EFFECTS OF SUPPLIMENTATION OF FISH MEAL WITH SOYBEAN MEAL ON THE GROWTH AND GONAD DEVELOPMENT OF

Transcription

1 i EFFECTS OF SUPPLIMENTATION OF FISH MEAL WITH SOYBEAN MEAL ON THE GROWTH AND GONAD DEVEPMENT 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

2 i TITTLE PAGE EFFECTS OF SUPPLEMENTATION OF FISH MEAL WITH SOYBEAN MEAL ON THE GROWTH AND GONAD DEVEPMENT OF THE AFRICAN CATFISH (CLARIAS GARIAPINUS) By Ugwoke Cyprian Chukwuma PG M Sc 07/43383 MARCH 2013

3 ii 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 Prof. Dr. S. O. C. Ugwu.. Head of Department Dr. A. E. Onyimonyi.. External Supervisor

4 iii 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.

5 iv 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

6 v LIST OF FIGURES Figure 1 Transverse section of gonads from female fish fed the different diet at week Figure 2 Transverse section of gonads of female catfish fed the different diets at week Figure 3 Transverse section of gonads of female catfish fed the different diets at week 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 Figure 8 Transverse section of male gonad of catfish fed the different diets at week Figure 9 Transverse section of gonads from female fish fed the different diets at week 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) 69

7 vi 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

8 vii Title page TABLE OF CONTENT i Certification ii Acknowledgement iii List of figures iv List of Tables v Table of content vi ABSTACT vii Chapter 1: Introduction Background of study Statement of the Problem Objectives of the study Justification 5 Chapter 2: Literature Review Protein Quality and Fish Reproduction Patterns of Gonad Development in Fishes Nutrition and Gonadal Development in Fish 11 Chapter Catfish Nutrition and Nutrient Requirements Energy requirement of catfish Proteins and Amino Acid Requirements of Catfish Vitamins and mineral requirements of catfish Fish Meal as a component of Fish Diet Combined Effects of Fish Meal with other Protein Sources in fish Diet Soybean Meal as Source of Protein for Fish Diet 22 Materials and Methods 3.1. Location and Duration the work study Experimental fish The Experimental Diets Experimental Design Data collection Sampling of fish for Dissection Histological Sectioning and Analysis 28

9 viii Fixing and embedding of tissue samples (gonads) Histological Sectioning Staining Statistical analysis 30 Chapter 4 Results and Discussion 4.1. Main Effect of Diet on Weight Gain and Final Body Weight of African Catfish Main Effect of Diets on the Gonad Weights and Development of African Catfish The Development pattern of the female gonad (the ovary) of the fish fed the different diets Effects of Sex (Block) on the Body and Gonadal Weight of the African Catfish Correlation between the Age, body weight and gonad weight of the Male African catfish 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

10 ix 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 and grams for males and females respectively. The mean final body weight values for the males of treatments B, C, and D were 96.60, 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.

11 1 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

12 2 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 ( 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 ( 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 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

13 3 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 aquacultural 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).

14 4 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

15 5 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.

16 6 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).

17 7 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

18 8 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 (E 2 ). 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

19 9 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 (E 2 ) (Fontainhas-Fernandes et al., 2000). Estradiol (E 2 ) 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 E 2 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

20 10 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 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).

21 11 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 postfertilization. These outgrowths later fused to form an ovarian cavity. In males, development of signs of testicular differentiation appeared by day 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)

22 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 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

23 13 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 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 kcal/g, kcal/g and 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%.

24 14 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 % 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

25 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.

26 16 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 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 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 monogastric 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

27 17 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.

28 18 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 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.

29 19 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 antinutritional 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

30 20 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 antinutritional 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

31 21 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

32 22 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 mineralbinding substances (CRC, 1983; Liener, 1994). Fish fed diets containing levels of soybean lecitins experienced reduction in growth and insulin production, while some species showed

33 23 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

34 24 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

35 25 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 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.

36 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 Defatted soy meal Full-fat soy meal Maize Wheat offal Animal fat Binder Bone meal Salt Vita./Min. Mix D-L Methionine Lysine Total Proximate composition of the experimental diets Crude protein Crude fibre Crude fat Ash NFE

37 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 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: γ іјk = µ + τ і + βj + (τβ)ij +Σ іјk Where γ іјk = any individual observation µ = the population mean τ і = effects of the i th level of soybean meal on the observed data βj = effect of the j th sex

38 28 (τβ)ij= the effect of interaction between the i th and j th sex on the individual observation Σ іјk = random error due to observation Data collection 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 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

39 29 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: 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 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) 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:

40 30 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.

41 31 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 Parameters Treatments (Diets) Sex SEM Weight gain(g) Male NS Female NS Final body weight (g) Male NS NS = not significant (P > 0.05) Female NS 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 g and 85.43g for males and females respectively. The males in other treatments had final body weight 96.60, 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

42 32 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.

43 33 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

44 34 differed significantly from treatment B. Treatment B differed significantly and D in their gonad weights. from treatments C Table 4.2: Main Effect of Treatment on the Body and Gonad Weights of the African Catfish Treatment Parameter Mean Body Weight (g) Mean Gonadal Weight (g) Gonado-Somatic Index (GSI) SEM Male NS Female NS Male 0.30 a 0.22 a 0.16 b 0.12 b 0.11 ** Female 0.53 a 0.35 b 0.23 c 0.22 c 0.18* Male 0.30 a 0.26 a 0.21 b 0.16 c 0.02** Female 0.60 a 0.43 a 0.31 b 0.30 b 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

45 35 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 (E 2 ), when other factors (food nutrients, health condition) are favourable any thing that affects the activities of estrogen, 17β-estradiol (E 2 ) 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);

46 36 interfering with the growth factor receptor; inhibiting the G protein-coupled receptor in ERdeficient 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

47 37 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 nonsteroidal 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 ( 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

48 38 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 18 th week

49 39 a B c d 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 12 th and 14 th weeks. There were no structural differences in the ovaries during the 12 th and 14 th 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.

50 40 From 18 th 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.

51 41 a b c d 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.

52 42 a b a c d 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.

53 43 a b c d 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

54 44 a b c d 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.

55 45 a b c d 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

56 46 a b c d 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

57 47 a b c d 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

58 48 a b c d 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

59 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 correlationcoefficients 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.

60 50 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 ** 0.77 ** Body weight ** Gonad Weight -- ** Significant at 1% (p < 0.01)

61 51 Table 5: Correlation coefficients between age, body weight and gonadal weight of the Female African Catfish Age Body weight Gonad Weight Age Body weight ** 0.78 ** ** 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.

62 52 Table 6: Effect of Interaction between Treatment and sex (Block) on Body and Gonadal Weight of African catfish Parameter Sex Treatments SEM Body Weight Male NS Female NS Gonadal Weight Male * Female * 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.

63 53 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 antinutritional 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.

64 54 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: Adams, N. R. (1995). Organizational and Activational Effects of Phytoestrogens on the Reproductive Tract of the Ewe Proc. Soc. Exp. Biol. Med. 208, 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 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) 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: 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): , 2011

65 55 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, 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, Bakke-Makellep, A.M., E.O. Kopping, G. Gunnes, M. Sanden, G.I. Hemre, T. Landsverk and A. Krogdahi Historical, digestive, metabolic, Hormonal and Immune factors in Atlantic salmon, Salmo salar L., fed genetically modified soybeans. J. Fish Dis., 30: 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): 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, 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, Brown, P. (2005) Soy-in-Aquaculture Partnership Studies Soybean Meal Use in Fish Feeds Cai, Y.J. and G.J. Burtle. (1996). Methionine requirement of channel catfish fed soybean mealcorn-based diets. Journal of Animal Science 74(3): Castell, A. G., W. Guenter, and F. A. Igbasan. (1996) Nutritive value of peas for nonruminant diets. Anim. Feed Sci. Technol. 60: 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: Cek, S. (2006). Early gonadal development and sex differentiation in rosy barb (Puntius conchonius). Anim. Biology. 56:

66 56 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) 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: 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: Cho, C. Y., and S. J. Slinger Effect of water temperature on energy utilization in rainbow trout. Pp 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) 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 Claude L. Hughes, Jr. (1988) Phytochemical Mimicry of Reproductive Hormones and Modulation of Herbivore Fertility by Phytoestrogens Environmental Health Perspectives Vol. 78, pp , 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: 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 Japan pp

67 57 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): 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) de Wet L. (2007) Use of soya in aqua-feeds 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: Delbert M. Gatlin III Use of Soybean Meal in the Diets of Omnivorous Freshwater Fish Evonic Industries (2012) Animal Nutrition Protein and Amino Acids 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): 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: (2005)

68 58 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 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, 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: , 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: 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, Green Facts (2012) Fisheries Latest data: How much fish is consumed worldwide?

69 59 Gunasekera RM, and Lam TJ (1997) Influence of dietary protein level on ovarian recrudescence in Nile tilapia, Oreochromis niloticus. Aquaculture 149: doi: /s (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: 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: 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: 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 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: 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: 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

70 60 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, 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, Johnson LA, Suleiman TM, Lusas EW (1979). Sesame protein: a review and prospects J. Am. Oil Chem. Soc., 56: 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 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): , Kinnberg, K., Korsgard, B. & Bjerregaard, P. (2000) Concentration-dependent effects of nonylphenol on testis structure in adult platyfish Xiphorus maculatus. Marine Environmental Research 50, 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: doi: /2009/

71 61 Krogdahl, A., Bakke-McKellep, A.M., Soybean in salmonid diets: antinutrients, pathologies, immune responses and possible solutions. Book of Abstract. Aquaculture 2001, Lake Buena Vista, FL, USA, p Krogdahl, A., Bakke-McKellep, A.M., Roed, K., Baeverfjord, G., 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, 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 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 pp Liener, I.E., (1994) Implications of antinutritional components in soybean foods. Critical reviews in food science and nutrition, 34(1): Lovell, T.T., (1976) Fish feed formulation and nutrition. In: Commercial fish farmer and Aquaculture. New York, pp: Maack, G. and H. Segner (2003) Morphological development of the gonads in zebra fish Journal of Fish Biology (2003) 62, Maclatchy, D. L. and Van Der Kraak, G. J.(1995) Toxicol Appl Pharmacol 1995 Oct;134(2): Madsen, T. G. (2008) Amino acid content in fishmeal shows great variation. Mahboob & Sheri (2011) 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,

72 62 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, Miles, R.D. and F.A. Chapman (2009): The Benefits of Fish Meal in Aquaculture Diets. 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, ( ) MSU (2006) Mississippi State University Extension Service: Catfish Nutrition: Nutrient Requirements. Mississippi State University Research and Extension System Bulletin March Nakamura, M.; Kobavashi, T.; Chang, N. and Nagahama, Y. (1998) Gonadal Sex Differentiation in Teleost Fish. The Journal of Experimental Zoology 281: Nakamura, M; T. Kobavashi; N. Chang; and Y Nagahama (1998) Gonadal.sex differentiation in teleost fish The Journal of Experimental Zoology 281: Pp 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 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 NRC, 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

73 63 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): 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: 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 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, 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 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

74 64 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 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, 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 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) 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 , 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) Santti, R.; Makela S.; Strauss, L.; Korkman, J. and Kostian, M. L. (1998) Toxicol Ind. Health 1998 Jan-Apr; 14(1-2): 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

75 65 and Responsiveness to Gonadotrophin-Releasing Hormone Biology of Reproduction 53, (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: 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, 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: 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: 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: 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: (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): 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 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

76 66 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 , 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: 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: 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 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: 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: Wee, K.L. and Wang, S.S. (1987) Nutritive value of Leucaena leaf meal in pelleted feed for Nile Tilapia. Aquaculture, 62:

77 67 West, M. C.; Anderson, L.; McClure, N. and Lewis, S. E. Dietary oestrogens and male fertility potential Hum Fertil (Camb) 2005 Sep;8(3): Wikipedia, the free encyclopedia (2012) 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): Yoshikuni, M. and Nagahama, Y. (1991) Endocrine regulation of gametogenesis in fish. Bulletin of Institute of Zoology. Academic Sinica Monograph 16, 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: Zhao E. and Mu Q. (2010) Phytoestrogen Biological Actions on Mammalian Reproductive System and Cancer Growth Sci. Pharm March 79(1): 1-20 Published online 2010 December 31. Doi: /scipharm

78 68 APPENDIX I RO GP TT 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; left ovary; TT tubular tract; and GP genital papilla

79 69 APPENDIX II GP VD EP TT GP VD EP TT 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 GP EP TT