i
EFFECTS OF SUPPLIMENTATION OF FISH MEAL WITH
SOYBEAN MEAL ON THE GROWTH AND GONAD
DEVELOPMENT OF
THE AFRICAN CATFISH (CLARIAS GARIAPINUS)
M.SC. RESEARCH RESULT
BY
UGWOKE C. C.
PG MSC 07/43383
PRESENTED TO THE DEPARMENT OF ANIMAL SCIENCE
UNIVERSITY OF NIGERIA NSUKKA IN PARTIALFULFILMENT FOR
THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN
ANIMAL SCIENCE
SUPERVISOR: PROF. DR. S. O. C. UGWU
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TITTLE PAGE
EFFECTS OF SUPPLEMENTATION OF FISH MEAL WITH
SOYBEAN MEAL ON THE GROWTH AND GONAD DEVELOPMENT OF
THE AFRICAN CATFISH (CLARIAS GARIAPINUS)
By
Ugwoke Cyprian Chukwuma
PG M Sc 07/43383
MARCH 2013
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CERTIFICATION
We certify that the work was done by Ugwoke Cyprian C (PG/M
SC/07/43383) and approved by the Department of Animal Science, University
of Nigeria Nsukka
…………………………. ……………………………..
Supervisor Head of Department
Prof. Dr. S. O. C. Ugwu Dr. A. E. Onyimonyi
………………………………………..
External Supervisor
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DEDICATION
This work is dedicated to God Almighty under whose care and protection I was able to
start and complete the project despite some logistic and other resources problems I
encountered during the course of the work.
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AKNOWLEDGEMENT
My profound gratitude goes to my project supervisor Prof. Dr. S.O.C. Ugwu who gave me the
motivation and encouragement that made me to persevere until the end; My special thanks also
go to Prof. Dr. J. E. Eyo, the Head, Department of Zoology University of Nigeria Nsukka who
designed this work, guided me in the management of the experimental fish and provided some
important equipment and materials I used for the work. I cannot forget his advice that made the
work easy and safe. To him I say thank you for the pain he took in reading through my write up
at different stages and making necessary corrections. He gave me fatherly guidance that made
me to be able to complete this work with less stress.
I use this opportunity to thank the attendants of the Department of Zoology University of Nigeria
Nsukka for their help and support through out the period of the work.
I can not conclude here if I do not thank in a special way Dr. S. N. Machebe for his contribution
and guidance during the course of this work. Despite his tight schedule he took time to read
through the script and made necessary corrections and gave me some pieces of advice.
I also want to use this opportunity to thank various authors and researchers whose works I
consulted during the course of this research. Finally I will like to express my gratitude to the
technical staff of Histopathology Laboratory Faculty of Veterinary Medicine University of
Nigeria Nsukka especially Dr. E. O. Onuoha
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LIST OF FIGURES
Figure 1 Transverse section of gonads from female fish fed the different diet at week 14. 39
Figure 2 Transverse section of gonads of female catfish fed the different diets at week 16 41
Figure 3 Transverse section of gonads of female catfish fed the different diets at week 18 42
Figure 4 Transverse section of gonads of female catfish fed the different diets at 20 weeks 43
Figure 5 Transverse section of gonads of female catfish fed the 4 different diets at 22 weeks 44
Figure 6 Transverse section of gonads of male catfish fed the different diets at 20 weeks 45
Figure 7 Transverse section of the testis of male catfish fed the different diets at week 18 46
Figure 8 Transverse section of male gonad of catfish fed the different diets at week 16 47
Figure 9 Transverse section of gonads from female fish fed the different diets at week 14 48
Figure10 The Internal structures of a Female Catfish showing the Reproductive Organ (the two
ovaries with developing eggs and the tubular tract down to the opening at the anal
region) 68
Figure 11 Reproductive Organ of a Male Catfish showing: TS- a pair of serrated Testes, EP - the
Epididymides, VD - Vasa Deferetia and GP - the genital papilla (external Copulatory
Apparatus)
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LIST OF TABLE
Table 1: Diet Formulation with different levels of soybean; All values are in kg per 100kg diet 26
Table 2: Main Effect of Treatment on the Body Weight Gain of the African catfish 31
Table 3: Main Effect of Treatment on the Body and Gonad Weights of the African Catfish 34
Table 4: Main effects of Sex (Block) on the body and gonadal weight of the fish 43
Table 5: Correlation coefficient between age, body weight and gonadal weight of the
male African Catfish 50
Table 6: Correlation coefficients between age, body weight and gonadal weight of the
Female African Catfish 51
Table 7: Effect of Interaction between Treatment and sex (Block) on Body and Gonadal
Weight of African catfish 52
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TABLE OF CONTENT Title page i
Certification ii
Acknowledgement iii List of figures iv List of Tables v Table of content vi ABSTACT vii Chapter 1: Introduction 1 1.0. Background of study 1 1.1. Statement of the Problem 2 1.2. Objectives of the study 4 1.3. Justification 5 Chapter 2: Literature Review 6 2.1. Protein Quality and Fish Reproduction 6 2.2. Patterns of Gonad Development in Fishes 10 2.3. Nutrition and Gonadal Development in Fish 11
2.4. Catfish Nutrition and Nutrient Requirements 12
2.4.1. Energy requirement of catfish 12
2.4.2. Proteins and Amino Acid Requirements of Catfish 15
2.4.3. Vitamins and mineral requirements of catfish 16
2.5. Fish Meal as a component of Fish Diet 16
2.6. Combined Effects of Fish Meal with other Protein Sources in fish Diet 18
2.7. Soybean Meal as Source of Protein for Fish Diet 22
Chapter 3 Materials and Methods
3.1. Location and Duration the work study 25
3.2. Experimental fish 25
3.3. The Experimental Diets 26
3.4. Experimental Design 27
3.5. Data collection 28
3.5.1. Sampling of fish for Dissection 28
3.5.2. Histological Sectioning and Analysis 28
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3.5.2.1 Fixing and embedding of tissue samples (gonads) 28
3.5.2.2 Histological Sectioning 28 3.5.2.3 Staining 29
3.6 Statistical analysis 30
Chapter 4 Results and Discussion
4.1. Main Effect of Diet on Weight Gain and Final Body Weight of African
Catfish 31
4.2. Main Effect of Diets on the Gonad Weights and Development of
African Catfish 33
4.3. The Development pattern of the female gonad (the ovary) of the fish fed the different diets 38
4.4. Effects of Sex (Block) on the Body and Gonadal Weight of the African
Catfish 49
4.5. Correlation between the Age, body weight and gonad weight of the
Male African catfish 49
4.6. Effects of Interaction between Treatment and Sex (Block) on Body and
Gonadal Weights of African catfish 51
Conclusion 53
Recommendation 53
References 54
Appendix 68
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ABSTRACT
This study was conducted to evaluate the effects of replacement of fish meal with soybean meal on the
development of the gonads and weight gain of the African catfish Clarias gariepinus. Four groups of fish
were fed four different diets made up of four different combinations of fish meal and soybean meal for 22
weeks. There were no significant (P > 0.05) differences in the daily weight gain and the final body weight
of the four treatments. Treatments A (control) had mean final body weights of 100.68 and 85.43 grams
for males and females respectively. The mean final body weight values for the males of treatments B, C,
and D were 96.60, 76.30 and 72.14g respectively while the females had 81.84, 74,30 and72.14 for
treatments B, C and D respectively. The mean daily weight gain values followed the same trend. The
mean daily weight gain values for the males were 2.06, 1.93, 1.84 and 1.35 respectively for treatments
A, B, C and D. The females had mean daily weight gains of 1.68, 1.66, 1.56 and 1.26 respectively for
treatments A, B, C and D. The gonad weights were significantly (P < 0.05) affected by level of
substitution of fish meal with soybean meal. In the male, the highest gonad weight of 0.30 was recorded
for treatment A. In the males there were no significant differences between treatment A and treatment B
in their gonad weights but treatments A and B differed significantly (P < 0.05) from treatments C and D.
There were no significant (P > 0.05) differences between treatments C and D in their mean gonad
weights. In both sexes, the histological inspections of the gonads revealed that the gonads of the fish fed
diets A and B were better developed than those in treatments C and D. Fish fed diets A and B had
significantly heavier and more developed gonads compared to those on diets C and D. In females, fish
fed higher levels of soybean (diets C and D), had fewer numbers of oocytes with some atretic oocytes
compared to the other two groups. The mean gonad weights of 0.30mg and 0.53mg for males and females
respectively obtained in the fish fed diet A did not differ significantly (P > 0.05) from 0.22 values
obtained from the fish treatment. Histological inspection of the gonads further revealed that in all the
treatments the female gonads developed earlier than the male gonads. It was concluded that 20%soybean
is the best level of substitution of fishmeal in diet of catfish used as brood stock. At this level both growth
performance and gonad development are not affected. Higher levels up to 30% and above of soybean
substitution may not affect the growth performance but will significantly reduce gonad development
thereby lowering the reproductive potential of catfish.
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CHAPTER 1
INTRODUCTION
BACKGROUND OF THE STUDY
Pond culture practice in most African countries was introduced after the Second World War (de
Graaf and Janssen, 1996). By this time only tilapia species were reared. This had a very poor
performance record which initiated action to find out more suitable species for better
performance. Catfish was identified as a more suitable species for rearing in tropical Africa.
Catfish is recorded to have very rapid growth, strong resistance to stress and handling and
feasible reproduction in captivity, though by artificial methods (de Graaf and Janseen, 1996;
Ceks and Yilmaz, 2007).
Intensive methods of culture are faced with numerous problems including environmental,
technical and economic. Intensive rearing of any animal can not be successful without good
reproductive performance in captivity. Considerable efforts have been made to improve the
productivity and reproductive performance of the catfish in captivity. Nevertheless, some
degrees of success have been achieved but one of the major problems which have persisted till
date is the problem of feeds and feeding in terms of cost and availability of the feed raw
materials. Animal feed materials are the major components in fish feed manufacture particularly
fishmeal. This is as a result of the high protein and amino acid requirement of fish.
During the last few years the price of fishmeal has more than doubled, thereby initiating research
interests for alternatives to fishmeal (Madsen, 2008). Such research efforts may not be aimed at
dropping fishmeal completely but includes developing supplemental alternatives that will reduce
level of addition in feeds. Fish meal is preferably used in many animal and aqua culture diets
because of the good amino acid profile in the protein which has rarely any close substitute in
plant sources. For any diet to be regarded as complete diet it must contain some protein. It is not
just enough to contain protein but the nutritional value of the protein must be high in terms of its
amino acid composition and digestibility. Amino acids are the building blocks of proteins, and
are released for absorption into the blood stream following protein digestion. Individual animals
have different requirements for specific amino acids. In other words, a food substance may have
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high level of protein but may not be suitable for the animal in question because of inadequate
amounts and availability of some amino acids in their protein. Food protein sources are simply
vehicles for providing amino acids in the diet. Animals need about 22 different amino acids to
build proteins. Some of these amino acids can be synthesized in the body of the animal while
some cannot be synthesized by the animal. Those amino acids that cannot be synthesized in the
animal body and therefore must be supplied in the diet are called essential amino acids (Li et al.,
2008). Ten essential amino acids must be contained in the diet of fish and they include arginine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and
valine (Li et al., 2008).
Nutritionists refer to food materials like fish meal that supplies a sufficient amount of the ten
essential amino acids as complete protein (http://www.new-fitness.com/nutrition/protein.html)
Virtually all proteins from animal foods are complete. While foods that lack or are short in one
or more of the essential amino acids - such as some fruits, grains, and vegetables - are called
incomplete proteins (http://www.new-fitness.com/nutrition/protein.html).
1.2 Statement of the problem
The high price of fishmeal makes it unaffordable by fish farmers. Also, fishmeal is usually very
scarce especially in the Sub-Saharan Africa. FAO (2003) predicted that the annual consumption
of fish per person in Africa, where the consumption is already low may go down further by about
3 percent by 2030. According to Green Facts (2012) per capita fish consumption figures for
Africa in 2005 was 8.3kg compared to 26.1kg, 24.5kg, 24.1kg and 20.8kg for China, Oceania,
North America and Europe respectively This will increase the competition for fishmeal by man
and animals making the price to rise further beyond the reach of the average fish farmer in this
region. The proportionate contents of the essential amino acids like lysine, methionine,
threonine and tryptophan are relatively high in most fishmeal samples when compared with plant
protein sources.
Fishmeal has been the major source of proteins in fish diets especially in the diets of carnivorous
fishes like catfish (Fontainhas-Fernandes et al., 2000). In most developing countries fish meal is
scarce and as a result of the high price of the product it may not be economical to use fishmeal
alone in fish feed especially with the high level of inclusion in the diet. This has made
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researchers to lay emphasis on substituting fishmeal with alternative cheaper and more readily
available plant proteins (Cumaranatunga and Thabrew, 1989; Fontainhas-Fernandes et al., 2000).
Studies to investigate the potentials of some plant nutrient sources for the fish diet have focused
on such criteria as digestibility, feed conversion, growth performance, energy supply,
bioavailability of nutrients, presence and availability of essential nutrients and the presence and
effects of anti-nutritional factors (Appler and Jauncey, 1983; Tacon et al., 1983; Viola and
Arieli, 1983; Appler, 1985; Wee and Wang, 1987). However, information on the effect of plant
materials on the reproductive performance of fish, particularly catfish is quite sparse. There is
need then to study the effect of the different levels of substitution of fishmeal with plant nutrient
sources on productivity in terms of growth and reproductive performance of particularly the
African catfish. Nutrition has been shown to affect gonad development, fish fecundity, sperm
and egg quality in rainbow trout Oncorhynchus mykiss (Bromage et al., 1995)
African cat fish is one of the most popular species reared in the Sub-Saharan Africa and aqua-
cultural production of this species depends on improved seed production. According to reports,
seed farmers are confronted with poor spawning response and poor quality spawn resulting from
a variety of factors which include nutrition and weather conditions (Clay 1979; Van den Hurk et
al, 1984; Richter et al, 1987; Bromage et al., 1995).
The poor nutritional condition is as a result of the high cost of feed materials especially fishmeal
making it unaffordable to farmers. This usually leads to poor feeding regime which results when
producers compare the cost of feed with the return in terms of growth and weight gain. Most
commercial feed manufacturers tend to reduce cost by going for the cheap materials without
considering the possible effects on the fish stock. There is fear then that apart from affecting the
growth performance such cheap feed may have other adverse effects on the fish stock especially
in the reproductive life of the fish.
Very few studies have been conducted to examine the effect of different levels of crude protein
on the reproductive performance and fry production of some fish species (Satiago et al., 1983;
Cisse, 1988; De Silva and Radampola, 1989; Washburn et al., 1990).
Specifically, little or no studies have been published on the effects of plant protein sources on the
development of the gonads of the African catfish (Clarias gariepinus).
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This study was designed to assess the effects of different levels of replacement of fish meal with
Soya bean meal in the diet of catfish on their body growth and gonad development. The major
goal is to identify the best replacement level suitable for the development of reproductive
potential of the catfish under tropical climate. This study intends to compare fish meal which has
many advantages with other products in reproductive functions.
1.3 Objectives of the Study
The main objective of this study is to investigate the capacity of the African catfish in this
region in their capacity to grow and reproduce successfully on diets containing fish meal
and an alternative protein source (soya bean meal)
The specific objectives of this study are:
1) To determine the effects of different levels of replacement of fish meal with
soybean meal on the development of the gonads of the African catfish (Clarias
gariepinus)
2) To compare the rate of growth of the gonads of the male and the female African
catfish under the different treatments.
3) To determine the extent of body growth and the relationship between body
weight, gain in body weight and the gonad weight (rate of development) in the
African catfish fed diets containing different amounts of soybean meal.
1.3 Justification
Generally, fish diets contain high proportion of fish meal as the major source of protein.
The proportion may range from 30 – 50%. However, due to the reduction in the global
cached fish in recent times amounting to scarcity and high cost of fish meal, coupled with
the high rate of consumption of fish by man, fish feed manufacturers are trying to reduce
the level of inclusion or even avoid fishmeal in fish feed compounding. The pressure is
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more in the sub-Saharan Africa where fish consumption mainly depends on imports.
Finding a suitable cheap protein material for the purpose of reducing dependence on fish
meal is of top priority in the development of aquaculture in the sub-Saharan Africa. Even
though research efforts are focused on the use of other protein sources in fish feed the
attention of most of the studies are directed to the effects of these protein sources on feed
conversion, growth rate and final body weight. Fish farming and generally aquaculture
can be more beneficial if high growth performance in terms of weight gain and feed
conversion are combined with high reproductive performance in captivity. Plant protein
sources are generally cheaper and more common but the fear of possible adverse effects
on the reproductive potential of the stock make them not to be used in large proportions
in feeds especially in diets meant for brood stock. Soybean meal has been graded as the
best plant protein both for man and other animals. But some reports have shown that it
may have effects on the reproductive performance of many species of animal including
man (West et al., 2005; Zhao and Mu 2010). However, determination of which level of
soybean meal will be better for gonad development and high reproductive potential of the
African catfish will help to educate fish farmers, aquaculturists and feed manufacturers
on the value of the feedstuff as a major protein source for the fish considering its low
price and ready availability when compared with fish meal.
The total or partial replacement of fish meal by soya bean meal can reduce the production
cost, increase sustainability of catfish production and lower the competition on the use of
fish for food.
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CHAPTER 2
Literature Review
2.1. Protein Quality and Fish Reproduction
The sustainability of aquaculture in any place depends absolutely on the availability and stability
of supply of good quality and affordable feedstuffs. Cho and Slinger (1979) identified feed costs
as accounting for more than half of operating costs of aquaculture operations. Fishmeal (FM) and
soybean meal (SBM) have been the main components of manufactured fish feeds as the major
protein sources. Though soybean meal has been rated as a good protein source for most animals
including man some studies have shown that the use of soybean meal especially in large
quantities has some problems associated with digestibility, availability of some micro nutrients
mostly as a result of the presence of some anti-nutritional factors (depending on the method of
processing) and the possible effects of phytohormones which are present in soybean.
The profitability in the production of any aquaculture species can only be achieved when its
qualitative and quantitative feed requirements are known making the formulation of nutritionally
balanced low-cost diets possible (Tacon et al., 1983b; Hashim et al., 1992; James et al., 1993).
The quantity and quality of protein in the diet have been shown to affect the reproductive
performance of different species of fish, (Shim et al., 1989). At least in the female fish, adequate
level of protein is necessary for the formation of egg, embryo development and reproduction.
Protein is also needed for the formation of follicles and other ovarian tissues. The absence of
adequate levels and quality of protein in the diet will result in abnormal or incomplete
development of the reproductive organ.
Shim et al., (1989) reported that the quantity of the dietary protein in the fish diet has profound
effect on the reproductive performance of fish. In the female fish adequate quantity of dietary
proteins is required for egg/embryo development and general reproductive performance. Quality
of protein is important for the formation of follicles and the growth of the ovarian tissues and the
production of important reproductive hormones in all animal species. Inadequate levels of
quality protein can affect the survival of the offspring (James and Sampath, 2003).
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The source of protein has been identified to affect reproductive performance in some species of
fish studied (Pereira et al, 1998; Fontainhas-Fernandes, et al. 2000)
Hash environmental conditions like excessive high temperatures, poor nutrient status and the
immediate water condition or exposure to some noxious substances caused germ cell loss in
some species of fish and exposure to such conditions from hatching until juvenile stage resulted
in failure of the germ cells to develop (Strussmann et al., 1996). Similar reports on the effects of
environmental factors on the reproduction of African catfish (Clarias gariepinus) were given by
Clay (1979); Schulz, et al. (1994) and Ofor (2005); for Asian catfish by Utiah (2002); for
Oreochromis niloticus by Cumaranatunga and Thabrew (1989) and Fontainhas-Fernandes et al.
(2000). Effects of different levels of protein and protein sources were reported by Pereira et al
(1998). The authors indicated that rainbow trout fed only plant materials as the only source of
protein showed less efficiency in reproductive indices than those fed on diets based on fishmeal.
This report was in agreement with the work of Fontainhas-Fernandes et al. (2000) who showed
that in the Nile Tilapia diets containing only plant protein were less efficient in terms of growth
and ovarian development. Fish meal has been the main source of proteins in fish diets
(Fontainhas-Fernandes et al. 2000). However, due to the high cost and scarcity of fish meal
particularly in developing countries, much emphasis has been placed on researches aimed at
substituting fish meal with alternative more readily available plant sources of protein
(Cumaranatunga and Thabrew 1989; Fontainhas-Fernandes et al. 2000). The potential benefits of
soybean meal as a good source of protein in the diets of many animals are undeniable but some
problems still need to be overcome before it can be fully utilized in aquaculture industries
especially when high levels of incorporation are to be used.
Brown (2005) reported that trout and salmon, showed reduced growth responses when soybean
meal was incorporated at 15-25% in the diet, even when the diets were formulated to meet
essential amino acid requirements. They did not test for the effects of soya bean meal on the
reproductive performance of the fish.
Studies to investigate the potentials of some plant nutrient sources for use in fish diet have
focused on such criteria as digestibility, feed conversion, growth performance, energy supply,
bioavailability of some essential nutrients and the presence and effects of anti-nutritional factors
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on some body parts (Appler and Jauncey , 1983; Tacon et al. 1983; Viola and Arieli, 1983; De
Silva et alal. 1983; Appler, 1985; Wee and Wang 1987; Francis et al. 2001). However,
information on the effect of these materials on the reproductive performance of fish, particularly
African catfish, is quite sparse. There is need then to study the effect of the different levels of
substitution of fish meal with plant nutrient sources on productivity in terms of reproductive
performance of particularly the African catfish. Very few studies have been conducted to
examine the effects of different levels of crude protein on the reproductive performance and fry
production of some fish species (Satiago et al.1983; Cisse, 1988; De Silva and Radampola, 1989
and Washburn et al. 1990)
Most studies on gonad development of catfish focused on the seasonal growth of mature gonads
of either the male or the female. Reports are very scanty on the development of the fish gonads
from the post hatching stage up to maturity. As such, reports are rare on the actual effects of the
plant protein sources (which many producers have been using to substitute animal protein –
fishmeal – in fish diet) on the growth and initial development of the gonad of fish especially the
African catfish.
Reports showed that oocyte growth in the fish is elicited during the vitellogenic stage of
development as a result of the sequestration of protein from the plasma (Norberg and Haux,
1985; Tyler et al., 1991; Fontainhas-Fernandes et al., 2000). These proteins are mainly
vitellogenin (VTG) – a high molecular weight glycolipophosphoprotein. Vitellogenin (VTG) is
produced by the liver under the influence of estrogen 17β-estradiol (E2). The source of amino
acids for the synthesis of these proteins is the diet. So it is necessary to study the effect of the
protein source on the development of the sex structures as the source of amino acid for the sex
tissue building.
Fishmeal has been noted for having the amino acid level that is best suited for the growth and
development of the fish body tissues. There is no other source of dietary protein known to have
the same amount and types of amino acids suitable for the fish as the fishmeal.
The extent to which fishmeal can be replaced in the fish diet by another feed stuff without
producing an appreciable negative effect depends on the availability of the necessary amino
acids in the particular stuff and the absence of anti-nutritional factors. The availability of these
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nutrients will determine the normal functioning of the body organs of the fish. The extent of
replacement of fishmeal by this plant protein sources can partly be investigated by looking at the
effect of the product on the development of the reproductive organ of the fish and the effects on
the reproductive performance of the mature fish when such material is used in the fish diet.
As stated above, vitellogenin is produced by the liver under the influence of circulating estrogen,
particularly 17β-estradiol (E2) (Fontainhas-Fernandes et al., 2000). Estradiol (E2) is synthesized
by the theca and granulosa cells which form the outer layer of the oocytes, (Wallace, 1985;
Yoshikuni and Nagahama, 1991; Kwom et al., 1993). Increasing levels of E2 stimulates gene
transcription and translation by the liver cells, (Specker and Sullivan, 1994). Pellissero et al.,
(1991) showed a strong relationship between diet and plasma levels of sex steroids which have
been known to determine the amount of VTG in the plasma. Fernandez-Palacios et al. (1996)
took it further in their study on the nutritional quality and viability of fish eggs. They reported a
strong relationship between viability of fish egg and the nutritional quality (nutrient sources and
amount). Work on Siberian sturgeon (Acipenser baeri) by Pellisero et al. (1991), showed that
diet influenced plasma sex steroids and consequently caused an increase in plasma vitellogenin
(VTG) levels. Sex steroids play important roles in the differentiation of gonads in all vertebrate
animals.
Besides, Phytoestrogens have been found to be present in plants like Gramineae and
Leguminosae family. The phtoestrogens that have been identified include isoflavonoids and
coumestans. These classes of compounds are known to structurally or functionally mimic
circulating estrogen in the vertebrate reproductive system. They function to induce or antagonize
estrogenic activities in the brain-pituitary-gonad axis. This is the principal endocrine system
involved in reproductive regulation in vertebrates and peripheral reproductive organs.
Total or partial replacement of fishmeal by soybean meal decreased the level of plasma
cholesterol in female salmonids while VTG levels were not affected (Bromage et al., 1992;
Robin and Kaushik, 1994; Kaushik et al., 1995). Though, the plasma cholesterol decreased with
increasing levels of replacement of fishmeal by soybean meal, the report of Fontainhas-
Fernandes et al., (2000) showed that the gonadosomatic index (GSI) and hence the VTG levels
in the plasma decreased with decreasing levels of replacement of fishmeal by soybean meal in
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the Nile Tilapia. Soy bean has also been known to contain isoflavones (phytoestrogens). Some
isoflavones have been reported as capable of interfering with reproduction in many species of
vertebrates.
2.2. Patterns of Gonad Development in Fishes
Initial studies on the early gonadal sex differentiation in fish showed that in most species, sex
differentiation occurs much earlier in ovaries than in testes (Strussmann et al., 1996; Strussmann
and Nakamura, 2002). Early observations on the sex differentiation showed that ovarian
differentiation has greater abundance of germ cells in the putative ovaries as compared to the
putative testes that had lesser number of germ cells (Nakamura et al., 1998).
Other ways of determining the sex differentiation include observing the ovarian cavity which can
take one of several different paths of development. In one path, the somatic tissue of the gonad
starts growing and elongating from the point of the primordial germ cell to form the proximal
and distal sides of the gonads. These developing outgrowths extend back across the proximal
axis towards each other. They eventually fuse together to form the ovarian cavity. The second
pathway follows the formation of an aggregation of cells that develop on the peritoneal wall
adjacent to the elongated edge of the gonadal structure. These cells continue to grow and
multiply in this structural framework and eventually fuse with the elongated portion of the
gonadal structure to form an ovarian cavity between the peritoneal wall and the gonad. The third
pathway involves the fusion of the proximal and distal elongations with the coelomic wall. This
fusion leaves a cavity, the ovarian cavity, dorsal to the ovary (Michael, 2003). In salmonids such
as trout and salmon, an ovarian cavity develops in the anterior part of the ovary. This makes the
ovary to ovulate mature oocytes directly into the coelomic cavity which is passed out via the
genital pore at maturity unlike many other fish species, which pass oocytes into the gonoduct at
ovulation, which then carries the oocytes to the genital pore (Nakamura et al., 1998).
Testicular development takes much longer time than the ovary. So, it is much more difficult to
determine testicular differentiation by means of germ cells, because germ cells in testes may not
show signs of differentiation or may remain undeveloped for long periods (Nakamura et al.,
1998).
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The efferent duct in the fish develops in a pattern in which narrow spaces first appear on the
connective tissue stroma of the gonad. Aggregation of these connective tissue cells in the distal
region of the gonad is a good criterion for the onset of testicular differentiation, especially in
cichlid fishes (Nakamura et al., 1998; Michael, 2003).
Patiňo et al. (1996) reported earlier sex differentiation in females than in males of channel cat
fish. In this report, they noted small tissue outgrowths developing at the proximal and distal ends
of the gonads in normal females and putative sex reversed females at day 19 – 21 post-
fertilization. These outgrowths later fused to form an ovarian cavity. In males, development of
signs of testicular differentiation appeared by day 90 – 102 post-fertilization. This supports the
reports that in most farm animals, females are born with thousands of primary oocytes but in the
male spermatogenesis starts at pubertal age and continues until death.
Spermatogenesis goes on in the male from puberty almost until death. In females, oogenesis goes
on from before birth until menopause. Females are born with about 40,000 primary oocytes, so
they have already begun maturation
2.3. Nutrition and Gonadal Development in Fish
It has been indicated that there is strong interaction between nutrition, growth and reproductive
performance in fish (Gunasekera et al. 1996; Mokoginata et al. 1998; Cek and Yilmaz 2007).
Information on the interaction between nutrition and reproduction has focused mainly on
salmonids and marine fishes (Washburn et al. 1990; Bromage, et al. 1992). Information is scanty
on tropical fishes like the African catfish Clarias gariepinus.
The sources and level of protein in the diet are known to regulate the growth and consequently
affect the ovarian development in tilapia (Fonthainhas-Fernandes et al. 2000). James and
Sampath (2003) showed that animal protein based diets induced gonad development earlier than
plant protein sources. In their experiment they noted that Female B. splendens fed 35% animal
protein based diet had higher gonad weight than those fed diet with the same level of plant
protein at the same age. Also the ovary of the fish fed with animal protein diets produced higher
number of eggs with higher hatchability than the ones fed plant protein based diets.
We have not found such reports on the African catfish (Clarias gariepinus)
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2.4. Catfish Nutrition and Nutrient Requirements
Diets are meant to supply all the essential nutrients and the energy required by the animal for
carrying out all the vital physiological functions such as growth, reproduction and the
maintenance of normal health condition. Different species require different nutrients at different
levels to carry out these functions effectively. Besides, in aquaculture as in other animal
production systems, a major issue is that of ensuring quality of the end product (flesh) and stock
replacement (reproductive performance), both of which can be affected by nutrition.
Although the precise nutritional requirements in terms of amounts and types for gonadal
development and maturation in catfish and many other species of fish have not been established,
there has been some consensus that the nutritional requirements of brood-stock during gonadal
development differs from those of young fish.
Fish size, metabolic function, management, and environmental factors have slight to profound
effects on dietary nutrient levels for optimum performance.
It has been reported that levels of protein has direct influence on the size of the gonads in the
fish. Many reports have shown that increased level of protein has resulted in increased weight
and size of the ovary in several species of fish, (Dahlgren, 1980; Pathmasothy, 1985 and Shim, et
al. 1989).
Catfish like any other animal has specific requirement for the different nutrients. They have
requirements for energy both from carbohydrate and lipid sources; proteins (different amino
acids) for body building and repairs; vitamins and minerals for other metabolic processes of life.
2.4. 1 Energy requirement of catfish
Energy is one of the most important parts of the diet of the catfish like all other species of
animal. Feeding standards of animals are based on the energy needs, MSU (2006). In their view,
the authors of the paper stated that the feed intake for catfish is more a function of how much the
fish is allowed to take rather than the energy concentration in the feed. Though it may not be
wise to say that catfish feed is strictly regulated by the energy content, a good balance of energy
is necessary in the diet as part of the high quality protein in the diet may be converted to energy
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if there is not enough of the non-protein energy components in the diet. On the other hand, if the
diet has too much dietary energy, the fish may not be willing to take enough quantities of the diet
to obtain required amounts of the other essential nutrients. At the same time, excess energy in the
diet may result to deposition of excess fat which will reduce the value of the final product. The
energy sources for fish diets may be from carbohydrate but fishes prefer energy from lipid
sources and more preferably from animal sources. Though the absolute energy requirements for
catfish has not been established, some researchers have suggested using the method of measuring
weight gain or protein gain of catfish fed diets with known amount of energy to estimate the
actual energy requirement MSU (2006). Some other authors have expressed energy requirements
of catfish as a ratio of digestible energy (DE) to crude protein (P). MSU (2006) suggested that
DE/P ratio of 8.5 - 9.5 kcal/g is adequate for use in commercial catfish feeds.
ADCP, (1983) reported that best growth rates and food conversion ratios are achieved when cat
fish are fed diets containing 35-42% crude protein and a calculated digestible energy level of 12
kJ/g. The authors gave the energy levels for catfish Fry and fingerlings as 3.0-4.0 kcal/g, 2.5-3.5
kcal/g and 3.0-4.0 kcal/g for growers and brood stock respectively.
Studies show that energy in fish nutrition is not only important for supplying the energy
requirement for carrying out the live activities but that low energy in the ration means that
protein may not be fully utilized to the fullest potentials ( Lovell 1976, NRC1993 and Bakke-
MaKellep et al. 2007). Energy in fish feed are mostly got from lipid and carbohydrate sources.
A. Dietary Carbohydrate
Carbohydrates are forms of energy stored in plant seeds, roots and tubers. Starch is the major
form of carbohydrate in these materials. Animals can utilize this form of energy for their energy
needs. The amount of carbohydrate in the diet varies with fish species and the productive
function. Consideration should be given to the ability of the fish species to use carbohydrate as
an energy source, and the processing requirements.
Some nutritionists have argued that catfish does not require carbohydrates in its diet, yet a typical
catfish diet contains considerable amounts of carbohydrates in the form of starch from grains or
grain by-products to the level of about 25%.
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B. Dietary Lipids (fats and oils)
Lipids (also called fats or oils) are biochemical compounds generally soluble in organic solvents
and usually insoluble in water. They are essential sources of easily digestible high grade energy
for animals. The most important factor to be considered when selecting the type and amount of
lipids (fats) to be used in the diet is the essential fatty acid (EFA) and energy requirements of the
fish. A unit of lipid can provide twice the amount of energy which the same amount of
carbohydrate will provide. Lipids or fats serve as energy stores for the body. Fats (or lipids) are
important in animals for insulating body organs against shock, and for body temperature
regulation. Studies have shown that fats are important for promoting healthy cell function. Fats
are broken down in the body to release glycerol and free fatty acids. The glycerol can be
converted to glucose by the liver and thus used as a source of energy. The fatty acids are the
main source of energy in fish, especially for many tissues, such as heart and skeletal muscle.
Another important function of fats is that it is useful for vitamin absorption. Vitamins A, D, E,
and K are fat-soluble, meaning that they can only be digested, absorbed, and transported and in
conjunction with fats and excess of these vitamins are stored in fat deposits.
Inclusion of lipids in the diet of catfish is essential as this may increase feed intake, MSU,
(2006). The amount and type of lipids in the tissue, like in the muscle tissue, has effect on the
flavour of the flesh. The level of inclusions and the type of lipids used in the catfish diets should
be based on the essential fatty acid requirements of the fish, the final cost of finished feed, and
the quality of the fish product (flesh) desired as the final product of the aquaculture practice.
Fish feed formulators have used different levels of inclusion ranging from 1.00 – 5.00% of either
animal or vegetable oils for different types of fish at different stages. Davies and Ezenwa (2011)
used 2.5% palm oil in the diet of Clarias gariapinus fry. In their work with Clarias gariapinus
fingerlings, Anyanwu et al. (2011) used 1.00% inclusion of palm oil in the diet of catfish
fingerlings. Fish oil has been used at 5.0% level in fry food, and at 2.0% and 1.0% levels
respectively in fingerling and food fish diets
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2.4.2 Proteins and Amino Acid Requirements of Catfish
In fish diet, like in most animal diets, protein is usually the most critical nutrient considered and
usually forms the basis for formulation and as such given stricter attention. Protein has been
identified as the basic nutrient that cannot be compromised in the choice of ingredients for feed
formulation (Zeitler et al., 1984). The energy level in the diet is adjusted to give the optimum
ratio for optimum utilization. The nutritional value of the protein relates directly to its amino acid
composition and digestibility (Rama-Rao et al., 1983; Wikipedia, 2011)
The muscle tissue of the fish is made up of about 70% protein. A continual supply of good
quality protein in terms of the essential amino acid is necessary for the catfish. These nutrients
must be provided in the diet. Cat fish, like other animals, need certain levels of protein and the
right types and amounts of amino acids for body building and repairs. Fish can utilize protein for
the supply of energy but usually protein materials are very expensive for this purpose.
Several experiments have been conducted for many years to find out the actual protein levels
most suitable for the fish, but there is yet no consensus as to the best levels of protein for fish at
different stages of development and physiological conditions. Use of high levels of protein in the
diet may be cherished as the fish can use protein as source of energy with ease but usually
protein feed stuffs are very costly. The best level of protein for the highest economic gain may
vary according to the cost of the ingredients and the types and availability of such ingredients
used in the location of production.
The type of protein ingredient is most important as the amount and types of amino acids
available depend on the ingredient. Some ingredients though have high levels of nitrogen or
proteins but can not be utilized properly by the fish. This may be as a result of the presence of
some anti-nutritional factors in these plant materials or the absence of some necessary amino
acids (essential amino acids) which must be present for proper utilization of the others. It is a fact
that animal protein has more complete protein contents with better amino acids profile and it is
known to have a full complement of the essential amino acids than the plant protein. Major
protein sources used in fish feed are fish meal, meat and bone meal, blood meal, soybean meal,
cotton seed meal, ground nut meal, sunflower meal and poultry by-product.
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Most commercial feeds for growing fish contain 28 – 32% crude protein. Some fish growers
have used diets with lower protein levels but some scientists argue that such diets may increase
body fats.
2.4.3. Vitamins and mineral requirements
Vitamins are organic compounds required in small quantities by the animal for normal body
functions, health and reproduction.
The vitamin requirement for fish diet is mostly supplied from a supplemental vitamin/mineral
premix in a ready made form. This has to be included as supplement because of uncertainty over
content and bioavailability of some of the vitamins that may exist in the feedstuffs
Mineral contents of feedstuffs are more consistent, so mineral supplementation usually is made
on the basis of the composition of the major ingredients. Over-fortification of labile nutrients in
processed fish feeds is necessary as a safety factor. Amino acids, several vitamins, and inorganic
nutrients are relatively stable to heat, moisture, and oxidation that occur under normal processing
and storage conditions. Some of the vitamins are subject to some processing and storage losses.
These are compensated for by the use of amounts in excess quantities of the requirement.
Several studies have been conducted to verify the amount and quality of vitamins required by
catfish. Although there are many types of vitamins, some of them can be synthesized in the
animal body. Only those that can not be synthesized in the body should be supplied in the diet.
These are called essential vitamins. Some of the feed stuffs used in making fish feed contain
some of the essential vitamins, but the major source of vitamins in the diet of most domestic
animals is in the ready made form – vitamin premix.
2.5. Fish Meal as a component of Fish Diet
Fishmeal is the fish product/by-product known by animal nutritionists as a high quality and very
digestible feed ingredient usually added to the diet of most reared animals, especially mono-
gastric species and fish, as a major source of protein in the diet. Fishmeal is a generic term for a
nutrient-rich feed ingredient used primarily in diets for domestic animals, sometimes used as a
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high-quality organic fertilizer (Miles and Chapman 2009). They described fishmeal as a material
that carries large quantities of energy per unit weight, an excellent source of protein, lipids (oils),
minerals, and vitamins but has very little amount of carbohydrate. Therefore, fishmeal is a good
stuff for species like fish that rely on lipid sources for their energy needs especially when the diet
has the required protein level.
Fish meal has been widely used as the major protein source for fish in the aquaculture industry
since the inception of fish culture. This is because no other material has shown the same nutrient
combination suitable for fish like fishmeal.
The amino acid profile of fishmeal is what makes it attractive and important as a feed ingredient
and a protein supplement in the diets of farm animals, especially mono-gastric species. Plant
proteins i.e. proteins from plant materials (cereal grains, legumes and other plant concentrates)
do not contain complete amino acid profiles and usually are deficient in the essential amino acids
especially lysine and methionine. Soybean and other legumes widely used in the diets of most
farm animals such as pigs and chicken are good sources of lysine and tryptophan but are limiting
in the sulfur-containing amino acids, methionine and cysteine (Miles and Chapman 2009).
High quality whole fishmeal provides a balanced amount of all essential amino acids,
phospholipids, and fatty acids for optimum tissue growth, development, and reproductive
processes. This is more critical in larvae and brood stock. The nutrient profile in fishmeal, i.e.,
good amino acid profile and fatty acid combination (polyunsaturated fatty acids which have
obvious health benefits are known to be present in fish oil) help to maintain functional immune
system and increase disease resistance.
Fish meal is also used in the fish diet because of the acceptability and high digestibility
coefficient. The nutrients digestibility of fish meal is indicated to be between 75% and 90%. The
other good aspect of fish meal is that unlike the plant protein materials fish meal has no record of
anti-nutritional and toxic materials if handled properly.
For a diet to be regarded as complete it must contain reasonable amount of protein with good
amino acid combination (Wikipedia 2011 and Evonik Ind. 2012) coupled with the presence of
the right amount of other nutrients like carbohydrates and lipids for energy supply, vitamins and
minerals for body functioning.
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Fishmeal is used by feed manufacturers as a good protein supplement because of its amino acid
profile. High quality fishmeal contains between 60 – 72% crude proteins (Wikipedia, 2011).
Fish meal is used in the diets of many species of animals, especially mono-gastric animals and in
aquaculture diets because it is acceptable and agreeable to the taste (palatable) (Wikipedia 2011).
This quality induces appetite and makes the feed to be ingested very rapidly thus reducing
wastage and loss of materials.
Fish lipids are known to be very rich in the essential poly-unsaturated group of fatty acids mostly
the omega-3 and omega-6 families of fatty acids. Docosahexaenoic acid (DHA), linolenic acid
and eicsapentaenoic acid that are very important for reproductive functions are the predominant
omega-3 fatty acids in fish oil. Generally essential fatty acids are necessary for normal growth,
and the maintenance of normal body immune system. Studies have shown that polyunsaturated
fatty acids present in fish oil are important for the maintenance of the nervous system and help to
reduce stress. They are also known to play major role in reproductive process and larval
development.
Fish meal is known to have a high digestibility coefficient. High digestibility index or standing
of fishmeal accounts for the high feed efficiency recorded when fishmeal is used in feed
compounding. The oil portion of the meal serves as the energy source in the feed. In a case
where the feed does not have enough of the energy components in the form of lipids or
carbohydrates, a part of the proteins will be broken down to supply energy. The problem that
may arise under this condition is the increase in the production of toxic ammonia within the
environment of the fish. Apart from this condition which can arise, there are no recorded bad
aspects of fish meal in terms of anti-nutritional or poisonous compounds unless that introduced
by handling. The important attributes of fish meal in the diets of animals is enormous which
explain why it is the preferred protein source in fish and mono-gastric animal feeds.
2. 6. Combined Effects of Fish Meal with other Protein Sources in fish Diet
Fish meal has been used as a major component in the diet and as the major source of dietary
protein for most species of fish especially carnivorous fishes.
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Fishmeal is known to contain a complete set of essential amino acids that is needed to meet the
protein requirement of most fish species (Sogbesan and Ugwumba, 2008). Since fishmeal is
expensive as a feed ingredient, using other feedstuffs has been the subject of research for some
time now in order to ascertain the effect of these alternative protein sources on the productivity
of various species of fish (Eyo, 1994). The results can better be realized if there is any evidence
of good reproductive performance in the species. In some cases fish meal constitute up to 60% of
the total diet. Higher levels are included for starter and fingerling diets. A good number of other
materials have been used for fish feed manufacturing but the choice of such materials depends on
the cost, constant availability and the nutrient status of such material. Apart from these factors
another factor that is always considered while selecting feed stuff is the presence of anti-
nutritional factors which play down the digestibility and availability of the nutrients in some of
the feed stuffs especially vegetable materials. Usually animal products have better nutrient
profile particularly protein and some important vitamins, but these products apart from being
costly have short and unstable supply. As earlier indicated, other animal products commonly
used in fish feed compounding are meat and bone meal, blood meal, poultry by-product meal.
The commonly used plant protein sources for fish diet manufacturing include soybean meal,
cottonseed meal, sunflower meal and groundnut meal. All these plant protein sources are
products of oil extraction. These products have crude protein ranging from 20 – 50%.
Fish feed manufacturers have used these plant protein sources in combination with animal
products to make diets for different species of fish. Also, various researchers have studied the
effects of different levels of combinations of these materials with fish meal and other animal
protein sources on the productive performance of some species of fish (de Wet 2007, Sullivan
2008 and Piccolo, et al 2011). Different results have been obtained using plant protein sources
only and using plant protein in combination with animal products in the diets of fish. Alam et al.,
2008 (unpublished data) reported no significant difference in growth rates between fish fed diet
with up to 60% of the fish meal replaced by soybean meal and those fed a control fish meal diet
Sullivan, (2008) also reported no significant differences in growth, liver tissue proximate
composition, muscle tissue moisture, protein and ash, and whole body moisture and protein
between fish fed diets replacing 60% or 70% of the fish meal protein with soybean meal
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compared to fish fed the fish meal diet. The author also reported no significant difference
between fish fed 30% replacement of fish meal by poultry by-product meal and meat and bone
meal respectively, and fish fed fish meal diet.
The actual levels of inclusion of some of these plant materials that will not affect productivity
adversely especially reproductive performance have not been clearly established.
The utilization of protein feedstuffs of plant origin in diet formulation has been limited as a result
of the presence of some substances generally referred to as anti-nutritional factors, despite their
nutrient values and low cost implications (New, 1987; Sogbesan et al., 2006). These anti-
nutritional substances include alkaloids, glycosides, oxalic acids, phytates, protease inhibitors,
haematoglutinin, saponin, momosine, cyanoglycosides and linamarin. These anti-nutritional
factors have been shown to produce negative effects on growth, reproduction and other
physiological activities particularly when included at very high levels (Oresegun and Alegbeleye,
2001).
Soybean meal has been the choice of plant protein source for diets of many animals because of
its high level of protein and good amino acid profile and has been used as a source of dietary
protein for most species of reared animals, especially mono-gastric animals. Although, plant
materials may contain high level of protein, research findings show that most of these materials
have some major problems in terms of digestibility and nutrient utilization by the animal. The
percentage nutrient composition of soybean has been estimated at 40% of protein and 18% of fat
for whole Soya (Jauncy and Ross 1982). Apart from the protein component which has been
shown as the best plant protein in terms of amino acid profile, the fat can be used by the feed
formulator to add appreciable amounts of essential fatty acids to the diet, and can also be used as
a source of protein, thereby sparing its energy supply in diet (Jauncy and Ross, 1982).
Amongst the available plant protein feed stuffs, oilcakes are the most important and are the most
promising alternative to fishmeal or animal protein sources in the diets of many reared animals
including fish. Oilcakes are the residues after the industrial removal of the greater part of oil
from oil seeds. The amount of oil in the residual material depends on the method of extraction.
Most of these oilcake feedstuffs have tropical origin and are known to be very rich in protein.
The percentage crude protein may range from 20 to 50 percent. Soybean has also been
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suggested as a very good material under description. Others are cottonseed, groundnut, sun
flower seed, etc. (Jauncy and Ross 1982)
Protein materials are graded depending on the level at which they provide the nutritional
amounts of the essential amino acids needed for overall body health maintenance, and growth
Several studies have shown that addition of fishmeal to the diets of many species of reared
animal increases feed efficiency and growth and general productive performance through better
food palatability, digestibility and good nutrient profile. It has also been reported that fishmeal
enhances nutrient uptake, digestion, and absorption (Miles and Chapman, 2009). The balanced
amino acid composition of fishmeal complements and provides synergistic effects with other
animal and vegetable proteins in the diet to promote fast growth and reduced feeding costs,
(Miles and Chapman, 2009).
High quality whole Fishmeal provides essentially all the nutrients required by the animal
including fish for normal growth and development and for other productive functions including
reproduction in a balanced amount. Almost all the essential amino acids, phospholipids, and fatty
acids e.g. docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are present in
reasonable quantities for optimum development and growth, especially of larvae and for
reproduction in brood stocks. The nutrients in fishmeal also aid in disease resistance by boosting
and helping to maintain a healthy functional immune system. Also, high-quality fishmeal imparts
a natural characteristic to the feed giving almost all the nutrients required by the fish and to the
final product the wholesome characteristics such as that provided by wild fish (Miles and
Chapman, 2009).
James and Sampath (2003) in their work on ornamental fish, Betta splendens indicated that fish
fed fish meal and other animal protein diets performed better than those fed plant protein diets in
their growth performance.
The authors indicated that on day 84, fish that were fed animal protein at the 10%, 15%, 25%,
35% and 45% levels had 3.02, 3.38, 4.72, 5.82 and 5.62 mg gain per g live fish per day
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respectively compared with 2.36, 3.30, 3.73, 4.76 and 4.55 mg gain per g live fish per day for
fish that were fed plant protein.
On the reproductive performance, studies have shown that fish fed fish meal based diets perform
better than those fed plant protein based diets (James and Sampath, 2003; Fagbenenro et al.,
2010). James and Sampath (2003) reported that at day 112 fish that were fed animal protein diet
had 79% higher gonad weight than those fed the same level of plant protein diets. The same
trend was obtained for the gonado-somatic index. They also indicated that fish meal based diets
induced gonadal development earlier than plant based diets as fish that were fed fish meal
developed gonads at 56 days while no gonads were seen in the fish that were fed plant protein
based diets
2.7. Soybean Meal as Source of Protein for Fish Diet
Soybean meal (SBM) has been a commonly used vegetable protein source in animal diets in
most parts of the world. Soybean contains protein with the best amino acid profile compared to
other oilseeds (FAO, 2010). However, like other legume seeds, it is limiting in some essential
amino acids. Soybean meal has been shown to be limiting in methionine and lysine when fed to 6
weeks old calves in the form of corn-soybean starter diets (Abe, et al., 1998). For catfish, Cai
and Burtle (1996) reported that Soybean meal-corn based diets were limiting in methionine.
It has been recognized that there are several compounds in soybeans that can potentially inhibit
high level of incorporation in diets of many species of animal. These compounds are generally
referred to as anti-nutritional factors because they have different types of negative impacts on
nutrient digestion, absorption and utilization in the body system of the animal. The lectins,
oligosaccharides, saponins, and trypsin inhibitors in soybeans have been associated with negative
nutritional effects in fish (Hart and Brown, 2005).
The nutritive value of soybean, however, is limited by the presence of these anti-nutritional
factors, including the indigestible non-starch polysaccharides (NSP) (Castell et al., 1996). Other
anti-nutritional factors associated with soybean meal include glucinins, goitrogens and mineral-
binding substances (CRC, 1983; Liener, 1994). Fish fed diets containing levels of soybean
lecitins experienced reduction in growth and insulin production, while some species showed
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increased mortality, decreased production of trypsin and poor digestion of protein. Soybean is
also known to contain phytoestrogens. The similarities, at molecular level, of estrogen and
phytoestrogens allow phytoestrogens to mildly mimic and sometimes act as antagonists of
estrogen. Phytoestrogens are plant-derived xenoestrogens functioning as estrogen (the primary
female sex hormone). These substances are not generated within the endocrine system of the
animal but are consumed by eating plant materials that contain them. They are also called
"dietary estrogens". It has been proposed that phytoestrogens can cause male infertility and that
some species of plants use phytoestrogens in them to control the overpopulation of herbivorous
species that feed on such plant species (Wikipedia, 2012) .
In spite of these negative effects whole Soybean (full fat soybean) contains 35 – 40% of protein
and about 18% of Fat. The fat fraction can be used by nutritionists and feed formulators as
source of essential fatty acids or as source of energy to prevent use of high cost protein for that
purpose (Jauncy and Ross 1982).
Incorporating soybean products in the aquaculture diets has become the main focus of protein
substitution in fish feed the world over. The high protein level makes it a key ingredient for
aquaculture and mono-gastric animal feeds. This is more so since soybean meal is far cheaper
than the traditionally used marine animal meals – fishmeal which supply is not assured in most
parts of the Sub-Saharan Africa and the supply is dependent on importation.
Incorporating Soybean meal in fish diets has been on the increase since the past few decades.
It is critical that the problems associated with soybean meal should be treated before it can be
used successfully in the diet. Proper heat processing prior to being incorporated in the fed has
been suggested as means of removing most of the anti-nutritional factors. However, overheating
should be avoided to ensure that the protein is not denatured. FAO (2012) reported that
digestibility and utilization of the protein fraction was high when well processed soybean meal
was fed to fish. Apparent protein digestibility of soybean meal according to FAO (2012) by
trout, carp and red sea bream was 90-93%. Soybean meal has better amino acid profile when
compared with cereal grain and even other legume seeds Proteins in cereal grains and other
plant concentrates generally do not contain complete amino acid profile and are said to contain
incomplete proteins. They are usually deficient in most of the essential amino acids particularly
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lysine and methionine. Hence using them in animal diets may need supplementation for these
essential amino acids.
It is clear that soybean is limiting in some critical amino acids and that the amino acid content is
not of the same level as in fish meal. Soya bean meal has been graded as almost the most
excellent source of dietary protein of plant origin for many species of animals.
Depending on the method of processing the soybean product may have high crude protein
content. According to Jauncy and Ross (1992) full fat or whole Soya bean contains crude protein
ranging from 33 to 40% and has about 18% of Fat. The fat portion is important because it serves
the important function of supplying appreciable amounts of essential fatty acids to diet which is
used as energy source for the animal.
The nutritional value of soybean for various species of fresh water fish species is variable (Gatlin
III, 2003). Though soybean has bean used extensively in monogastric feed formulation, studies
have shown that it has some negative effects on some species of fish. For instance,in Atlantic
salmon and rainbow trout, soybean meal has been found to cause distinct morphological
alterations in the intestine (Krogdahl and Bakke-McKellep, 2001). They also indicated that
soybean inclusion in the feed for these specie resulted in impaired growth and protein utilization
and that the effects escalate with increasing dietary level. There may be other effects that have
not been established. This work was conducted to determine the amount of soybean meal that
could replace fish meal in formulated diets without reducing reproductive performance
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Chapter Three
Materials and methods
3.1. Location and Duration of the study
This study was done at the Wet/Aquaculture Unit of the Department of Zoology, University of
Nigeria, Nsukka, Enugu State, South Eastern Nigeria.
The experiment lasted for four months.
3.2. Experimental fish
A total of five hundred and sixty Clarias fish (C. gariapinus) were used for this experiment. The
fish stock was purchased as juveniles at about 6 weeks of age. Initially they were kept together in
four ponds for about four weeks for acclimatization. During this period they were fed ad libitum
with the control diet (diet A) at the rate of 3 – 3.5% of body weight
At the end of the acclimatization period the fish were randomly sorted into four groups
(treatments). Each of the groups was divided into two (replicates) and kept in separate ponds
making a total number of eight ponds. Four diets were formulated; one of the diets, the control
diet 1, was formulated using fish meal alone as the major protein source and other two diets,
diets 2 and 3 were formulated using two different combinations of fish meal and soybean meal
and the fourth diet was formulated using soybean alone as the major source of protein in the diet
(Table 1). The four diets were randomly assigned to the groups. Each of the groups was fed
within the range of 3% - 3.5% of the body weight with the diet assigned to it.
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3.3. The Experimental Diets
Four experimental diets were formulated containing different levels of fish meal and soybean
meal. Diet A which is the control diet contained 44.99% fish meal as the major protein source in
the diet. The other formulations B, C and D contained 34.91%, 24.05% and 0% fish meal
respectively as the major protein source in the diets. The soybean fraction in the diets A, B, C
and D were 0%, 16.54%, 33.18% and 66.77% as the protein source respectively. The percentage
ingredients compositions of the diets are shown in table 1.
Table 3.1: Diet Formulation with different levels of soybean; All values are in kg per 100kg
diet.
Feed Stuff A B C D
Fish meal 44.98 34.91 24.95 – Defatted soy meal – 14.58 25.23 61.35 Full-fat soy meal – 4.96 12.95 23.83 Maize 24.01 19.65 14.26 3.28 Wheat offal 21.86 17.65 13.36 2.29 Animal fat 3 3 3 3 Binder 2 2 2 2 Bone meal 2 2 2 2 Salt 1 1 1 1 Vita./Min. Mix 0.25 0.25 0.25 0.25 D-L Methionine 0.5 0.5 0.5 0.5 Lysine 0.5 0.5 0.5 0.5
Total 100 100 100 100 Proximate composition of the experimental diets Crude protein 35.01 35.01 35.01 35.00 Crude fibre 6.18 6.62 6.27 6.90 Crude fat 5.04 5.60 5.73 5.92 Ash 6.17 6.13 6.18 7.01 NFE 47.60 46.64 46.81 45.17
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3.3.1. Diets evaluation:
The proximate compositions of the experimental diets are shown at the bottom of Table 3.1
The experimental diets were analyzed in the Department of Animal Science, University of
Nigeria, Nsukka, Nutrition and Biochemistry Teaching and Research Laboratory by proximate
method. The proximate composition of the experimental diets did not show much variation in the
nutrients of the various diets.
The two forms of soybean meal used in the diets were the commercial forms got from Phinoma
Nig. Ltd., a vegetable oil processing company situated in Ngwo Enugu State Nigeria. The full fat
form as the name implies has its full compliment of fat (or oil) while greater part of the oil has
been removed from the defatted form by mechanical method.
The defatted form has higher crude protein but lower amount of oil. Though, we want feed that
will provide the required level of quality protein but this should not be sought at the expense of
quality energy which a very important component of any good diet. Though fish can use energy
from carbohydrate sources but fishes prefer energy from lipid sources. Oils/lipids are noted to
give flavour to the diet and hence help to increase palatability of the feed.
3.4. Experimental Design
The experiment was carried out in a randomized complete block design (RCBD). The diets
served as the treatments and sex was the block. The statistical model of the design is given
below:
γіјk = µ + τі + βj + (τβ)ij +Σіјk
Where γіјk = any individual observation
µ = the population mean
τі = effects of the ith level of soybean meal on the observed data
βj = effect of the jth sex
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(τβ)ij= the effect of interaction between the ith and jth sex on the individual observation
Σіјk = random error due to observation
.
3.5. Data collection
3.5.1. Sampling of fish for Dissection
Data collection commenced from the second week following separation into treatments. From
each of the treatments sixteen fish were sampled for dissection – eight males and eight females in
line with the method by James and Sampath (2003).
The sampled fish from each replicate were weighed together first and then individually and their
weights recorded. The fish were killed for dissection to get the gonad. After dissection of each of
the sampled fish, the severed gonads were weighed fresh and recorded. The gonads of each
replicate were preserved in a separate container of 10% formalin solution.
3.5.2. Histological Sectioning and Analysis
This was carried out in the Histology Laboratory of the Faculty of Veterinary Medicine,
University of Nigeria, Nsukka, following the standard procedure described by Onuoha, (2010)
Data were collected on a biweekly basis using different samples of fish for four months. This
means that data were collected eight times during the period of this experiment. The body weight
data of fish used were collected by weighing the fish samples live, individually and in group. Six
fish were sampled per treatment with three fish per replicate. The gonads were weighed
immediately after dissection to obtain the fresh weight.
The tissues were processed for further studies by following the procedure described by Onuoha,
(2010). The first step involved tissue dehydration by fixing the tissue in graded levels of ethanol
starting with 70% ethanol for 1hour 30minutes. This was followed by fixing in 80%, 90% and
finally in absolute ethanol for 1hour 30minutes respectively. After this the next step was tissue
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clearing. Clearing was done by putting the tissue in chloroform to remove the ethanol from the
tissue. After which the tissues were transferred to molten paraffin. This procedure is summarized
in the list below:
3.5.2. 1 Fixing and embedding of tissue samples (gonads)
Fixed gonad samples were dehydrated and embedded in graded solutions as listed below:
a. fixing
1. The tissue samples were fixed in 70% ethanol and left for about 90 minutes; then moved
to 80% ethanol, to 90% and finally to absolute ethanol and kept for 90 minutes in each
case.
b. clearing
The tissue samples were cleared of alcohol using graded level of Chloroform. They were put in
chloroform I and left for 90 minutes then transferred to Chloroform II and also left for 90
minutes
c. embedding
The tissue samples were ready and kept in Paraffin wax for 90 minutes and then moved to the
next, Paraffin wax II and left for 90 minutes each time for paraffin infiltration of the tissue.
These tissue samples were transferred into fresh molten wax in square wooden boxes with metal
base. The blocks were put in tray with the face in contact with ice to cool.
3.5.2.2 Histological Sectioning Sections of the tissues were taken from the embedded tissues at 5µm thickness using a microtone and
placed on consecutive slides. Each of the samples was sectioned at 5µm using standard
histological procedures as described by (Luna, 1992, Cek et al., 2001, Cek, 2006).
3.5.2.3 Staining
The sections were stained with Mayer's haematoxylin and eosin Y according to the procedure
modified from Bancroft and Stevens (1991), for histological evaluation. The procedure is as
described below:
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The first step involved putting the sections in Xylene for 3 minutes, then put again for 2 minutes.
They were put in absolute ethanol for 2 minutes and then soaked in methylated spirit for 1
minute. The samples were rinsed in water for 30 seconds before they were mixed with
Haematoxylin solution and left for about 5 minutes. They were rinsed again in water for about 30
seconds. They were dipped in 7. 1% Acid alcohol solution in 3 quick successions then washed in
water for 30 seconds.
They were soaked in Eosin Y for about 5 minutes washed in water for 30 seconds and
Methylated spirit for 1 minute. They were first soaked in absolute alcohol for 2 minutes and then
for 1 minute. They were placed in Xylene and left for 5 minutes
The slides were held in xylene until cover-slipping using Pertex mountant.
Gonad development was determined using MOTIC IMAGE 2.0 micro-photograph software
connected to a computer and high power microscope with images being captured with the aid of
zoom lens linked to a computer using the image capture software.
The gonad tissues of the fish were evaluated accordingly to the stage of gametogenesis. The
gonads were grouped into 4 stages: stage 1, stage 2, stage 3 or stage 4 gonads depending on the
stage and cell type predominating in the tissue.
3.6 Statistical analysis
Data collected on body weight and gonad weight were analyzed using the analysis of variance
(ANOVA) to find out the differences between the treatments in terms of body weight and gonad
weight. Correlation was done to find out whether there was any interaction between the diets
and sex of the fish on the gonad or body weight of the fish using SPSS statistical software for
Windows 1996, Standard Version SPSS Inc. London.
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RESULTS AND DISCUSSION
4.1 Main Effect of Diet on Weight Gain and Final Body Weight of African
Catfish
Table 4.1 Main Effect of Treatment on the Body Weight Gain of the African catfish
NS = not significant (P > 0.05)
Table 4.1 shows the main effect of diet on Body weight gain of African catfish. There were no
significant differences among the treatments (P > 0.05) in the weight gain and the final body
weight of the African catfish fed different diets. Catfish in treatment A had final body weight
values of 100.68g and 85.43g for males and females respectively. The males in other treatments
had final body weight 96.60, 76.30 and 74.09g respectively for treatments B, C and D. The final
body weight values for the females followed the same trend. The values obtained here for 120
days catfish were similar to the mean weight of 74.2g obtained by Kenan et al., (2008) for 105
days old European catfish. A similar trend was reported by Isicheli (2008). He obtained figures
in the range of 40.2 g and 85.5 g for 24 weeks (168 days) Clarias spp although he did not group
the fish into males and females.
This result is in agreement with the results of (Oso, et al., 2011 and Nyirenda et al, 2000) who
reported that weight gain (W.G), percentage weight gain (% WG) and specific growth rate
(SGR) of Clarias gariepinus fingerlings were not significantly different (P>0.05) when fish
Parameters Treatments (Diets)
Sex 1 2 3 4 SEM Weight gain(g) Male 2.06 1.93 1.84 1.35 0.44NS
Female 1.68 1.66 1.56 1.26 0.44NS
Final body weight (g)
Male 100.68 96.60 76.30 74.09 9.16NS
Female 85.43 81.84 74.30 72.14 9.16NS
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specimen were fed diets with different levels of substitution of fish meal with various plant
protein sources. Udo et al. (2012) also showed that growth performance parameters were not
significantly different (P>0.05) when different plant protein sources were included at different
levels in the diets of C. gariepinus. However, in an experiment with trout and salmon, Hart and
Brown (2005) found that growth responses diminished when soybean meal was incorporated at
15-25% in the diets of trout and salmon, even when the diets were formulated to meet essential
amino acid requirements. This suggests that apart from the protein and amino acid profile there
are other factors that affect growth and development in the treated fish. Several reports show that
increased growth in trout and salmon is usually not associated with an increase in protein
deposition but on the increase in lipid deposition rate (Sunde, et al., 2001; Rungruangsak-
Torrissen & Fosseidengen, 2007; Krisna et al., 2009) and on the physiological condition of the
body. For example, in females in their vitellogenic stage when yolk deposition is usually very
rapid and in pubertal males when the rate of nitrogen retention is high weight gain is usually
accelerated.
This result disagrees with the result of Eyo (1999) who reported that there was significant
difference in growth performance of Clarias gariapinus when juvenile fish were fed different
levels of plant protein. Hernandez et al. (2007) also found a decrease in final weights of sharp
snout sea bream (Diplodus puntazzo) as the soybean meal content of the diets increased starting
from 40% substitution rate. Eyo (1999) obtained poor growth rate when C .anguillaris was fed
soybean based diets. Fafioye et al. (2005) observed higher percentage increase in body weight
of fish fed heat treated soybean over fish fed raw soybean. So the growth difference was as a
result of a factor in the soybean which was reduced by processing. Faromi (2002) in an
experiment with African catfish noted that the growth of the group of fish fed complete plant
protein as main protein source were poorer than those fed complete fish meal because fish meal
contains some amino acids not present in tissues from terrestrial plant; but when lysine and
methionine were added to the diets there was improvement in all the diets. He did not show the
extent of improvement.
Davies and Ezenwa (2011) suggested that supplementing plant based diets with lysine and
methionine improves growth performance and attributed the poor growth performance of C.
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gariepinus fry fed plant protein as the major source of protein to the deficiency of lysine and
methionine. Lysine and methionine were included in all the diets used as shown in Table 1. This
was probably the reason for non-significant difference in growth performance.. The lower body
weight recorded for the fish that were fed diets C and D, though not statistically different, may
be attributed to reduced palatability of the diets which resulted in lower feed intake. The report
of Liener and Kakade (1980) shows that feeding animals with raw soybean retards the growth
due to the unavailability of proteins to the animal body system resulting from indigestibility of
proteins because of the presence of trypsin inhibitors. Lovell (1976) observed that heating
soybean improved the digestibility of polysaccharides and metabolizable energy portion and
increases the utilization of protein as a result of inactivation of trypsin inhibitors.
4.2 Main Effect of Diets on the Gonad Weights and Development of African
Catfish
Table 4.2 presents gonadal weights of C. gariepinus fed the experimental diets;
There was a highly significant difference (P < 0.01) between the treatments in the gonadal
weight. The figures show that treatment A (0% soybean) had higher gonadal weight for both
males and females followed by the treatment B. Treatments C and D were similar.
The significant differences in gonadal weight of fish among the treatments may be due to the
effect of treatments on gonadal development. The soybean had inherent properties that had
negative effects on the gonadal growth and development. Diet A, (0% soybean diet), came up as
the best fish diet for overall gonad development among the four treatments as shown in the
Table.
Variance analysis shows that in the males the gonad weight of the fish in treatments A and B
were similar. The gonad weights of the fish in treatments A and B were higher than that of
treatment C which is higher than those in treatment D. This shows that the effects of soybean
increases as the level of replacement of fish meal with soybean increases from 20% level. The
20% inclusion of soybean meal in diet B had no effect on the gonad weight of the male fish. For
the females, the gonad weights of the fish in treatments C and D were similar. Treatment A
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differed significantly from treatment B. Treatment B differed significantly from treatments C
and D in their gonad weights.
Table 4.2: Main Effect of Treatment on the Body and Gonad Weights of the African
Catfish
NS - Not significant (p < 0.05)
** - highly significant (P < 0.01)
a, b, c – Means on the same row with different superscripts are significant 1% (p < 0.01)
Fish that were fed diets C and D had poor gonadal growth performance because of plant protein
source and also the effects of anti-nutritional factors in the soybean which forms greater part of
the protein portion of the diets. Also fish meal has good fatty acid profile containing mostly the
poly-unsaturated fatty acids. Fishmeal and fish oil contain more omega-3, than omega-6 fatty
acids and most plant lipids contain higher concentrations of omega-6 fatty acids, Miles and
Chapman (2009). Omega-3 fatty acids play important structural roles in reproductive process as
components of phospholipids in fish bio-membranes, being related with the membrane fluidity
and correct physiological relationship with bound membrane enzymes and cell functions
(Izquierdo and Fernandez-Palacios, 2009).
Soybean like most legumes contains phyto-hormones. The most common phyto-hormones are
flavons, coumestanes and isoflavons. Phytoestrogens are plant-derived xeno-estrogens
functioning as the primary female sex hormone. Xeno-estrogens are hormone-like substances not
Parameter
Treatment
1 2 3 4 SEM
Mean Body
Weight (g)
Male 100.6 96.60 76.30 74.09 6.48NS
Female 85.43 81.84 74.30 72.14 6.48 NS
Mean Gonadal
Weight (g)
Male 0.30a
0.22a
0.16b
0.12b
0.11**
Female 0.53a 0.35
b 0.23
c 0.22
c 0.18*
Gonado-Somatic
Index (GSI)
Male 0.30a 0.26
a 0.21
b 0.16
c 0.02**
Female 0.60a 0.43
a 0.31
b 0.30
b
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generated within the endocrine system but consumed by eating plants materials that contain these
substances. These phyto-hormones sometimes called "dietary estrogens" are diverse group of
naturally occurring non-steroidal plant compounds that have structural similarity with estradiol
(17-β-estradiol). They have the ability to cause or antagonize estrogenic activities in the animal.
Causing estrogenic activities means it can induce oocyte growth in the fish which is elicited
during the vitellogenic stage of development as a result of the sequestration of protein -
vitellogenin (VTG) - from the plasma, (Norberg and Haux, 1985; Tyler et al., 1991; Fontainhas-
Fernandes et al., 2000) or antagonize this process. Since Vitellogenin is produced by the liver
under the influence of estrogen, 17β-estradiol (E2), when other factors (food nutrients, health
condition) are favourable any thing that affects the activities of estrogen, 17β-estradiol (E2) in the
animal affects this reproductive process. Estrogen is critical and fundamental to vertebrate
reproduction and the requirement for estrogens in the reproductive process in female is
persuasively universal (Claude, 1988). Any factor that affects the function of estrogen affects the
entire reproductive life of at least the female vertebrate.
Hughes (1988) noted that early exposure of the male or female fish to sex steroids permanently
alters the sexual behavior of fish, amphibians, reptiles, birds, and mammals although there may
be species differences in the response of the animal to the role of sex steroids in the
differentiation of gonads. Apart from the source of protein which determines the amino acid
profile the result can also be attributed to the presence of some organic substances that are
capable of producing negative influences on the development of the reproductive organs of the
fish. From the foregoing soybean is not only deficient in some amino acids necessary for
building strong reproductive health in the fish but the presence of phyto-hormones can be
detrimental to the development of the reproductive structures.
It was observed in this study that at week 20 when the fish of the treatments A and B had mature
follicles, the ovaries of the fish of treatment D and some fish in treatment C had few oocytes and
young meiotic oocytes. This result suggests that exposure of the fish to the phyto-chemicals
contained in soybean alters the normal development of the germ cells of the fish resulting from
the condition of estrogen stress. Phytoestrogens have been shown in an earlier study to interfere
with estrogenic activities by blocking the nuclear and membrane estrogen receptors (ER);
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interfering with the growth factor receptor; inhibiting the G protein-coupled receptor in ER-
deficient cells, and activating apoptosis and nullifying anti-apoptotic signals (Zhao and Mu
(2010). In addition, Fontainhas-Fernandes, et al. (2000) showed that in the Nile Tilapia, diets
containing only plant protein were less efficient in terms of growth and ovarian development.
Also Pereira et al. (1998) found that rainbow trout fed only plant materials as the only source of
protein showed less efficiency in reproductive indices than those fed on diet based on fishmeal.
In contrast, the report of Adewumi (2006) who showed that fish meal had adequate nutrients
required for the formation of genital products that produced strong offspring in C. gariepinus
brood-stock culture.
Gonad development in all species of animal is influenced by gonadotropic hormone produced by
the pituitary gland as a response of hypothalamus to environmental cues such as photoperiod,
feeding, and environmental temperature. In fish, rainfall, water temperature and fluctuation of
the water level are also stimulatory to gonadal development.
The result of this work suggests that replacement of fish meal with plant protein sources affects
gonadal development. Inclusion of soybean above 20%, to replace more than 10% of fish meal in
the diet of African catfish will affect the gonad development in the African catfish. The protein
source is not the only factor that produced the negative impact since, according to Davies and
Ezenwa (2010), the major limiting amino acids in the protein were included which should have
improved the gonadal development. This suggests that there are other unnamed factors which
have serious impacts on gonadal growth. There are some reports indicating negative association
between exposure to phytoestrogens and poor gonadal development in some species including
human. Mitchell et al., (2001) indicated that dietary consumption of soy-based infant formula
reduces the neonatal surge in testosterone and increases Leydig cell number in the testes of male
marmosets. However, Sertoli or germ cell numbers were not affected. The human health
implications of these results are unclear. They did not show the method of processing and the
effects of the methods of processing on these indices.
It has been established that sexual development can only be effected if certain levels of necessary
hormones are available when required. There are specific hormones necessary for normal
development of the sex structures and any interference with these activities may be detrimental
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to normal development. Giwercman (2011) suggested a negative trend in male reproductive
function due to exposure to some chemicals interfering with the action of sex hormones. These
chemicals include phytoestrogens which are of plant origin. Phytoestrogens which are non-
steroidal estrogens of plant origin are potent endocrine disruptors as they modulate normal
physiological functions (West et al., (2005). They pointed out soybean as a common source of
phytoestrogens and indicated that the increasing use of soybean particularly in infants is of
concern since the most vulnerable periods for estrogenic insult are thought to be the pre- and
neonatal periods when irreversible damage can be inflicted on the developing germinal
epithelium.
The diets had higher effect on the gonadal development than it had on the growth and body
weight. This result suggests that phyto-estrogens which is a major factor that is very critical for
the development of the reproductive structures than for other tissues. Apart from the
development of the sex structures they may also have effects on the sexual behaviour of the
individual fish (Hughes (1998). Phytoestrogens given in high doses or at critical stages of
development in rodents result in severe reproductive tract disorders (Mitchell et al, 2001) and
temporary infertility syndromes in domestic animals have been related to high phytoestrogen
consumption in grazing animals (Adams, 1995).
The present study demonstrates that inclusion of soybean meal especially at high levels (above
35%) in the catfish diet had negative effects on the gonadal development and long term
reproductive performance.
There were no significant differences among the treatments in the GSI values of fish fed diets A
and B in both the males and the females.
The gonadosomatic index, usually abbreviated as GSI, is the expression of the gonad weight as a
proportion of the total body weight of the animal. GSI is calculated as:
GSI = [Gonad Weight / Total body Weight] x 100. It is a tool for measuring the sexual maturity
of the animals in correlation to ovary or testes development
(http://en.wikipedia.org/wiki/Gonadosomatic_Index).
Fish fed diets C and D showed poor gonad development. Mean gonado-somatic index
significantly decreased with increasing level of replacement of fish meal with soybean meal. . In
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the female, the fish fed diets C and D had significantly lower gonado-somatic index compared to
those that were fed diets A and B. Mean separation showed that diets A and B were not different
in both males and females. Also, in the males, there was no significant difference between the
fish in treatments C and D. But in the female, the fish in diet C had significantly lower
gonadosomatic index than those in diets A and B. Fish in diet D had lowest mean GSI.
4.3 The Development pattern of the female gonad (the ovary) of the fish fed
the different diets
In both sexes, the histological inspections of the gonads did reveal differences among the fish fed
diets with different levels of soy bean. Fish fed diets A and B had significantly heavier and more
developed gonads compared to those on diets C and D. Fish fed diets C and D with higher levels
of soy bean had fewer numbers of yolky oocytes compared to other groups. Figure 1 show the
gonadal tissues from fish of different treatments. This difference became clearer from the 18th
week
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Plate1 Transverse section of gonads from female fish fed the different diets at week 14. A. showing the ovarian organization of the female fish fed diet A; b Shows the cross section of the ovary of the fish fed Diet B; c. Shows the cross section of the ovary of the fish fed Diet C d. Shows the cross section of the ovary of the fish fed Diet D
Plate 1 shows the micrographs of the histological sections of the ovaries of catfish fed the
different test diets A – D at week 14. The ovaries of the fish did not show much difference on the
12th and 14th weeks. There were no structural differences in the ovaries during the 12th and 14th
week period. At this time the ovaries were seen as smooth colourless to translucent brown
tubular structures. Histological examination shows that there were small developing oocytes seen
as light transparent sports in the ovary.
a B
d c
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From 18th week small semi transparent oocytes and larger opaque oocytes were observed in the
ovaries of more than 50% of the female fish of treatments A and B, a few in treatment C and
very scanty in treatment D.
From this period the colour changes were observed in the ovaries. There colour of some ovaries
changed from translucent light brown to dark opaque brown. These changes were very clear in
treatments A and B and less conspicuous in treatments C and D. Cek and Yilmaz (2009)
attributed these colour changes which they observed in sharptooth catfish to stages of yolk
formation in the ovaries. The result of this work shows that there was delay in yolk formation
and ovarian growth and development in the female fish fed diets (C and D) with high levels of
soybean.
The number of oocytes per ovary was also low for the fish that were fed diets with increased
levels of soybean. Histological observation shows that there were signs of germ cell loss in the
ovaries of the fish that were fed diets with high levels of soybean
Higher levels of inclusion of soybean in the fish diet caused a reduction in oocyte number and
development. The magnitude of these effects was reflected in the increase in germ cell loss
observed in treatments C and D between weeks 20 and 22 (fig. 4 c and d). This treatment
significantly decreased ovarian size.
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Plate 2. Transverse section of gonads of female catfish fed the different diets at week 16: a. shows the ovary of the fish of treatment A with developing oocytes. The oocytes at this stage appear like shiny pin points in the ovary; b. cross section of the ovary of the fish of treatment B this ovary compares favourably treatment A; c. cross section of the ovary of the fish of treatment C there were no oocytes seen in these ovaries. d. the cross section of the ovary of the fish in treatment d shows a clear transparent tubular structure filled with fluid. no oocytes were identified in these ovaries.
a b
c d
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Plate 3. Transverse section of gonads of female catfish fed the different diets at week 18: a. shows the ovary of the fish of treatment A with numerous semi-transparent oocytes and opaque brown oocytes giving the ovary shiny brown coloration; b. cross section of the ovary of the fish of treatment B this ovary compares favourably with the ovary of the fish in treatment A; c. shows cross section of the ovary of the fish of treatment C. ttransparent oocytes dominate in the ovary makng the ovary to appear as colourless lobular structure. It has fewer number of developing oocytes than that of treatments A and B There is also evidence of atretic oocytes in the ovary. d. shows cross section of the ovary of the fish of treatment D. the number of oocytes is very small and also there is clear evidence of oocyte atrecia in the ovary.
a
d
a
b
c
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Plate 4. Transverse section of gonads of female catfish fed the different diets at 20 weeks; a shows the ovary of the fish in treatment A with normal yolk formation in the ovary b. shows cross section of the ovary of the fish of treatment B, there is normal yolk formation as in treatment A, the oocytes do not differ from those in the ovary of treatment A; c. Shows cross section of the ovary of the fish of treatment C. there is delay in the yolk formation. also the number of oocytes in the ovary is less than the number of oocytes in treatments A and B The spaces between the oocytes show eveidence of germ cell loss in these ovaries; d. Shows cross section of the ovary of the fish of treatment D. the number of oocytes is very small compared to A and B. the size of individual oocytes is smaller when compared with treatments A and B
c d
b a
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Plate 5. Transverse section of gonads of female catfish fed the 4 different diets at 22 weeks: a shows the ovary of the fish of treatment A with normal ovarian structures with yolky oocytes. b. shows cross section of the ovary of the fish of treatment B with yolky oocytes it does not differ from the ovary of treatment A. c. Shows cross section of the ovary of the fish of treatment C. the number of oocytes in the ovary is less than that of treatments A and B There is also evidence of atretic oocytes in the ovary. d. Shows cross section of the ovary of the fish of treatment D. the number of oocytes is very small and also there is clear evidence of oocyte atrecia in the ovary.
c d
b a
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Plate 6. Transverse section of gonads of male catfish fed the different diets at 20 weeks; a shows the internal structures of the testis with spermatozoa at different stages of development; b. shows cross section of the testis of catfish of treatment B, the sperm cells in this testis do not differ from those in testis of the fish in treatment A; c. Shows cross section of the testis of the fish of treatment C; d. Shows cross section of the testis of the fish of treatment D
b a
c d
c
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Plate 7. Transverse section of the testis of male catfish fed the different diets at week 18: a. shows the ovary of the fish fed diet A . The oocytes at this stage appear like shiny pin points in the ovary; b. cross section of the ovary of the fish of treatment B this ovary compares favourably treatment A; c. cross section of the ovary of the fish of treatment C there were no oocytes seen in these ovaries. d. the cross section of the ovary of the fish in treatment d shows a clear transparent tubular structure filled with fluid. no oocytes were identified in these ovaries
c d
a
b
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Plate 8 Transverse section of male gonad of catfish fed the different diets at week 16. a shows the lobular testis of the male catfish with developing germ cells; b Shows the cross section of the testis of the fish fed Diet B; c. Shows the cross section of the testis of the fish fed Diet C d. Shows the cross section of the testis of the fish fed Diet D
d c
a b
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Plate 9 Transverse section of gonads from female fish fed the different diets at week 14. A. shows the collection of germ cells in the developing gonad; b Shows the cross section of the ovary of the fish fed Diet B; c. Shows the cross section of the ovary of the fish fed Diet C d. Shows the cross section of the ovary of the fish fed Diet D
a
b
c d
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4.4 Effects of Sex (Block) on the Body and Gonadal Weight of the African
catfish
There were no significant differences between the males and the females in their final body
weight and body weight gain. This indicates that the sex had no effect on the body weight gain
and growth performance, though the figures showed lower weights for the female. This is normal
as it is known that males usually tend to have higher weights than their female counterparts (Cek
and Yilmaz, 2007).
The results in Table 4 show that males had significantly lower weights of gonad than females
even though the average body weight of the female was lower than that of the males. This result
is in agreement with the findings reported by Memis and Gun (2004). They implied that female
gonads had consistently higher weights than the male gonads in all the treatments.
The females had higher gains in gonad weight when compared with their male counterparts.
Naturally, ovaries have higher weights than testes. Also, reports have shown that female gonads
develop earlier than male gonads in most species.
Maclatchy and Van Der Kraak (1995) show that exposure of male fish to phytoestrogens reduces
the gonadal steroid biosynthetic capacity through effects on cholesterol availability in the male
thus contributing to the reproductive dysfunction in fish exposed. Gonadal steroid is a very
important factor in gonadal growth and development. This is a factor contributing to the slow
rate of development of the gonads of the male fish fed soybean. Low level of sex steroids for this
reason means poor development since sex steroid hormones are the regulators of sexual
development and maturation in all species (Roy Dahle et al., 2003).
4.5 Correlation between the Age, body weight and gonad weight of the Male
African catfish
The data in Tables 4.3 and 4.4 show the correlations between the age, body weight and gonad
weight in male and female catfish respectively. There were very strong positive correlations
between the age, body weight and gonad weight of the African catfish. The correlation-
coefficients are close to unity (i.e. they were very close to 1). This indicates that the body weight
increases with the age and the gonad weight also increases as both the males and the females age.
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The body weight of the catfish also has very strong relation with gonad weight as shown in the
Tables 4. and 5.
The results of the present study are substantiated by the findings of Delahunty and De Vlaming
(1980), and also by Mahboob and Sheri (2011) who indicateded that gonadal weight increases as
the body weight increases. Reports show that in most species of teleost, gonadal weight has
direct relation with the body weight (Gaikwad et al., 2009).
The result of this work indicates that, although there were no significant differences among the
treatments in the body weight, fish that were fed diet A and diet B had apparently heavier
gonadal weights than those on the other diets while gonadal weights increased with body weight.
The gonadal weights decreased as the level of replacement of fishmeal with soybean increased.
The level of replacement had no effect on the body weight but it had significant effect on the
gonadal weight. This can be attributed to the level of phyto-hormones which have direct effect
on the reproductive system of the fish. Also, this may be partially attributed to the availability of
necessary amino acids and fatty acids in fish meal that are not in soybean meal. Omega 3 group
of fatty acids have been indicated to play important roles in sexual development in many species
of animal including fish.
Table 4. Correlation coefficient between age, body weight and gonadal weight of the male
African Catfish
Age Body weight Gonad Weight
Age
--
0.92**
0.77**
Body weight
--
--
0.89**
Gonad Weight
--
** Significant at 1% (p < 0.01)
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Table 5: Correlation coefficients between age, body weight and gonadal weight of the
Female African Catfish
Age Body weight Gonad Weight
Age
--
0.95**
0.78**
Body weight
--
--
0.84**
Gonad Weight
--
** Significant at 1% (p<0.01)
4.6 Effects of Interaction between Treatment and Sex (Block) on Body and
Gonadal Weights of African catfish
Table 6 presents the result of the effects of interaction between treatments and sex on the body
weight and gonad weight. There were no significant interactions (P > 0.05) between the sex and
treatment on the body weight. On the other hand there were significant interactions between the
treatments in gonadal weight. The gonadal weight in the males decreased with increasing levels
of soybean but the reduction was more in the females as the level of soybean increased in the
diets Females responded to the treatments more than the males in gonadal weight.
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Table 6: Effect of Interaction between Treatment and sex (Block) on Body and Gonadal
Weight of African catfish
Parameter Sex
Treatments 1 2 3 4 SEM
Body Weight
Male 100.68 96.60 76.30 74.09 9.16 NS
Female 85.43 81.84 74.30 72.14 9.16 NS
Gonadal Weight
Male 0.30 0.22 0.16 0.12 0.44 *
Female 0.53 0.35 0.23 0.22 0.44*
Table 6 shows that there was significant interaction between the treatments and the
sex of the fish on the gonadal weight. The treatments had more effect on the
female than on the male catfish.
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Conclusion
The results of this study have demonstrated that fish meal can be replaced by soybean meal in
African catfish diets up to 30 - 35% rate without negatively affecting the growth performance.
However, it is advised that soybean should not be used for fish meant for breeding because this
will have negative effects on the reproductive performance of the fish in the long run. Inclusion
of soybean meal up to 20% is recommended considering the cost of fish meal and the
observations that this level of inclusion does not have negative effect on the reproductive
development of the fish brood stock. At levels higher than this the gonadal development will be
significantly reduced.
However, more research is needed to fully elucidate the physiological mechanisms by which
plant protein sources, such as soybean, the anti-nutritional components they contain and the
amino acid profile affect the reproductive development of the fish. It will also be necessary to
provide a complete characterization of the effects of soybean in fish nutrition and physiology so
as to improve the understanding of the potential application and methods of handling and
overcoming the short-cmings of this feed stuff in aquaculture. This will help fish
producers/nutritionists increase the use of soybean in aquacultural diets considering the high cost
of fish meal and other animal materials especially in the Sub-Saharan Africa.
Recommendation:
Based on the results obtained in the present study, it was recommended that:
i. soybean can be included in the diet of growing fish not meant for breeding at up to 30 –
35% provided it is properly treated by heating or other methods to reduce the anti-
nutritional factors to the barest minimum;
ii. to reduce the cost of producing fish feeds, soybean meal can be included in catfish brood
stock diet at up to 20% without any deleterious effect on fish reproductive capacity.
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References: Abe, M., T. Iriki, M. Funaba and S. Onda, (1998) Limiting amino acids for a corn and soybean
meal diet in weaned calves less than three months of age. Journal of Animal Science 76:628-
636.
Adams, N. R. (1995). Organizational and Activational Effects of Phytoestrogens on the
Reproductive Tract of the Ewe Proc. Soc. Exp. Biol. Med. 208, 87–91
ADCP Aquaculture Development and Co-ordination Programme, (1983) Fish feeds and feeding
in developing countries. An interim report on the ADCP feed development programme.
FAO-ADCP/REP/83/18:97 pp, Rome, Italy.
Adewumi, A. A. (2006) The Growth and Gonadal Maturation of the African catfish, Clarias
gariepinus (Burchell) Broodstock Fed Differently Heated Soybean-based diets
http://www.mendeley.com/research/growth-gonadal-maturation-african-catfish-clarias-
gariepinus-burchell-broodstock-fed-differently-heated-soybeanbased-diets/
Ai, Q. and Xie , X. (2005) Effects of Replacement of Fish Meal by Soybean Meal and
Supplementation of Methionine in Fish Meal/Soybean Meal-based Diets on Growth
Performance of the Southern Catfish Silurus meridionalis Journal of the World Aquaculture
Society 36 (4) 498-507 2005 http://fishnutrition.uoguelph.ca/content/effects-
replacement-fish-meal-soybean-meal-and-supplementation-methionine-fish-
mealsoybean-m
Alam, M. S., Watanabe, W.O., Carroll, P.M. & Rezek, T. (2008) Effects of dietary protein and lipid
levels on growth performance and body composition of black sea bass Centropristis striata
(Linnaeus 1758) during grow-out in a pilot-scale marine recirculating system. Aquaculture
Research, in press.
Anyanwu, D.C., E.O. Faturoti and C.C. Kekeocha, (2005) Comparative study of substitution
effect of fish meal with different plant protein on growth of koi carp fingerlings. J. Occup.
Training, 2: 111-117
Anyanwu, E.O. Nwoye and J.I. Offor (2011) Effects of Dietary Levels of Jackbean (Canavalia
ensiformis) Meal on Body Composition of Clarias gariepinus Fingerling Pakistan Journal of
Nutrition 10 (11): 1066-1068, 2011
55
LO
AOAC (Association of Official Analytical Chemist), 1990 Official methods of Analysis, 15th
Ed., Virginia, USA.
Apler, H. N. and Jauncey, K. (1983): The Utilization of Filamentous Green Algae (Cladophora
glomerata) (L, Kutzin) as protein source in pelleted feeds for Saratherodon niloticus Journ.
of Fish Biol. 24, 315-321
Appler, H. N. (1985) Evaluation of Hydrodictyon reticulatum as protein source in feeds of
Oreochromis (Tilapia) niloticus and Tilapia zillii. Journ. of Fish Biol. 27, 327-334.
Bakke-Makellep, A.M., E.O. Kopping, G. Gunnes, M. Sanden, G.I. Hemre, T. Landsverk and A.
Krogdahi 2007. Historical, digestive, metabolic, Hormonal and Immune factors in Atlantic
salmon, Salmo salar L., fed genetically modified soybeans. J. Fish Dis., 30: 265-279.
Bichi, A .H. and M.K. Ahmad (2010) Growth Performance and Nutrient Utilization of African
Catfish (Clarias gariepinus) Fed Varying Dietary Levels of Processed Cassava Leaves
Bayero Journal of Pure and Applied Sciences, 3(1): 118 – 122
Bromage, N. (1995) Broodstock Management and Seed Quality-General Considerations In:
Bromage, N.R, Roberts, R.J. (Editor), Broodstock Management and Egg and Larval Quality
University Press, Cambridge, UK, 1-24, 1995.
Bromage, N.; Jones, J.; Randall, C.; Trush, M.; Davies B.; Springate, J.; Duston, J. and Baker, G.
(1992) Broodstock Management Fecundity egg quality and the time of egg production in
rainbow trout (Oreochromis mykis) Aquaculture 100, 141 – 166.
Brown, P. (2005) Soy-in-Aquaculture Partnership Studies Soybean Meal Use in Fish Feeds
http://www.soyaqua.org/sites/default/files/reports/sbmuseinfishfeed05.pdf
Cai, Y.J. and G.J. Burtle. (1996). Methionine requirement of channel catfish fed soybean meal-
corn-based diets. Journal of Animal Science 74(3):514-521.
Castell, A. G., W. Guenter, and F. A. Igbasan. (1996) Nutritive value of peas for nonruminant
diets. Anim. Feed Sci. Technol. 60:209–227
Cek, S, Bromage, N.R., Randall, C. and Rana, K. (2001). Oogenesis, hepatosomatic indexes, and
sex ratio in Rosy barb (Puntius conchonius). Turk. J. Fish. Aquat. Sci. 1: 133-141.
Cek, S. (2006). Early gonadal development and sex differentiation in rosy barb (Puntius
conchonius). Anim. Biology. 56: 335-350.
56
LO
Cek, S. and Yilmaz E. (2007) Gonad Development and sex ratio of sharptooth catfish (Clarias
gariepinus Burchell, 1822) cultured under laboratory conditions. Turkey Journal of Zoology
31 (2007) 35-46.
Cek, S., Bromage, N.R., Randall, C. and Rana, K. (2001). Oogenesis, hepatosomatic indexes,
and sex ratio in Rosy barb (Puntius conchonius). Turk. J. Fish. Aquat. Sci. 1: 133-141.
Cek, Sehriban and Erdal Yilmaz (2009) The effect of varying dietary energy on gonad
development at first sexual maturity of the Sharptooth catfish (Clarias gariepinus Burchell,
1822) Aquacult Int (2009) 17:553–563
Cho, C. Y., and S. J. Slinger. 1979. Effect of water temperature on energy utilization in rainbow
trout. Pp. 287-291 in Proceedings of the Eighth Symposium on Energy Metabolism in Farm
Animals, S. E. Mont, ed. Cambridge, U.K.: Cambridge University Press.
Chou R.L., B.Y. Her, M.S. Su, G. Hwang, Y.H. Wu, H.Y. Chen (2003) Substituting fish meal
with soybean meal in diets of juvenile cobia Rachycentron canadum Aquaculture 229 (2004)
325–333 http://www.scsagr.com/upimg/2008530133918.pdf
Cisse, A. (1988) Effects of varying protein levels on spawning frequency and growth of
Sarotherodon melanotheron Proceeding of the second on Tilapia Aquaculture Manila,
Philippines pp. 329 333.
Claude L. Hughes, Jr. (1988) Phytochemical Mimicry of Reproductive Hormones and
Modulation of Herbivore Fertility by Phytoestrogens Environmental Health Perspectives Vol.
78, pp. 1 71-1 75, 1988
Clay, D. (1979) Sexual maturity and fecundity of the African catfish (Clarias gariepinus) with an
observation on the spawning behaviour of the Nile catfish (Clarias lazera). Zool. J. Linn. Soc.
65: 351-365.
CRC, (1983) CRC handbook of naturally occurring food toxicants. CRC Press, Boca Raton, FL,
USA.
Cumaranatunga, P. R. T. and Thabrew, H. (1989): Effects of Legume (vigna catiang) substituted
diets on ovarian development of Oreochromis niloticus (L) Proc. Third Inter. Symp.on
Feeding and Nutrition in Fish, Toba Aug. 28-Sept. 1 1989. Japan pp. 333-344.
57
LO
Davies, O.A and Ezenwa, N.C (2011) Groundnut Cake as Alternative Protein Source in the Diet
of Clarias Gariepinus Fry International .Journal of Science and Nature VOL. 1(1): 73-76
De Graaf G. and Janssen, J. (1996): A Handbook on the Artificial Reproduction and Pond
Rearing of the African Catfish Clarias gariepinus in Sub-Saharan Africa FAO, Fisheries
Technical Paper 362 Rome
De Silva, S.S. and Radampola, K., (1989) Effects of dietary protein levels on the reproductive
performance of Oreochromis niloticus L. Proceeding of the second Asian Fisheries Forum:
1989 (Japan) 233 - 2.38
de Wet L. (2007) Use of soya in aqua-feeds http://en.engormix.com/MA-aquaculture/use-of-
soybean.htm
Delahunty, G. and V. L. DeVlaming (1980) Seasonal relationships of ovary weight, liver weight
and fat stores with body weight in the goldfish, Carassius auratus (L.) J. Fish. Biol. 16:5-13.
http://www.ajas.info/Editor/manuscript/upload/15_115.pdf
Delbert M. Gatlin III Use of Soybean Meal in the Diets of Omnivorous Freshwater Fish
http://www.soymeal.org/pdf/omnivorousfwfishtechreview.pdf
Evonic Industries (2012) Animal Nutrition Protein and Amino Acids http://feed-
additives.evonik.com/product/feed-additives/en/about/healthy-nutrition/animal-
nutrition/pages/default.aspx
Eyo, A. A. (1999): The effect of different methods of soybean processing on the growth and food
utilization of African mud catfish Clarias anguillaris (L) fingerlings. Nigerian Journal of
Biotechnology 10(1): 9 – 18.
Eyo, A.A. (1994) The requirements for formulating standard artificial feeds. A paper presented
at the 11th Annual Conference of the Fisheries Society of Nigeria (FISON), 22nd-24th
February, Ikeja Lagos, Nigeria 15 pp.
Fafioye, O.O.; S. O. Fagade, A.A.; Adebisi, Jenyo-Oni, and G.A.K. Omoyinmi (2005) Effects of
Dietary Soybeans (Glycine max (L.) Merr.) on Growth and Body Composition of African
Catfish (Clarias gariepinus, Burchell) Fingerlings Turkish Journal of Fisheries and Aquatic
Sciences 5: 11-15 (2005)
58
LO
FAO (2003) Food and Agriculture Organization of the United Nations Aquaculture rapidly
growing Half of world marine stocks fully exploited - State of World's Fisheries and
Aquaculture 2002 published 20 February 2003, Rome
FAO (2010) Animal Feed Resource Information, Soybean Meal
http://www.fao.org/ag/Aga/agap/frg/afris/Data/736.htm
Faromi A. O. (2002) Effect of partial or complete replacement of fish meal by soybean meal in
catfish diets (unpublished article)
Flammarion, P., Brion, F., Babut, M., Garric, J., Migeon, B., Noury, P., Thybaud, E., Tyler, C.
R. & Pallazzi, X. (2000). Induction of fish vitellogenin and alterations in testicular structure:
preliminary results of estrogenic effects in chub (Leuciscus cephalus). Ecotoxicology 9, 127–
135.
Fontainhas-Fernandes, A.; Monteiro, M.; Figueiredo, E; Gomes, E.; Coimbra, J. and Reis-
Henriques, M. A. (2000): Partial and total replacement of fish meal by plant protein affects
gonadal development and plasma 17β-estradiol levels in female Nile tilapia. Aquaculture
International 8: 299-313, 2000.
Francis G, Makkar HPS, Becker K. (2001) Antinutritional factors present in plant-derived
alternate fish feed ingredients and their effects in fish. Aquaculture 2001; 199: 197–227.
Gaikwad, S.L. ;T.S. Pathan, M.V. Gaikwad, P.M. Davne, D.K. Hiwrale, Y.K. Khillare (2009)
Gonadal development and differentiation of germ cells during larval growth of Cyprinus
carpio Journal of Ecobiotechnology, Vol 1, No 1 (2009)
Gatlin G. M. III (2003) Use of Soybean Meal in the Diets of Omnivorous Freshwater Fish
(unpublished material) Gimeno, S.; Komen, H.; Gerritsen, A. G. M. and Bowmer, T., (1998) Feminisation of young
males of the common carp, Cyprinus carpio, exposed to 4-tert-pentylphenol during sexual
differentiation. Aquatic Toxicology 43, 77–92.
Green Facts (2012) Fisheries Latest data: How much fish is consumed worldwide?
http://www.greenfacts.org/en/fisheries/l-2/06-fish-consumption.htm
59
LO
Gunasekera RM, and Lam TJ (1997) Influence of dietary protein level on ovarian recrudescence
in Nile tilapia, Oreochromis niloticus. Aquaculture 149:57–69. doi:10.1016/S0044-
8486(96)01441-X
Gunasekera, R.M., K.F. Shim and T.J. Lam. (1996)a Effect of dietary protein level on spawning
performance and amino acid composition of eggs of Nile tilapia, Oreochromis niloticus.
Aquaculture 146:121-134.
Gunasekera, R.M., K.F. Shim and T.J. Lam. (1996)b Influence of protein content of broodstock
diets on larval quality and performance in Nile tilapia, Oreochromis niloticus. Aquaculture
146:245-259.
Hardy, R.W., (1995) Current Issues in Salmonid Nurtrition. In: Nutrition and Utilization
Technology in Aquaculture, Lim, C.E. and D.J. Sessa (Eds). AOCS Press, Champaign, IL,
USA, pp: 26 – 35.
Hart Steven and Paul Brown (2005) Soy-in-Aquaculture Partnership Studies Soybean Meal Use
in Fish Feeds Purdue University Department of Forestry and Natural Resources 715 West
State Street West Lafayette, Indiana www.soyaqua.org/.../soy-aquaculture-partnership-studies-
soybean-meal-use-fish-feeds
Hashim R., Ahyaudin A. and N.A.M. Saat, (1992). Improvement of growth and feed conversion
of hybrid catfish (Clarias gariepinus x C. macrocephalus) fry fed with diets supplemented
with live Tubifex. J. Aqua. Trop. 7:239-248
Hernández, M.D., Martínez, F.J., Jover, M. García García, B., (2007) Effects of partial
replacement of fish meal by soybean meal in sharpsnout seabream (Diplodus puntazzo) diet.
Aquaculture 263:159-167
http://en.wikipedia.org/wiki/Fish_meal#Nutrient_composition#Nutrient_composition
http://en.wikipedia.org/wiki/Gonadosomatic_Index
Isicheli F. I. (2008) The Growth Performance Of Heteroclarias In Polyculture With
Oreochromis Niloticus Unpublished material
Izquierdo and Fernandez-Palacios (2010) Nutritional requirements of marine fish larvae and
broodstock http://ressources.ciheam.org/om/pdf/c22/97605925.pdf
60
LO
James R., Muthukrishnan J. and K. Sampath, (1993) Effects of food quality on temporal and
energetics cost of feeding in Cyprinus carpio (Pisces: Cyprinidae). J. Aquat. Trop., 8:47-53
James, R and K. Sampath (2003) Effect of Animal and Plant Protein Diets on Growth and
Fecundity in Ornamental Fish, Betta splendens (Regan). The Israeli Journal of Aquaculture –
Bamidgeh 55(1), 2003, 39-52.
Jauncy, K and B. Ross (1982) A guide to Tilapia feeds & feeding.Institute of Aquaculture Univ.
of Stirling, Scotland.
Jobling, S., Sheahan, D., Osborne, J. A., Matthiessen, P. and Sumpter, J. P. (1996) Inhibition of
testicular growth in rainbow trout (Oncorhynchus mykiss) exposedto estrogenic
alkylphenolic chemicals. Environmental Toxicology and Chemistry 15, 194–202.
Johnson LA, Suleiman TM, Lusas EW (1979). Sesame protein: a review and prospects J. Am.
Oil Chem. Soc., 56: 463-68.
Kaushik, S.J.; Cravedi, J.P.; Lalles, J.P.; Smpter, J.; Fauconneau, B. and Laroche, M. (1995)
Partial or Total Replacement of fish meal by soya bean protein on growth, protein utilization
potential estrogenic or antigenic effects, cholesterolemia and flesh quality in rainbow trout
Oncoorhynchus mykis Aquaculture 133 257 – 274.
Kenan Gullu, Yusuf Guner, Edis Koru, Ersin Tenekecioglu and Hulya Saygi (2008) Artificial
Spawning and Feeding of European Catfish (Siluris glanis L.,) in Turkey Journal of Animal
and Veterinary Advances 7 (10): 1285 – 1291, 2008.
Kinnberg, K., Korsgard, B. & Bjerregaard, P. (2000) Concentration-dependent effects of
nonylphenol on testis structure in adult platyfish Xiphorus maculatus. Marine Environmental
Research 50, 169–173.
Krisna, R. T.; Lars, H. S.; Britt, S. D.; Tone, V.; Grethe, B. T.; Declan, T.; and Ossi, R. (2009)
Different Dietary Levels of Protein to Lipid Ratio Affected Digestive Efficiency, Skeletal
Growth, and Muscle Protein in Rainbow Trout Families Scholarly Research Exchange
Volume 2009 (2009), Article ID: 709529
doi:10.3814/2009/709529 http://syrexe.com/biology/2009/709529.pdf
61
LO
Krogdahl, A., Bakke-McKellep, A.M., 2001. Soybean in salmonid diets: antinutrients,
pathologies, immune responses and possible solutions. Book of Abstract. Aquaculture 2001,
Lake Buena Vista, FL, USA, p. 340.
Krogdahl, A., Bakke-McKellep, A.M., Roed, K., Baeverfjord, G., 2000. Feeding Atlantic salmon
Salmo salar L. soybean products: effects on disease resistance (furunculosis), and lysozyme
and IgM levels in the intestinal mucosa. Aquacult. Nutr. 6, 77–84.
Kwom,, H.C.; Hayashi, S. and Mugiya, Y. (1993) Vitellogenin induction by estradiol-17β in
primary hepatocyte culture in rainbow trout Oncohynchus mykis Comparative Biochemistry
and Physiology 104B 381 – 386.
Liener, I. E. and Kakade, M. L. (1980) Protease inhibitors In: Proceeding of Aquaculture, Feed
Processing and Nutrition Workshop Report, Academic Press Inc., Thailand and Indonesia
pp184 -241.
Liener, I.E., (1994) Implications of antinutritional components in soybean foods. Critical reviews
in food science and nutrition, 34(1):31-67.
Lovell, T.T., (1976) Fish feed formulation and nutrition. In: Commercial fish farmer and
Aquaculture. New York, pp: 42-43.
Maack, G. and H. Segner (2003) Morphological development of the gonads in zebra fish Journal
of Fish Biology (2003) 62, 895–906 http://groups.exeter.ac.uk/eabrg/pdf/maack20032.pdf
Maclatchy, D. L. and Van Der Kraak, G. J.(1995) Toxicol Appl Pharmacol 1995
Oct;134(2):305-12. http://www.ncbi.nlm.nih.gov/pubmed/7570607
Madsen, T. G. (2008) Amino acid content in fishmeal shows great variation.
www.aquafeed.com/docs/fiaap2008/Madsen.doc
Mahboob & Sheri (2011) http://www.ajas.info/Editor/manuscript/upload/15_115.pdf
Metcalfe, T. L.,Metcalfe, C. D., Kiparissis, Y., Niimi, A. J., Foran, C.M. & Benson,W. H. (2000)
Gonadal development and endocrine responses in Japanese medaka (Oryzias latipes) exposed
to o,p0-DDT in water or through maternal transfer. Environmental Toxicology and
Chemistry 19, 1893–1900.
62
LO
Michael, Todd Byerly, B.S. (2003) Temperature Effects on Gonadal and Somatic Growth in
Channel Cat Fish, M SC Thesis in Fishery Science submitted to Graduate Faculty of Texas
Technical University. December, 2003.
Miles, R.D. and F.A. Chapman (2009): The Benefits of Fish Meal in Aquaculture Diets.
http://edi.ifas.ufl.edu/pdffiles/FA12200.pdf
Mitchell, J. H.; E. Cawood; D. Kinniburgh; A. Provan; A. R. Collins and D. S. Irvine (2001)
Effect of a Phytoestrogen Food Supplement on Reproductive Health in Normal Males:
Clinical Science (2001) 100, (613–618)
MSU (2006) Mississippi State University Extension Service: Catfish Nutrition: Nutrient
Requirements. Mississippi State University Research and Extension System Bulletin March
2006.
Nakamura, M.; Kobavashi, T.; Chang, N. and Nagahama, Y. (1998) Gonadal Sex Differentiation
in Teleost Fish. The Journal of Experimental Zoology 281: 362-372.
Nakamura, M; T. Kobavashi; N. Chang; and Y Nagahama (1998) Gonadal .sex differentiation in
teleost fish The Journal of Experimental Zoology 281: Pp.362-372.
Narasinga RMS (1985). Nutritional aspect of oil seeds in oil seed productions – constraints and
opportunities: (Srivastava, H.C.; Bhaskaran, S.; Vatsya, B.; and Menon K.K.G. Eds.) p. 625-
34. New Delhi: Oxford and IBH.
New, M.B. (1987) Feed and feeding of fish and shrimp. A manual on the preparation and
presentation of compound feeds for shrimp and fish in aquaculture. FAO ADCP/REP/87/26.
Rome, 275 pp
Norberg, B. and Haux, C. (1985) Induction, Isolation and Characterization of the lipid content of
plasma vitellogenin for two salmon species: rainbow trout (Salmo gairdnen) and sea trout
(Salmo trutta) Comparative Biochemistry and Physiology 81B 869 – 876.
NRC, 1993. Nutrient requirements of fish (Nutrient requirement of domestic animals). National
Research Council, National Academy Press, Washington DC., pp: 114.
Nyirenda, J; M. Mwabumba; E. Kaunda and J. Sales (2000) Effect of Substituting Animal
Protein Sources with Soybean Meal in Diets of Oreochromis karongae (Trewavas 1941)
Naga, The ICLARM Quarterly (Vol. 23, No. 4) October-December 2000
63
LO
Ofor C. O. (2005) Gametogenesis in Pubescent Heterobranchus longifilis (Teleostei:
Clariidae)(Val 1840) Attaining Puberty Under Artificial Conditions; and Under Rainy and
Dry Season Conditions in Tropical Earthen Ponds Asian Fisheries Science 18(2005): 139 –
151 Asian Fisheries Society, Manila, Philippines.
Oresegun, A. and Alegbeleye, W.O. (2001) Serum and tissue thiocynate concentration in tilapia
(Oreochromis niloticus) fed cassava peels based diets supplemented with D,L-methionine.
In: AA. Eyo (Ed.), Fish Nutrition and Fish Feed. Published by FISON, Nigeria: 107-115.
Oso, J. A., E. O. Idowu and O. F. Agoi (2011) Growth response of Clarias gariepinus fingerlings
fed Parkia biglobosa diet as protein source Indian Journal of Science and Technology Vol. 4
No. 2 (Feb 2011)
Patiňo, R.; Davis, K. B.; Schoore, K. E.; Uguz, C.; Strussmann C. A.; Parker, N. C.; Simco, B.
A. and Goudie, C. (1993) Sex Differentiation of channel catfish gonads: Normal
development and effects of temperature, Journal of Experimental Zoology 276. pp. 209 –
218.
Pellisero, C.; LeMenn, F. and Kaushik, S. (1991) Estrogenic Effect of Dietary Soybean Meal on
Vitellogenesis in Culture Siberian Sturgeon (Acipenser baeri) General Comparative
Endocrinology 83, 443 – 457
Peng Li, Kangsen Mai, Jesse Trushenski & Guoyao Wu (2008) New developments in fish amino
acid nutrition: towards functional and environmentally oriented aquafeeds Springer-Verlag
2008
Pereira J. O. B.; Reis-Henriques, M. A.; Sanchez, J.L. and Costa, J. M. (1998) Effects of protein
source on the reproductive performance of female rainbow trout Onchorhynchus mychis
Aquaculture Research 29 751 – 760.
Piccolo G, Gerardo Centoducati, Stefania Marono, Fulvia Bovera, Raffaella Tudisco, Antonino
Nizza (2011) Effectsof the partial substitution of fish meal by soy bean meal with or without
mannanoligosaccharide and fructooligosaccharide on the growth and feed utilization of
sharpsnout seabream, Diplodus puntazzo (Cetti, 1777): preliminary results
http://www.aspajournal.it/index.php/ijas/article/view/ijas.2011.e37/html
64
LO
Rama Rao, P. B.; H. W. Norton and B. Connor Johnson (1983) The Amino Acid Composition
and Nutritive Value of Proteins J. Nutrition 82: 64
http://jn.nutrition.org/content/82/1/88.full.pdf
Reidel, A., (2007) Níveis de energia e proteina na alimentação do jundiá (Rhamdia quelen)
criados em tanques-rede. Campus Jaboticabal, Universidade Estadual Paulista, Centro de
Aqüicultura da UNESP. 87 p. (Dissertação de doutorado).
Richter, C.J.J., Viveen, W.J.A.R., Eding, E.H., Sukkel, M., Rothuis, A.J., van Hoof, M.F.P.M.,
van der Berg, F.G.J. and van Oordt, P.G.W.J., (1987) The significance of photoperiodicity,
water temperature and an inherent rhythm for production of viable eggs by the African
catfish, Clarias gariepinus kept in subtropical ponds in Israel and under Israeli and Dutch
hatchery conditions. Aquaculture, 63, 169-185.
Robin, J. and Kaushik, S. (1995) Nutrition and broodstock performance. Proceedings of the
second fish Nutrition Workshop Asian EEC Aquaculture Development Coordination
Programme, Singapore, pp. 415- 424.
Roy Dahle; Geir L. T.; Ørjan Karlsen; Olav S. K. and Birgitta Norberga (2003) Gonadal
development and associated changes in liver size and sexual steroids during the reproductive
cycle of captive male and female Atlantic cod (Gadus morhua L.) Comparative Biochemistry
and Physiology Part A 136 (2003) 641–653
Rungruangsak-Torrissen, K. and J. E. Fosseidengen (2007) Effect of artificial feeding on
digestive efficiency, growth and qualities of muscle and oocyte of maturing Atlantic
mackerel (Scomber scombrus L.) Journal of Food Biochemistry, vol. 31, no. 6, pp. 726–747,
2007.
Santiago, C.B.; Aldaba, M.B. and Laron, M.A. (1983) Effects of varying dietary crude protein
levels on spawning frequency and growth of Sarotherodon niloticus breeders. Fisheries
Research Journal of the Philippines 8(2) 9-18.
Santti, R.; Makela S.; Strauss, L.; Korkman, J. and Kostian, M. L. (1998) Toxicol Ind. Health
1998 Jan-Apr; 14(1-2):223-37. http://www.ncbi.nlm.nih.gov/pubmed/9460177
Schulz, R.W., Renes I. B., Zandbergen M. A., van Dijk W., Peute J and Goos H. J. T. (1995)
Pubertal Development of Male African Catfish (Clarias gariapinus) Pituitary Ultrastructure
65
LO
and Responsiveness to Gonadotrophin-Releasing Hormone Biology of Reproduction 53, 940
– 950 (1995)
Schulz, R.W., Van der Corput, L., Janssen-Dommerholt, J. and Goos, H.J.Th. (1994) Sexual
steroids during puberty in male African catfish (Clarias gariepinus): serum levels and
gonadotropin stimulated testicular secretion in vitro. J. Com. Physiol B. 164: 195-205.
Schuulz, R. W.; Renes, I. B.; ZandbergenM. A.; van Dijk, W.; Peute, J. and Goos, H.J.T. (1995):
Pubertal Development of Male African Catfish (Clarias gariepinus). Pituitary Utrastructure
and Responsiveness to Gonadotropin- Releasing Hormone. Biol. Of Reprod. 53, 940-950.
Shim K.F., Landesman L. and T.J. Lam, (1989). Effect of dietary protein on growth, ovarian
development and fecundity in the dwarf gourami Colisa lalia (Hamilton). J. Aquat. Trop.,
4:111-123
Shimeno, S., Hasokawa, H., Kumon, M., Masumoto, T. and Ukawa, M., (1992) Inclusion of
defatted soybean meal in diet for fingerling yellow tail. Nippon Suisan Gakk. 58:1319-1325.
Siddiqui AQ, Al-hafedh YS and Ali SA (1998) Effect of dietary protein level on the reproductive
performance of Nile tilapia, Oreochromis niloticus. Aquacult Res 29:349–358
Sogbesan A. O. and A. A. Ugwumba (2008) Nutritional Values of Some Non-Conventional
Animal Protein Feedstuffs Used as Fishmeal Supplement in Aquaculture Practices in Nigeria
Turk. J. Fish. Aquat. Sci. 8: 159-164 (2008)
Sogbesan, O.A, Adebisi, A.A., Falaye, B.A., Okaeme, B.N.and Madu, C.T. (2006). Some aspects
of dietary protein deficiency diseases in semi-intensive cultured fishes. A review. J. of Arid
Zone Fish, 2(1): 80-89.
Specker, J.L. and Sullivan C.V. (1994) Vitellogenesis in fishes: status and perspectives: In:
Perspectives in comparative Endocrinology (K. G. Davey, R.E. Peter and S.S. Tobe eds.)
National Research Council of Canada, Ottawa, pp 304 – 315.
Strussman C.A., T. Saito, F. Takashima and K. Toda (1996) Sex differentiation and hormonal
feminization in pejerrey Odontesthes bonariensis. Aquaculture 139:345
Sullivan, K. B. (2008) Replacement of Fish Meal by Alternative Protein Sources in Diets for
Juvenile Black Sea Bass A Masters of Science Thesis Submitted to the University of North
Carolina Wilmington Department of Biology and Marine Biology
66
LO
Sunde, J., G. L. Taranger, and K. Rungruangsak-Torrissen, (2001) “Digestive protease activities
and free amino acids in white muscle as indicators for feed conversion efficiency and growth
rate in Atlantic salmon (Salmo salar L.),” Fish Physiology and Biochemistry, vol. 25, no. 4,
pp. 335–345, 2001.
Tacon A.G.J., Stafford E.A. and C.A. Edwards, (1983) A preliminary investigation of the
nutritive value of three terrestrial lumbricid worms for rainbow trout. Aquaculture, 35:187-
199.
Tacon, A.G.J.; Jauncey, K.; Falaye, A.; Pantha, M.; Mac Goan, I. and Stafford, E.A. (1983) The
use of meat and bone meal, hydrolysed feather meal and soybean meal in practical fry and
fingerling diets for Oreochromis niloticus. Proceedings of the International Symposium on
Tilapia, Nazareth, Israel.
Tuladhar, B. (2003) Comparative Study of Fish Yields with Plant Protein Sources and Fish Meal
Our Nature (2003) 1: 26-29
Tyler C.R., E.M. Santos, F. Prat, (2000) Unscrambling the egg—cellular, biochemical, molecular
and endocrine advances in oogenesis. In: Norberg, B., Kjesbu, O.S., Taranger, G.L.,
Andersson, E., Stefansson, S.O. (Eds.), Reproductive Physiology of Fish. John Grieg Forlag
AyS, Bergen, pp. 273–280
Utiah, A., (2002) Gonadal maturation of Asian catfish in captivity with stagnant water pond
system. Individual Paper for Science Philosophy Graduate Program Institut Pertanian Bogor
November 2002 (PPs 702)
Van den Hurk, R., W.J.A.R. Viveen and P.G.W.J Van Oordt (1984) The natural gonadal cycle in
the African catfish Clarias gariepinus: A basis for applied studies on its reproduction in
farms. Israeli Journal of Zoology. 33:129-147
Viola, S., and Arieli, Y., (1983) Nutritional studies with Tilapia (Saratherodon) Replacement of
fish meal by soybean meal in feeds for intensive tilapia culture, Bamidgeh, 35: 9 – 10
Wallace RA and K Selman (1981): Cellular and dynamic aspects of oocyte growth in teleosts
Am. Zool. 21: 325-345.
Wee, K.L. and Wang, S.S. (1987) Nutritive value of Leucaena leaf meal in pelleted feed for Nile
Tilapia. Aquaculture, 62: 97 – 108.
67
LO
West, M. C.; Anderson, L.; McClure, N. and Lewis, S. E. Dietary oestrogens and male fertility
potential Hum Fertil (Camb) 2005 Sep;8(3):197-207
Wikipedia, the free encyclopedia (2012) Phytoestrogens
http://en.wikipedia.org/wiki/Phytoestrogens
Yildirim, Y.B. and Turan, F. (2010) Effects of Exogenous Enzyme Supplementation in diets on
the growth and feed utilization in African Catfish, Clarias gariepinus Journal of Animal and
Veterinary Advances 9(2): 327-331.2010
Yoshikuni, M. and Nagahama, Y. (1991) Endocrine regulation of gametogenesis in fish. Bulletin
of Institute of Zoology. Academic Sinica Monograph 16, 139 – 172.
Zeitler M.H., M. Kirchgessner and F.J. Schwarz (1984) Effects of different proteins and Energy
supplies on carcass composition of carp (Cyprinus carpio, L.) Aquaculture, 36: 37-48
Zhao E. and Mu Q. (2010) Phytoestrogen Biological Actions on Mammalian Reproductive
System and Cancer Growth Sci. Pharm. 2011 March 79(1): 1- 20 Published online 2010
December 31. Doi:10.3797/scipharm.1007-15
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APPENDIX I
Figure 10: The Internal structures of a Female Catfish showing the Reproductive Organ (the two ovaries with
developing eggs and the tubular tract down to the opening at the anal region) RO – right ovary; LO – left
ovary; TT – tubular tract; and GP – genital papilla
RO
LO GP TT
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APPENDIX II
Figure 11: Reproductive Organ of a Male Catfish showing: TT- a pair of serrated Testes, EP - the Epididymides, VD - the Vasa Deferetia and GP - the genital papilla (external Copulatory Apparatus
VD
EP TT GP
TT EP
VD GP
GP EP TT
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