermented mechanically deboned
meat (MDM) over a 77-day growth study in tanks. Dietary treatments represented increasing
levels of FM replacement by MDM: 0%, 25%, 50%, 75% or 100%. Colored lines depict
average response time of two feedings plotted by day. Feeding response time is the period of
time (seconds) from when the feed was dropped into the tank until the first strike of feeding
behavior. Periods of significant treatment differences (100% versus other treatments)
illustrated by arrowed lines.

99

The effect of dietary treatment on feed response time, FI, SGR, FCR, BW and BL
throughout the experiment is summarized in Table 3.

Feed intake varied across the

treatments because fish were fed to apparent satiation at each feeding time. Dietary
treatments significantly affected feed intake, with a cubic regression response as the level of
dietary MDM increased. Fish fed with 25% and 50% replacement trended to consume the
most feed, but the only the fish consuming the 25% replacement diet consumed significantly
more feed than those fed the 100% replacement diet. The replacement of FM with fermented
MDM had significant cubic effects on the BW of HSB. Fish fed with 100% replacement had
the lowest final BW, which was statistically different from other treatments. SGR had a
linear inverse relationship to the level of inclusion of fermented MDM. In comparison to
those fish fed the diet containing 25% replacement, fish fed the diets containing 0% and
100% replacement had significantly lower SGR (0.015 vs 0.012, 0.012; p<0.05,
respectively), but neither of these parameters were significantly different from those fed with
diets containing 50% or 75% replacement of FM. There were no consistent dietary MDM
level effects on BL, although fish fed with the diet containing 100% replacement diet had
significantly greater BL than those fish fed the 75% replacement diet (215 vs 235; p<0.05
respectively), but neither of these treatment groups were significantly different from the other
treatment groups. There were no significant treatment effects on feed conversion ratio
(feed/weight gained).

100

Table 4. Effect of dietary replacement of fishmeal with fermented mechanically deboned

meat on the growth response parameters of Hybrid striped bass over a 77-day growth study
in tanks.1
% Dietary
Replaceme
nt of FM by
MDM
0
25
50
75
100
Source of
Variation
MDM
Level
Linear
Quadratic
Cubic
SEM(40)2

Feeding
response
Time

Feed
Intake

Specific
growth rate

Feed/
weight
gained
----------------------------- Days 1 - 77 ---------------------------

Body
Weight

Body
length

---------------- Day 77 -------------

gram/gram
seconds
gram
gram/day
Gram
Mm
b
ab
b
13
583.416.83
0.0120.001
0.970.12
138.208.30ab
222.674.26ab
220.2215.51ab
12 b
689.578.83a
0.0150.002a
0.980.05 146.5012.15a
b
ab
ab
a
11
667.99.04
0.0130.001
1.010.07
146.067.04
224.633.70ab
b
ab
ab
a
12
644.031.71
0.0140.001
1.010.07 157.1013.02
235.896.75a
a
b
b
b
17
534.430.52
0.0120.001
1.020.09 125.7512.13
215.837.60b
-------------------------------------------------------P-Value--------------------------------------------------

0.0001

0.0097

0.0261

0.9187

0.0023

0.0204

0.1262
0.0010
0.00153
0.4320

0.4507
0.0057
0.01934
18.47

0.04985
0.1497
0.0824
0.0004

0.3403
0.6398
0.8283
0.0400

0.3458
0.0073
0.00396
8.7500

0.4769
0.7750
0.1644
4.3100

Values are means SEM; N= 3 tanks/treatment; 5 fish/tank.

SEM = Pooled standard error of the mean with 40 degrees of freedom
3
2
3
Feeding response time = 13.63-(0.0051X)-(0.11X ) + (0.0086X ), where X= % FM replacement
4
2
3
Feed intake = 495.37+ (102.96X)-(6.75X )-(2.41X ), where X= % FM replacement
5
Specific growth rate = 0.012+ (0.0002X), where X= % FM replacement
6
2
3
Body weight = 162.75-(41.36X) + (20.95X )-(2.82X ), where X= % FM replacement
2

101

3.4.3 Radioimmunoassy- Plasma IGF-1 concentration and Hepatic IGF-1 mRNA Levels
and Compositional Indices.

Table 4 is a summary of the effect of dietary inclusion of fermented MDM at the expense
of FM on plasma IGF-1 concentration, the hepatic expression of IGF-1 mRNA, ASI and HSI.
In this study, the responses in plasma IGF-1 and hepatic IGF-1 mRNA expression to dietary
inclusion of fermented MDM followed were similar and paralleled those to growth. Plasma
IGF-1 concentrations increased by a significant cubic function as the level of dietary
inclusion of fermented MDM increased, with the maximum plasma IGF-1 observed among
fish fed diets that replaced 50% of the dietary FM by fermented MDM.. The replacement of
FM with fermented mechanically deboned meat did not significantly alter hepatic expression
of IGF-1 mRNA (Table 4). Conditional indices ASI and HSI were not affected by the
replacement of FM with fermented MDM in this study.

102

Table 5. Effect of dietary replacement of fishmeal with fermented mechanically deboned

meat on the plasma and hepatic IGF-1 expression and compositional indices of Hybrid
striped bass measured on day 77 of a 77-day growth study in tanks.1

% Dietary
Replacemen
t of FM by
MDM

Plasma IGF- 1
(ng/ml)

0
25
50
75
100
Source of
Variation
MDM Level
Linear
Quadratic
Cubic
SEM(40)4

0.0463
0.0283
0.0024
0.00265
2.5425

Hepatic IGF-1 mRNA

HSI3
ASI2
(gram/gram)
expression
(gram/gram)
(ngcDNA/gBW)
37.517.40a
0.15790.0318
3.242.49
1.690.17
ab
40.448.05
0.15570.0333
3.052.40
1.680.14
41.815.03a
0.18450.0260
3.002.37
1.600.08
41.118.45a
0.15290.0312
2.671.97
1.600.09
32.228.64b
0.11850.0293
2.972.32
1.460.09
------------------------------------------------P-Value-----------------------------0.6519
0.4093
0.3744
0.5529
0.0288

0.3375
0.1418
0.2022
0.2810
0.2292

Values are means SEM; N= 3 tanks/treatment; 5 fish/tank

ASI=Adiposomatic index = fat weight/body weight
3
HSI=Hepatosomatic index = liver weight/body weight
4
SEM = Pooled standard error of the mean with 40 degrees of freedom
2
3
5
Plasma IGF-1= 46.21-(12.59X)-(6.23X )-(0.87X ), where X= % FM replacement
2

103

0.3128
0.0505
0.1064
0.2147
0.1117

3.4.4 Carcass Composition

At the end of the 77-day experimental period, carcass composition was determined by
proximate analysis (Table 5). Regardless of dietary treatment, there was no significant
difference (p>0.05) among the experimental treatment groups on carcass dry matter, crude
protein, ash, and gross energy content. However, a quadratic response to increasing dietary
inclusion level of fermented MDM level was observed on crude fat content, with the
minimum carcass fat content observed among fish fed the diet that replaced 50% of the FM
with fermented MDM.

104

Table 6. Effect of dietary replacement of fishmeal with fermented mechanically deboned

meat on the dry matter, crude protein, ether extract, ash content and gross energy
composition of Hybrid striped bass measured at day 11 after a 77-day growth study in tanks.1

% Dietary
Replacement
of FM by
MDM

0
25
50
75
100
Source of
Variation
MDM Level
Linear
Quadratic
Cubic
SEM(40)2

Dry Matter

Crude
Protein

Crude Fat

ASH

Gross Energy

----------------------------- (%) ------------------------------(Kcal/g)

28.820.54
74.830.59
21.050.45 14.520.22
532237.72
28.570.52
74.110.85
19.071.04 14.230.38
5243104.91
28.770.44
76.600.54
18.671.20 13.990.25
513261.64
28.710.64
75.570.96
19.920.86 13.752.28
519779.75
28.010.94
75.230.57
20.681.45 13.960.78
4661464.41
----------------------------------------- P-Value --------------------------------------0.9483
0.4523
0.7496
0.6206
0.5546

0.0958
0.5233
0.2087
0.2906
0.5911

0.2268
0.8290
0.04083
0.0538
1.0999

Values are means SEM; N= 3 tanks/treatment; 5 fish/tank.

SEM = Pooled standard error of the mean with 40 degrees of freedom.
2
3
Crude fat =20.96+(0.09X)-(0.00092X ), where X= % FM replacement
2

105

0.4877
0.1262
0.2624
0.3817
0.3996

0.4280
0.2603
0.2173
0.3893
93.7129

3.4.5 Protein Efficiency Ratio

Protein efficiency ratio (PER) is an indicator of the quality of the dietary protein in
the diet, relative to the amino acid balance, digestibility, and bioavailability. High PER is an
indicator of good protein quality. The replacement of FM with fermented MDM had no
significant effect the PER of HSB (Figure 3, p>0.05).

106

Protein efficiency ratio(Body weight

gain over the period observed/
protein intake by fish)

Effect of dietary replacement of fishmeal with fermented

mechanically deboned meat on the protein efficiency ratio of Hybrid
striped bass
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0%
Replacment

25%
Replacement

50%
Replacement

75%
Replacemnet

100
Replacement

Dietary treatments

Figure 3. Protein efficiency ratio (mean SEM; N= 3 tanks/ treatment; 5 fish/tank) of

Hybrid strip bass over a 77-day growth study in tanks. Treatments were fed with diets either
containing 0%, 25%, 50%, 75% or 100% replacement of fishmeal (FM) with fermented
mechanically deboned meat (MDM). There was no statistically significant differences in the
protein efficiency ratio among the fish fed with different diets (p>0.05).

107

3.5 DISCUSSION
Fishmeal is a major dietary protein source for commercial HSB (Rumsey 1994;
Tacon 1994; Muzinic et al., 2006), but decreasing global FM supply relative to demand and
increasing costs threaten the economic sustainability and growth of the HSB industry
(Rumsey 1994; Tacon 1994; Muzinic et al., 2006). There is a need to remove or limit the
dietary inclusion of FM to reduce feed costs for HSB and decrease the reliance on FM.
The complete or partial substitutions of FM with alternative proteins that limit
potential effects on HSB growth performance has been demonstrated in past studies and are
similar to our observations (Webster et al., 1999; DAbramo et al., 2000; Muzinic et al.,
2006; Rawles et al., 2006; Pine at al., 2008). Complete replacement of FM with PBM has
been found to be suitable for HSB without adverse effect on the growth performances
(Webster et al., 1999, Rawles et al., 2006). Pine et al. (2008) observed no adverse effect on
the growth performance and body composition of sunshine bass after a complete replacement
of FM with PBM. DAbramo et al. (2000) performed a 175 d grow-out of phase III sunshine
bass (144 - 188 g fish), replacing up to 67% of FM with soybean meal (SBM) in the diet.
They observed no significant differences in growth performance when 67% of dietary FM
protein was replaced SBM protein. Muzinic et al. (2006) evaluated the use of turkey meal to
replace FM in a sunshine bass diet. They concluded that tank-grown sunshine bass can be fed
a diet containing 264 g/ kg turkey meal without FM in comparison to diets containing up to
300 g / kg FM, without adverse effects on weight gain, growth rate, feed conversion and

108

body composition. However, the growth performance of fish fed the diet with only turkey
meal was more variable than those fed diets containing FM, indicating possible differences in
feed palatability and attractiveness even though the diets satisfied the nutrient requirements
of HSB (Webster, 1999).

Feed costs are a significant expense in all aquaculture operations, and there is need to
maximize feeding rates, improve feed conversion efficiency, and reduce feed wastage,
thereby reducing production costs that are vital to sustained profitability. In the tank study
reported herein, total replacement of FM with fermented MDM had a profound effect on
behavioral response of HSB to the feeds. The behavioral response was observed as the period
of time between when feed was dropped into the tank and the first morsel of feed was
consumed; we considered this feeding response time as a measure of organoleptic palatability
and feeding behavior stimulation. Both feeding response time and feed intake data indicate
that increasing dietary levels of fermented MDM in place of FM positively affected the
organoleptic attraction and feeding stimulation property of the feed. Fish fed with 25%, 50%
and 75% replacement had similar feeding stimulation and response time compared to the
control diet. However, the rapid feeding stimulation and initiation was not proportionally
associated with continued feeding, as the fish offered feed containing 25% MDM
replacement had similar feeding response time as those fed diets containing 50% and 75%
MDM replacement of FM but they had greater feed intake and better FCR.

109

FCR is an important economy indicator in feed production industry. FCR is a marker

of how efficiently an animal utilizes feed, therefore minimizing feed wastage. Low FCR is
usually desired in feed production. In general, the FCR observed in our study with HSB was
lower, ranging from 0.97 to 1.01, than reported by other researchers that evaluated alternative
dietary protein sources to fish meal for bass species. Muzinic et al. (2006) reported and FCR
range of 1.70-1.78 when turkey meal was used at various replacement levels in diets for
Sunshine bass. Rawles et al. (2006) reported a FCR range of 1.58-1.72 when PBM was used
to replaced FM in diet for HSB. However, Webster et al. (1999) reported much higher FCR
for sunshine bass, ranging 2.06 to 2.92, when fed diets that replaced FM with protein from
both soybeans meal, PBM and MBM. Similarly, Pine et al. (2008) reported FCR of 2.312.55 for Sunshine bass fed diets containing different levels of PBM in place of FM. The
differences in FCR observed among these studies are likely due to differences in genetic
stock, water conditions, stocking density, feeding rate, and size of fish (stage of growth),

PER is an indicator of quality of protein content of feed. It shows how well the amino
acids profile of feed protein content matches the requirements of the fish. High PER is
usually desired. PER determined in our study ranged between 0.89-1.32, which is within the
range reported by other researchers working with HSB. Nematipour et al. (1992) reported a
PER ranging between 0.97 and 1.42 in a study that evaluated the effect of dietary energy and
protein ratio on growth characteristics and body composition of HSB. In contrast to the
studies sited above, a higher PER value of 1.80 was reported by Gallagher (1994) for HSB

110

fed a diet that replaced FM with soybean meal. Differences in PER values determined by
different studies are expected to be variable because they are composite values of FCR and
growth response to the quality of dietary protein. Therefore, it is necessary to compare PER
values among treatments within experiments than make any conclusions about treatment
response between experiments.

Growth of animals is a complex process influenced by genotype, environment,

nutrition and the hormonal status of the animal. Fish fed diets that replaced 50% and 75% of
the FM with MDM had similar or higher BW, SGR, and BL than fish fed the other
experimental diets, possibly because of differences in feed palatability, as measured by
feeding behavior response time and feed intake. Although the genetic potential for growth
may differ among fish, nutrition and hormonal factors are significant contributors to the
expression of that genetic potential for growth and efficiency of nutrient utilization. The main
hormone regulating growth is the adenohypophysial hormone, otherwise known as growth
hormone (Guyton and Hall, 2000). Thyroxine and insulin influence the anabolic effects of
the growth hormone and this activity is mediated by insulin-like growth factor-1 (IGF-1).
IGF-1 is secreted primarily from the liver in response to the elevated level of the growth
hormone released by the anterior pituitary gland into the blood stream; the secretion of IGF-1
can be retarded by under nutrition or insensitivity of growth hormone. The main function of
the IGF-1 is to mediate many of the anabolic biological actions of growth hormone,
including the effects on skeletal and muscle protein synthesis by causing the mitogenic

111

actions of growth hormone on bone, cartilage, and muscle and stimulating muscle-protein
synthesis. IGF-1 has a strong affinity for growth hormone plasma binding proteins, prolongs
the growth promoting effects of growth hormone (Hadley, 2000). Concentration of both
plasma IGF-1 and hepatic expression of IGF-1 are indicators of growth potential. Plasma
concentration and hepatic expression of IGF-1 is regulated by the quantity and nutritional
quality of dietary proteins (Phillips et al. 1978; Prewitt et al. 1982; Isley et al. 1983;
Takahashi et al, 1990; Miura et al, 1991; Davenport et al., 1995). In this study, the
replacement of FM with fermented MDM affected these growth indictors. There was a
positive relationship between the level of fermented MDM and the IGF-1 concentrations in
the plasma and the hepatic gene expression, with the maximum positive correlation observed
among fish consuming diets that replaced 75% of the FM protein with MDM. Dietary
treatment effects on IGF-1 concentration in the plasma and gene expression in the liver are
similar.
The ASI and HSI are often used as an indicator of condition and nutritional status of
fish (Rueda-Jasso et al., 2003). The replacement of FM with fermented MDM resulted in a
reduction in both ASI and HSI, which could have been influenced by the dietary fat content.
The reduction in the ASI and HSI was unexpected because MDM contain relatively more fat
than FM. However, Gaylord and Gatlin (2000) observed similar decrease in HSI when HSB
(Morone chrysops X M. saxatilis) were fed diets containing 20% lipids as compared to fish
fed diets containing 5% or 10% lipids. In contrast, Lee et al. (2002) reported significantly

112

higher HSI for Sebastes schlegeli fed 14% dietary lipid as compared to those fed 7% lipid
level. Steffens (1994) found increasing body lipid and HIS as the level of dietary inclusion of
PBM increased.

Similarly, Zoccarato et al., (1996) observed increasing HSI and

viscerosomatic index in rainbow trout as dietary inclusion level of PBM increased. The HSI
observed in our study increased as the dietary fermented MDM level increased. HSI was
within the range of 1.46-1.69, which is lower than the 2.05-2.43 range reported by Rawles et
al. (2006) after substituting FM with PBM in commercial diet of HSB with average initial
body of 87g. The range of ASI (2.97-3.42) observed in our study is lower than the ASI value
of 6.1 reported by Webb and Gatlin (2003), the 5.2 value reported by DAbramo et al.
(2000), and the 9.5 value reported by Pine et al., (2008). High HSI value, increases the
probability of fish developing fatty liver problems (Gaylord and Gatlin 2000; Lee et al.,
2002; Rueda-Jasso et al., 2003). Carcass proximate analysis of HSB in this study revealed no
significant treatment effects on percentage dry matter, crude protein, crude fat, ash and gross
energy content of whole body after the 77-d experimental period. Previous studies had
reported that replacement of FM with alternative proteins does affect the proximate
composition of HSB (Webster et al., 1999; Muzinic et al., 2006; Rawles et al., 2006; Pine at
al., 2008)

The results of our study confirms the results of other researchers that HSB can be fed
diets that replace part or all of the FM with alternative protein sources, such as fermented
MDM. However, the reduced performance observed among HSB fed diets that replaced

113

100% of the FM with fermented MDM may compromise organoleptic palatability, thus
adversely affecting stimulation to feeding behavior and feed intake. Complete replacement of
FM with fermented MDM may be possible after supplementing the diet with chemoattraction
substances, such as fish oil.

114

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CHAPTER 4

SUMMARY AND CONCLUSION

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4.1 RESEARCH SUMMARY

It has been widely documented that fishery play a vital role in improving food
security status of the developing countries. It is a vital source of protein, essential fatty acids,
vitamins and minerals for people in low income and food-deficient countries. An estimated
520 million people, nearly 8% of the world population, derive their sustenance from fisheries
and fish-related economic activities. Furthermore, with the continued increase in the
awareness of health benefits, the global demand for aquatic foods, even in the developed
countries, is expected to continue to rise. The world's population is expected to grow by more
than 30 % by 2050, resulting in an estimated 2.3 billion more mouths to feed, with the major
growth expected in the developing countries where fish is the main source of food protein
(UN, 2010).
Aquaculture is recognized as the only way to meet the increasing demands for aquatic
foods (Allan, 2004). Currently aquaculture is responsible for about 50% of the global fishery
production, and this figure is expected to rise as the demand for aquatic products increases.
Fishmeal is traditionally the favored protein source in production of aquaculture feed because
of its superior nutritional value. Moreover, the aquaculture feed industry competes in the
marketplace with other sectors of the animal feed industry for fishmeal use. However, the
continue decline in the fishmeal production relative to increasing demand is forcing the
market price of fishmeal upwards, thus threatening the profitability and economic
sustainability of the aquaculture industry. In particular, small holders and rural farmers in the

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low income food deficient countries are susceptible to this economic situation and the
resultant effect may further aggravate the level of food insecurity in such countries. The way
to sustain the aquaculture industry into the future is to limit or eliminate the use of fishmeal
in commercial aquaculture feeds.
Complete or partial substitution of fishmeal in fish feeds with alternative proteins has
been demonstrated in past studies without affecting the growth performance of several
species of fish. Dietary substitution of fishmeal with alternative protein ingredient sources is
evidently more feasible for omnivorous species than carnivorous species because they
require diets with lower protein content and can overcome digestibility constraints of proteins
from vegetable and animal sources. The general hypothesis of this thesis is as follows: the
replacement of fishmeal with alternative protein sources (particularly poultry processing byproducts) in aquaculture feed is biologically and economically feasible for the commercial
aquaculture production of both carnivorous and omnivorous fish species. In chapter 2, we
hypothesized that protein from yeast extract (Nupro, Alltech Inc), poultry mechanically
deboned meat and poultry by-product meal can be used to replace fishmeal in Nile tilapia
(Oreochromis niloticus) diets. Nile tilapia was specifically chosen as model in the
experiment because it is an omnivorous species and has biological advantages that enable it
to grow well in harsh environmental conditions that are typically encountered in low-income,
food-deficient countries and intensive commercial production operations. No significant
dietary treatment effects were observed on specific growth rate of tilapia, but the average

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weight of the fish fed diets containing fishmeal was significantly greater at the end of the 106
day trial than fish fed diets containing poultry by-product meal, fermented mechanically
deboned poultry meat (MDM), and yeast extract (P< 0.05). Apparently due to feed form
differences, replacement of FM with MDM significantly increased FCR by 12.5% (p<0.05)
due to increased feed pellet size and durability, but the other protein sources did not
adversely affect FCR in comparison to the FM control diet. In contrast, poultry by-product
meal resulted in significantly lower PER than fishmeal (p<0.05), whereas the other protein
sources, MDM and yeast extract, were not different. Finally, the replacement of fishmeal
with these alternatives did not affect the body carcass composition and plasma IGF-1
concentration (p>0.05). Although the dietary inclusion of protein sources tested as
alternatives to fishmeal resulted in slightly lower market weights, they resulted in greater
economic margin because of markedly lower costs. Moreover, MDM may offer additional
benefits as it has a PER similar to FM, but also may improve feed pellet binding
characteristics and pellet durability in aqueous environments.
Because fermented MDM was found to be a potentially cost effective alternative to
FM with high protein quality and functional properties in Tilapia feed, a follow up study was
conducted to determine the optimum level of dietary replacement of FM with MDM in
Hybrid Striped Bass (Morone saxatilis X M. chrysops), a carnivorous fish that requires
higher dietary inclusion levels of FM. HSB is a relatively more expensive fish to buy than
Nile tilapia and more preferred for consumption in the developed countries. HSB is used in

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this study as a model because fishmeal constitutes about 33% of its diet which is typical of
the carnivorous species (Gatlin, 1997). In chapter 3, we hypothesized that fermented
mechanically deboned meat can be used as an alternative protein source in replacing fishmeal
in Hybrid Striped Bass. Fermented MDM was tested at four dietary inclusion levels: 25%,
50%, 75%, and 100% at the expense of FM. All diets met the minimum requirement for all
essential nutrients to satisfy the needs of Hybrid striped bass. There was no significant
difference in the feeding behavior responses and growth performance of hybrid striped bass
when they were fed diets that replaced up to 75% of the dietary fishmeal with fermented
poultry MDM. However, there was a difference in the feeding behavior response, feed
intake, body weight, body length, specific growth rate, plasma IGF-1 concentration and
hepatic IGF-1 gene expression compared to fishmeal when the level of the replacement was
beyond 75% (p<0.05). We concluded that fermented mechanical deboned meat can be used
up to 75% to replace fishmeal in pellet hybrid striped bass diet.

4.2 CONCLUSION
The results obtained from these experiments demonstrated that yeast extract,
fermented mechanically deboned meat and poultry by-product meal proteins are feasible
alternatives to fishmeal in aquaculture diets. Fermented poultry mechanically deboned meat
is considerably cheaper than fishmeal. The lactic acid fermentation improves the
mechanically deboned meat stability and the organoleptic property. The feed conversion of

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the poultry MDM can be improved by adjusting the pellet processing condition to take
advantage of the MDM binding characteristic of MDM to produce more durable but smaller
pellets.

4.3. RECOMMENDATIONS

The search for fishmeal alternatives in aquaculture feed has been a subject of
extensive research in the recent years, from the use of animal rendering by-products and
plant protein to novel proteins such as yeast extract, algae, insect and bacteria as partial or
complete fishmeal replacers. The commercialization of the use of fermented mechanically
deboned poultry meat as an alternative to fishmeal in fish diets will improve the profitability
of the aquaculture industry. And because an extruded feed is more costly than pellet feed, an
investment in the production of pellet fermented mechanically deboned feed instead of the
traditional extruded aquaculture feed will definitely add to the profitability.

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