الفهرس 
INTRODUCTION & AIM OF THE WORKOnly 14 pages are availabe for public view
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Abstract
The aquaculture industry in Egypt has been developed and well sustained within the last few decades. It is evident that the aquaculture industry; in spite of its successes, still suffers from serious limitations, such as lack of fry, expensive feed (fishmeal) and low resistance to diseases.
In fish diets, fish meal is considered as an important ingredient due to its high protein quality, balanced amino acids profile, excellent palatability and digestibility as well as minimum anti-nutritional factors. On the other hand, due to the static production and growing demand, fish meal price has been multiplied. Therefore, many studies targeted at reducing or substituting fishmeal in diets utilizing food additives or alternative protein sources with valuable, feasible, renewable and biologically sustainable materials, which can improve the biological and physiological functions of fish.
Several studies postulated that soybean meal (SBM) is regarded as the most convenient alternative protein source to replace fishmeal. However, contradictory results have been noticed upon replacement of fishmeal by dietary SBM in aquaculture, because the levels of SBM that could be utilized without opposing with the growth rate are sensitive to culture systems and species. Moreover, phytic acid in SBM had adverse effects on the feed conversion ratio, fish growth and feed palatability.
On the other hand, corn gluten meal (GM); the product of the wet milling of corn, is another commercially attainable plant protein-based product. It contains about 60% crude protein, and has a sufficient essential amino acids profile except for lysine and arginine.
In aquaculture, numerous types of dietary supplements such as probiotics, and prebiotics are being used to enhance the disease resistance, immune responses, antibiotic replacement as well as the growth performance. Chitosan (CS) is considered as one of these smart, safe natural cationic biopolymers. It has remarkable properties including non-toxicity, biodegrability, biocompatibility, improved solubility, in addition to, immunorestorative properties. Chitosan is obtained from the deacetylation of chitin that is considered as an important component found in the exoskeletons of aquatic crustaceans. In Egypt, during early 1980’s, the crustacean crayfish Procambarus clarkii had been accidentally introduced to the River Nile via a private fish farm. The crayfish industry has increased during the last 5 years in Egypt, and the drastic problem for investors in this field is how they can get rid of solid wastes of the crayfish that reaches to hundreds tons, as the inedible part of the animal reaches to about 60-70 % of the total body weight. These wastes are considered as excellent and feasible sources of chitin. The present study addressed a solution for the problem of the crayfish solid wastes by using it in the production of chitosan.
In aquaculture, chitosan nanoparticles (CSNP) have attracted attention due to their distinctive properties and interesting applications. CSNP have effective antibacterial activity, enhancing growth performance and survival of fish.
The objectives of the present study were:
1. To get rid of the solid wastes of P. clarkii industry and reuse them in a sustainable form, such as: producing
chitosan and its derivatives that can support applications of the green chemistry.
2. To allocate the most optimum condition for CS and CSNP preparation to get the best physico-chemical characteristics.
3. To evaluate and compare the potential of CS and CSNP to enhance the properties of gluten meal and SBM (plant protein-based diets) for total or partial replacement of FM-based diet.
4. To study the immunostimulatory properties and antioxidant capacity of dietary CS and CSNP on Oreochromis niloticus and its growth performance.
5. To evaluate the impact of CS and CSNP on the histological structure of the ovaries, testes, gills and liver of O. niloticus.
Exoskeleton wastes of P. clarkii were obtained from a commercial crayfish processor company (Jiang’s Fish Processor Co., Ltd., Cairo, Egypt). In the Lab, chitin was extracted from the exoskeletons through three main steps, namely: demineralization, deproteinization, and decoloration. Chitosan was produced by deacetylation of the extracted chitin that was treated with 50 % NaOH at 100 °C. Chitosan nanoparticles (CSNP) were synthesized by the ionotropic gelation method. For the determination of the optimum condition for the prepared chitosan nanoparticles, three different ratios of chitosan to the polyanion TPP were used, 1:1, 3:1, and 1:3. A novel nanoparticle system composed of low molecular weight chitosan was successfully prepared by the ionotropic gelation technique under an aqueous-based condition. The physicochemical characteristics of chitosan and chitosan
nanoparticles were elucidated by Fourier-transform IR spectroscopy, nuclear magnetic resonance spectroscopy, elemental analysis, scanning electron microscopy, transmission electron microscopy, and zeta potential. The best physicochemical characteristics were obtained by adding chitosan to TPP in a ratio of 1:1. The small particle size and narrow range distribution of the obtained chitosan nanoparticles may increase their chance for easy and efficient manipulation for several applications in various fields.
Six isonitrogenous (30% crude protein) and isocaloric (4500 kcal/kg) diets were formulated according to the requirements for O. niloticus. The experiment was designed using a 2 x 3 factorial design; with two different based diets; fishmeal-based diet (FM-based diet) and gluten meal-based diet (GM-based diet); with three forms of chitosan [zero-chitosan as a control, chitosan (CS) and chitosan nanoparticles (CSNP), to evaluate the effect of dietary supplementation of CS and/or CSNP on O. niloticus performance. The experimental diets were prepared by blending the ingredients into a homogeneous mixture; then the mixture was passed through a pelletizer, dried overnight at room temperature and stored in plastic bags at 4 °C till used.
Fish were grown out under recirculating aquaculture system (RAS) with a flow rate of 0.4 Lmin-1. A total of 18 plastic tanks (55- L capacity) were provided a side flow (SF) opening and connected to a standpipe (SP) provided with a valve that regulates the quantity of water flowing out through the side drain. The water passed from the side over flow to a sump then to a mechanical filter (MF) to get rid of
the food remains and feces. Water was drained from the mechanical filter to biological filter 1 (BF1) then by gravity to biological filter 2 (BF2) through which the ammonia was disposed. Water in the biological filter 2 passed through a pump to a collecting tank (CT) and filtered water was drained back to the RAS tanks. Aeration was continuously provided to the tanks using an air pump and porous stones to maintain the oxygen supply above 5 mg L-1.
A total of 270 O. niloticus fingerlings were randomly distributed into 6 different treatments with a triplicate of 15 fish each. Fish were acclimatized to the experimental conditions for one week prior to the feeding trial. During the experiment, the fish were fed till apparent satiation, twice daily (10 a.m. and 4 p.m.). Fish weight was measured every 15 days. The experiment lasted for 82 days and the number of dead fish was recorded daily.
Growth performance parameters (Body weight gain, Specific growth rate, Survival rate) and Feed utilization ones (Feed intake, Feed conversion ratio, Protein efficiency ratio, Protein productive value, Relative length of gut) of O. niloticus fed different experimental diets were determined.
The crude protein, crude lipid contents, ash contents, fatty acids and amino acids of the fishmeal-basal diet and gluten meal-basal diet as well as fish carcass were determined. Heavy metals concentrations of the basal diets, CS, CSNP and fish carcass were also analyzed.
At the end of the experiment, fish were starved for 24 hours, then removed from the tanks and immersed into anesthetic solution for 10 minutes. Blood samples were collected by caudal severance. Total red blood cells (RBCs), Hemoglobin (Hb), Erythrocyte indices, and total
white blood cells (WBCs) have been measured. Phagocytosis, Antioxidant activity, total cholesterol levels, and the biochemical parameters of O. niloticus were determined. Moreover, small pieces of gills, liver, ovaries and testes were dissected out for histological studies.
Results indicated that, the addition of CS and CSNP to GM-based and FM-based diets has promoted and increased the innate immunity, health status, antioxidant activity and biochemical parameters of all experimental fish. The growth performance parameters and feed utilization of fish fed GM-based diets were improved by the addition of CSNP. On the contrary, the addition of CS and CSNP affected negatively the growth and utilization of the FM-based diets.
On the other hand, total substitution of fishmeal- based diet by gluten-meal-based diet affected the histological structure of both the gills and liver. However, chitosan nanoparticles (CSNP) fortification to GM-based diets improved the architecture of the gills and liver. Concerning the histological studies of ovaries and testes, neither the protein source nor the chitosan forms affected the reproductive status or stages of O. niloticus.
Note worthy of attention, is that chitosan nanoparticles increased the omega-3 level in all experimental fish that will make it healthy for human consumption. So, the present study recommends addition of supplementary CSNP to the plant-based protein diet of the Nile tilapia for better growth performance, health and disease resistance, as well as augmentation of nutritional value for fish, as well as human being.