UTILIZATION OF FISH SILAGE FERMENTED WITH ...

UTILIZATION OF FISH SILAGE FERMENTED WITH DATE FRUIT RESIDUES FOR FEEDING THE COMMON CARP Cyprinus carpio L. AND ITS PHYSIOLOGICAL AND HISTOLOGICAL EFFECTS

A THESIS SUBMITTED TO THE COUNCIL OF THE COLLEGE OF AGRICULTURE, UNIVERSITY OF BASRAH IN PARTIAL FULFILLEMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN FISHERIES AND MARINE RESOURCES (FISH CULTURE AND NUTRITION)

BY

SALAH MAHDI NAJIM AL-KANAANI B. Sc. Agricultural Sciences, Fisheries and Marine Resources, 1984 M. Sc. Fisheries and Marine Resources, Fish Ecology and Biology, 1989

SUPERVISION BY PROF. DR. SAJED S. AL-NOOR AND ASSIST. PROF. DR. BASIM M. JASIM

SEPTEMBER 2014

‫ﺻﺪﻕ ﺍ‪ ‬ﺍﻟﻌﻠﻲ ﺍﻟﻌﻈﻴﻢ‬ ‫۞ ﺳﻮﺭﺓ ﺍﻟﻨﺴﺎﺀ ‪۞ 113‬‬

DEDICATION

This thesis is dedicated to the memory of my beloved father who enriches my life with his wisdom and spontaneity and to my family for gracious care and sincere partnership in all the successes of my life.

ACKNOWLEDGMENTS

First of all, I must thank The Almighty Allah for the merciful guidance, graces and inspiration through all my life. I hereby express profound gratitude to Prof. Dr. Sajed S. Al-Noor and Assist. Prof. Dr. Basim M. Jasim for their supervision, support and kind assistance. I would like to thank the Dean and the staff of the College of Agriculture and the Department of Fisheries and Marine resources for acceptance in PhD program and support to complete this study. I am also very grateful to Prof. Dr. Alaa A.Kh. Sawad the chairman of the examining committee and the members for dignified participation and efforts. My thanks to all my professors and colleagues for their unlimited assistance especially Dr. Jalal M. Al-Noor, Assist. Prof. Dr. Adel AlDubaikel, Assist. Prof. Dr. Amina A. Hashim, Miss. Furat K. Jassim, Dr. Huda F. Saad and Miss. Balquess K. Hassan and Mrs. Raja A.A. AlMudhaffar. I also extend my appreciation to Assist. Prof. Dr. Abdulmotalib J. AlRudainy, Assist. Prof. Dr. Loay M. Abbas, Assist. Prof. Dr. Dhia F. AlFekaike, Assist. Prof. Dr. Majdy F. Majeed, Dr. Rashad A. Omran and Assist. Prof. Mr. Majed A.A. Banai for assistance with analytical and histological work. Finally, my true thanks for all the persons who have helped and supported me in my academic life from the first steps. These favors will always be remembered.

SUMMARY

SUMMARY This study is carried out to evaluate the utilization of marine fish by-catch silage fermented with date fruit residues as carbohydrate substrate in feeds for fry and fingerlings of the common carp Cyprinus carpio L. The study also aims to assess some physiological and histological effects of fish biosilage inclusion into fish feeds. Marine fish by-catch sample consist of 23 marine fish species belonging to 18 families. A series of experiments are conducted to determine the optimum conditions for fermented fish silage production. Evaluation is based on several biochemical analyses. Date fruit residues DFR are widely available agricultural waste which is added successfully at 10% to fermentation medium. Domestic vinegar was served as inoculum when added at 20% of the total fermentation medium. Citric acid at 2% provides adequate acidity to start ensiling process. The produced silage is further improved by adding an antioxidant and antimycotic agent to conserve lipid quality and decrease histamine content. Fish meal is prepared by the standard method from the same fish sample for comparison purposes. Amino acid profile of fish silage is closer to the raw fish than fish meal especially the essential amino acids and is considered acceptable for fish feeding according to the documented criteria. Fatty acid profile of fish silage oil has been very rich in unsaturated fatty acids especially PUFA which consisted 47% of fatty acids in fish silage comparing to 37% in fish meal and this makes it a valuable source for marine fish oil in fish nutrition. To evaluate fish biosilage as a fish meal alternative in carp fry feeds, it is included to replace 0, 25, 50 and 75% of fish meal protein in isonitrogenous (42% CP) and isocaloric (4600 Kcal/kg) feeds. Physical quality properties of the prepared feeds are tested to determine the effect of silage inclusion. Bulk

I

SUMMARY

density, pellet durability and water stability have been improved by increasing silage addition while settling velocity and floatability show an opposite trend in comparison with the fish meal feed. The results of feeding and growth performance study which is performed with carp fry (average weight 0.68 gm) indicate very close resemblance between the four different feeds as assessed by weight gain, specific growth rate SGR, thermal growth coefficient TGC, feed conversion ratio FCR, protein efficiency ratio PER and survival rate. Another feeding trial is carried out with carp fingerlings (average weight 5.81 gm) fed isonitrogenous (35% CP) and isocaloric (4400 Kcal/kg) with similar fish silage inclusion rates as for fry feed. Silage inclusion rate of 50% is very close in feeding and growth performance parameters to fish meal basal feed. Higher silage addition has significantly lowered many parameters except for feed intake which increase with silage addition rate. Water quality parameters in both feeding experiments with fish fry and fingerlings has not been influenced adversely by silage inclusion in feeds. Apparent digestibility coefficients of major feed nutrients were improved with silage addition especially protein ADC which reach 89.88% in 75% silage feed in comparison with 82.55% in fish meal feed. Proximate composition of fish fingerlings fed on different experimental feeds showed no significant differences. However, fatty acid profile has been improved with increasing silage inclusion especially ω3-PUFA which elevated significantly from 7.98 to 10.18% in fish meal and 75% fish silage feed groups, respectively. Lipid and glycogen contents in fish liver and muscle do not differ significantly with silage addition in feed. General haematological parameters show no significant differences between the four feed groups despite silage inclusion rate so as plasma proteins. However, plasma lipid profile is improved with silage inclusion

II

SUMMARY

because of fish oil content in silage especially the total cholesterol levels which decrease by 20% in fish meal and 30% in 75% fish silage feed groups. The histological study includes the measurement of some tissue and cellular components of fish intestine and liver. The results of this study show no significant differences between fish meal basal feed and silage containing feed groups as to the examined components. This result is confirmed by examining the gross histology of intestine and liver sections which show no abnormal signs. Glycogen detection in hepatic tissue by PAS stain reveals rather similar pattern of distribution among liver section of different feed groups. However, lipid detection by both osmium tetraoxide and Sudan Black B stains demonstrate gradual improvement in hepatic lipid stores with silage addition in feed although carp liver has proved not to be a primary storage site for lipids. No signs of disturbance or abnormality of lipid metabolism are detected in the examined hepatic tissue sections. The study concludes that fish biosilage can be easily prepared by using local raw materials such as by-catch fish, DFR and vinegar. It is also a good partial alternative for fish meal in diets for fry and fingerlings of the common carp for its suitable nutritional properties. However, more improvements are needed before its exploitation in practical feeds especially some important physical quality parameters of feed. The study recommends some aspects to further investigation of fish silage and its application in aquafeeds.

III

LIST OF CONTENTS

LIST OF CONTENTS

1 2 2.1 2.2 2.3 2.4 2.4.1 2.4.2 2.5 2.5.1 2.5.2 2.5.3 2.6 2.7 2.8 2.9 2.10 2.11 2.12 3 3.1 3.1.1 3.1.2 3.1.3 3.2 3.3

Summary List of tables List of figures List of Abbreviations Introduction Review of Literature Overview of world fisheries and aquaculture Global aquafeed needs and supply Fish meal replacement in aquaculture Applications of fish silage Fish silage as protein source in aquafeeds Other potential uses of fish silage Types of fish silage Chemical fish silage Biological fish silage Enzymatic fish silage Requisites for fermentation of biosilage Storage quality of fish silage Date fruit residues DFR as a novel ingredient in fish feeds

I VII IX X 1 4 4 6 9 12 12 13 14 15 15 16 17 19 20

Nutritional quality of fish biosilage Effects of fish silage on pelleted feed quality Nutritional requirement of the common carp Cyprinus carpio L. Biochemical and histopathological markers of fish health

23 27 29

Materials and Methods Raw materials Fish Date fruit residues DFR Domestic vinegar Chemical composition Determination of pH and Titratable acidity

39 39 39 39 40 40 41

32

IV

LIST OF CONTENTS

3.4 3.5 3.6 3.7 3.7.1 3.7.2 3.7.3 3.8 3.8.1 3.8.2 3.8.3 3.8.4 3.8.5 3.8.6 3.8.7 3.9 3.9.1 3.9.2 3.10 4 4.1 4.1.1 4.1.2 4.1.3 4.1.4 4.2 4.2.1 4.2.2 4.2.2.1 4.2.2.2 4.2.2.3 4.2.2.4 4.2.2.5 4.3

Determination of total sugars, fibers and TDS Microbiology Production of fish meal FM Preparation of fish biosilage Preliminary screening experiment Determination of optimum conditions for biosilage production Chemical characterization of fish meal and fish silage Biological evaluation of fish silage in fish diets Preparation of experimental feeds Physical quality of experimental feeds Biological Evaluation of fry feed Biological evaluation of fingerling feed Determination of feed intake Measurement of apparent digestibility Analysis of Lipid and glycogen contents Biochemical and histological evaluation General Hematology and Biochemical Parameters Histological study Statistical analysis Results and Discussion Raw materials Fish Date fruit residues DFR Domestic vinegar Microbiology of raw materials Production of fish biosilage Preliminary experiment Quality of biosilage from upscaled ensiling treatments Proximate composition The pH and titratable acidity TVB-N TBA and FFA Histamine Formulation of fish feed using fish biosilage as fish meal alternative

41 41 42 43 43 43 44 46 47 48 50 51 52 52 54 54 54 57 59 60 60 60 62 63 64 67 67 68 68 72 75 76 78 80

V

LIST OF CONTENTS

4.3.1 4.3.2 4.3.3 4.3.4 4.3.4.1 4.3.4.2 4.3.5 4.3.6 4.3.7 4.3.8 4.3.9 4.4 4.5 4.5.1 4.5.2 4.5.3 5 5.1 5.2 6 ‫أ‬

Quality of whole fish, fish meal and fish biosilage Amino acid composition of whole fish, fish meal and fish silage Fatty acid profiles of whole fish, fish meal and fish silage Preparation and examination of fry feeds Physical quality of formulated feeds Feeding and growth performance of common carp C. carpio fry Preparation and examination of fingerling feeds Apparent digestibility coefficients ADC of different nutrients in common carp fingerling feeds Proximate composition of common carp fingerlings fed different fish biosilage ratios Fatty acid profile of common carp fingerlings fed different fish biosilage ratios Lipid and glycogen contents in experimental fish General Hematology and Plasma Biochemistry Histological study General histology of intestine and liver Histological measurements of fish Intestine and Liver Components Histochemical detection of glycogen and lipids in fish hepatic tissue. Conclusions and Recommendations Conclusions Recommendations References ‫اﻟﺨﻼﺻﺔاﻟﻌﺮﺑﻴﺔ‬

80 82 86 89 90 97 103 111 113 115 120 123 128 128 141 146 159 159 160 162

VI

LIST OF TABLES

LIST OF TABLES No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14

15 16 17 18

Title

Page

Classification and weight percentage of by- catch fish sample Chemical composition of whole fish and DFR used for ensiling Characteristics of domestic date vinegar Microbiological quality of raw materials Results of pH determination for different treatments in the preliminary experiment for biosilage Characteristics of upscaled successful ensiling treatments from preliminary experiment Proximate composition and chemical quality of whole fish, fish meal and fish silage The specific and total essential and non-essential amino acid in whole fish, fish meal and fish silage used Fatty acid profile (%) of whole fish, fish meal and fish silage used in fish feeds. Fry feed formulation and proximate composition using fish silage as a partial fish meal alternative Physical quality of experimental feeds with different substitution ratios of fish meal by fish silage. Feeding and growth performance of common carp fry feed different experimental feeds Water quality parameters of rearing water for common carp fry during feeding experiment Fingerling feed formulation and proximate composition using fish silage as a partial fish meal alternative Feeding and growth performance of common carp fingerlings feed different experimental feeds Water quality parameters of rearing water for common carp fingerlings during feeding experiment Apparent digestibility coefficients of nutrients for common carp fingerlings fed different fish silage ratios Proximate composition of common carp fingerlings before and after feeding on experimental feeds

61 63 64 65 67 69 81 84 87 89 91 98 103 104

106 110 112 114

VII

LIST OF TABLES

19 20 21 22 23

24

Fatty acid profile of fish before and after feeding Lipid and glycogen contents in muscle and liver of common carp fingerlings before and after feeding General hematology of common carp fingerlings before and after feeding Blood plasma biochemistry of common carp fingerlings before and after feeding Measurements of some intestine tissue and cellular properties of common carp fingerlings before and after feeding Measurements of hepatocytes in livers of common carp fingerlings before and after feeding

116 121 124 126 142

144

VIII

LIST OF FIGURES

LIST OF FIGURES No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Title

Page

Time related variation in dry matter content during ensiling of the three studied biosilage treatments. The time course of pH variation during ensiling of the three studied biosilage treatments. Feed intake by common carp fingerlings fed on different experimental feeds. (A). Cross section in fish intestine (initial group) basic structure of villi. (B). Villus cellular components. (A). Cross section in fish intestine (FM2 group) basic structure of villi. (B). Villus cellular components. (A). Cross section in fish intestine (FSa2 group) basic structure of villi. (B). Villus cellular components. (A). Cross section in fish intestine (FSb2 group) basic structure of villi. (B). Villus cellular components. (A). Cross section in fish intestine (FSc2 group) basic structure of villi. (B). Villus cellular components. Fish liver histology. (A). Initial group. (B). FM2 feed group. Fish liver histology. (A). FSa2 feed group. (B). FSb2 feed group. Fish liver histology. (A). FSc2 feed group. (B). Enlarged hepatopancreas of the same section. Glycogen detection in hepatic tissue. (A) Initial group. (B). FM2 feed group. Glycogen detection in hepatic tissue. (A) FSa2 feed group. (B). FSb2 feed group. Glycogen detection in hepatic tissue. FSc2 feed group,. Lipid detection in hepatic tissue. Initial fish group. (A) Osmium tetraoxide stain (B) Sudan black B stain. Lipid detection in hepatic tissue. FM2 group. (A) Osmium tetraoxide stain (B) Sudan black B stain. Lipid detection in hepatic tissue. (A) FSa2 group. (B) FSb2 group. Osmium tetraoxide stain. Lipid detection in hepatic tissue. FSc2 group. (A) Osmium tetraoxide stain (B) Sudan black B stain.

70 73 107 131 132 133 134 135 138 139 140 147 148 149 154 155 156 157

IX

LIST OF ABBREVIATIONS

LIST OF ABBREVIATIONS AAB ADC AS BS BSE CP DFR DHA DM DPX EAA EPA ES FA FBS FCR FFA FM FO FPH Hb Hct HDL HE LAB LDL MPE MUFA MWG NEAA NFE NLFA PA PAS PCA PDI P/E ratio

Acetic Acid Bacteria Apparent Digestibility Coefficient Acid Silage Biological Silage Bovine Spongiform Encephalopathy Crude Protein Date Fruit Residues Docosahexaenoic Acid Dry Matter Distyrene Plasticizer in Xylene Essential Amino Acid Eicosapentaenoic Acid Enzymatic Silage Fatty Acids Fish Biosilage Feed Conversion Ratio Free Fatty Acid Fish Meal Fish Oil Fish Protein Hydrolysate Hemoglobin Hematocrit High Density Lipoprotein Hematoxylin-Eosin Lactic Acid Bacteria Low Density Lipoprotein Moringa Protein Extract Monounsaturated Fatty Acid Mean Weight Gain Non- Essential Amino Acid Nitrogen Free Extract Neutral Lipid Fatty Acids Plasma Albumin Periodic Acid-Schiff reagent Perchloric Acid Pellet Durability Index Protein/Energy ratio

X

LIST OF ABBREVIATIONS

PER PLFA PUFA RBC SBB SFA SGR SV TAG TBA TC TG TGC TP TPC TPG TSS TVB-N VLDL VO WBC

Protein Efficiency Ratio Polar Lipid Fatty Acids Polyunsaturated Fatty Acid Red Blood Corpuscle Sudan Black B Saturated Fatty Acid Specific Growth Rate Settling Velocity Triacylglycerol Thiobarbituric Acid Total Cholesterol Triglycerides Thermal Growth Coefficient Total Protein Total Plate Count Total Plasma Globulin Total Soluble Solids Total Volatile Basis-Nitrogen Very Low Density Lipoprotein Vegetable Oil White Blood Corpuscle

XI

CHAPTER ONE: INTRODUCTION

CHAPTER ONE: INTRODUCTION The current world population of 7.2 billion is projected to increase by 1 billion over the next 12 years and reach 9.6 billion by 2050 according to a United Nations (2013) paper, which points out that growth will be mainly in the developing countries, with more than half in Africa. World population growth could spur food shortage because the demands that will be placed on farming and agriculture during that time will become too extreme, given that we are currently struggling to meet food demands. In fact, it is predicted that by 2050 global agriculture production needs to increase by about 60-110% to provide food security (FAO, IFAD and WFP, 2013). The world’s waterways and oceans cover about two thirds of the surface of the planet and they are the earth’s most underutilized natural resource when it comes to food production. Fisheries and aquaculture could be vital participants in resolving world food crisis especially for high quality animal protein. Fisheries and aquaculture are important sources for food, labor and income for people along the world’s seashores and waterways (Smith et al., 2010), and they influence the livelihoods for more than one billion people. Both industries exploit renewable natural resources with a substantial potential for environmental degradation if the industries’ production practices are not sustainable, a feature that is not uncommon (Pauly et al., 2003). Although aquaculture continues to be the fastest growing animal foodproducing sector and to outpace population growth (FAO, 2014), it faces many challenges over the next decade, notably, combating diseases and epizootics, broodstock improvement and domestication, development of

1


appropriate feeds and feeding mechanisms, hatchery and grow-out technology, as well as water-quality management (World Bank, 2013). Nutrition and feeding will play an essential role in the sustained development of aquaculture. Therefore, it is imperative that fertilizers and feed resources continue to be produced and refined. Sustained development of aquaculture, however, must take into account and ensure that the needs of competing users are met, and that environmental integrity is protected. Therefore, sustainable aquaculture management should address allocation of inputs based on local circumstances, and balance maximizing profitability with social and environmental costs (Hasan and Halwart, 2009). Recycling of agricultural byproducts and wastes to recover valuable nutrients represents an attractive modern strategy for waste management. Fisheries by-catch and food processing byproducts are used efficiently to formulate animal feeds including aquafeeds. This could provide sustainable solutions for disposal and pollution problems in addition to the economical profits (Polprasert, 2007). The increased prices and fluctuated availability of fish meal, the primary animal protein ingredient in aquafeeds, stimulated the efforts to find effective and sustainable alternatives. Many animal and plant components have been experimented and gained different degrees of success (Rana et al., 2009). Fish silage is defined as a liquid product produced from the whole fish or parts of it, to which acids, enzymes or lactic-acid-producing bacteria are added, with the liquefaction of the mass provoked by the action of enzymes from the fish which degrade fish proteins into smaller soluble units. The acid helps to speed up their activity while preventing bacterial spoilage

2


(Arruda et al., 2009; Vazquez et al., 2011). Fish biosilage, commonly fermented with local carbohydrate substrates, was among the most successful fish meal alternatives in aquafeeds. According to various researches, it offers many advantages over other fish meal alternatives (Arruda et al., 2007; Arruda et al., 2009; Ramasubburayan et al., 2013). Preparation of fish silage does not need any special skills or equipment. It can be done by using any available quantity of by-catch, trash fish or even fish wastes with cheap organic acids and a carbohydrate source. This makes it especially valuable when the infrastructure and feed fish production from marine fisheries do not allow establishing of fish meal processing plants (Wassef et al., 2003), as it is the case in Iraq. Other encouraging factors are the availability of large quantities of carbohydrate byproducts like date fruit residues, beet pulp and sugarcane molasses which could be used for preparation of fish silage (Soltan and El-Laithy, 2008). Therefore, replacement of fish meal with cheap, sustainable and simply prepared alternatives could contribute significantly to improve aquaculture economics while exploiting otherwise neglected organic wastes (Nguyen et al., 2009). With the fast development of aquaculture sector in Iraq during the last few years, a necessity appears for exploring new feed ingredients from local resources. Therefore, this study is designed to prepare fish silage from marine by-catch fish with date fruit residues and using it as fish meal alternative in the common carp Cyprinus carpio fry and fingerling feeds. In addition, some hematological and histological features of fish were examined to determine the suitability of this alternative within the applied replacement rates.

3

CHAPTER TWO: REVIEW OF LITERATURE


2.1. Overview of World Fisheries and Aquaculture Capture fisheries and aquaculture supplied the world with about 158 million tonnes of fish in 2012 (with a total value of US$237 billion), of which about 136.2 million tonnes were utilized as food for people, and preliminary data for 2013 indicate increased production of 162 million tonnes, of which 140 million tonnes was destined as food. Asian countries supplied more than 53% of this global fish production. This makes an important contribution to the global food security (FAO, 2014). With the continuously increasing world population, the demand on aquatic food is expected to rise steadily. At the same time, world fish supplies from the capture fisheries sector will fail to meet the increasing global demand for seafood due to generally stagnant production from world capture fisheries. Most of the main fishing zones have reached their maximum potential (Briones and Arnulfo, 2014). On the other hand, the aquaculture sector is expected to make a considerable contribution to compensate for the increasing demand on seafood. Aquaculture is expanding more rapidly compared to all the other animal food-production sectors, from a production of less than I million tonnes per year in the early 1950s, to a reported level of 62.7 million tonnes in 2011 with an annual growth rate of approximately 8.8% during the last three decades (FAO, 2013). Aquaculture production uses freshwater, brackish water and full-strength marine water as culture media. Data available at FAO (FAO, 2014) show that, in terms of quantity, the percentage of production from freshwater rose

4


from less than 50 % before the 1980s to 62.9 % in 2012, with the share of marine aquaculture production declining from more than 40 percent to about 37 %. In 2012, freshwater aquaculture was the source of 58.1% of global production by value. Brackish water aquaculture yielded only 7.9 % of world production in terms of quantity but accounted for 12.8 % of total value because of the relatively high-valued marine shrimps cultured in brackish-water ponds. Marine water aquaculture accounted for about 29.2 % of world aquaculture production by value. The average annual growth rate for freshwater aquaculture production from 2000 to 2010 was 7.2 percent, compared with 4.4 percent for marine aquaculture production. Freshwater fish farming has been a relatively easy entry point for practicing aquaculture in developing countries, particularly for small-scale producers (FAO, 2013). Information about Iraq fish production is very scarce after 2003. The FAO fishery country profile about Iraq (FAO, 2004) presented relatively outdated statistics. It stated that annual marine fish production has declined from around 12 000 - 13 000 t per annum in the 1980s to an estimated 5 000t in 2002 with the principal species being shad (Tenuolosa spp), pomphret (Pampus spp) and mullet (Liza spp). The mean production from inland fisheries for 1981-1997 was 18 800 t/year, compared to an estimated 8 000t in 2001. The total production from aquaculture in 2001 has been estimated at 2 000t, produced from 1900 farms with a combined area of 7 500 ha. This compares with a mean annual production for 1986-1997 of 4 000 t. In 1998, production was reported to have increased to about 7500 t. The main species cultured is the common carp and to a lesser extent grass Ctenopharyngodon idella and silver carp Hypophthalmichthys molitrix while Barbus spp. is also

5


cultured in small quantities. There is hatchery production of common carp (FAO, 2004). However, a new report from Arab Federation of Fish Producers (AFFP) issued in 2013 gave new information claimed to be supplied by official bodies. As indicated, Iraq total fish production has increased from 28.6 thousand metric tonnes in 1997 to 59 thousand metric tonnes in 2011. Production from capture fisheries increased from 28.6 to 41.7 thousand metric tonnes from 2002 - 2011. Aquaculture production in Iraq increased dramatically from just 0.9 to 17.3 thousand metric tonnes from 2002-2011. However, Iraq is still importing more than 20 thousand tonnes of fishery products annually because the local fishery production covers about 74% of the market demand with per capita consumption of 2.3 kg/year (AFFP, 2013) in comparison with 18.6 kg/year for world annual per capita fish consumption in 2010 which is projected to exceed 20 kg/year in 2022 (OECD-FAO, 2013).

2.2. Global Aquafeed Needs and Supply Feeds represent the major contributor in the recurrent costs of aquaculture, often ranging from 30% to 60%, depending on the intensity of the operation. Aquafeeds are generally used for feeding omnivorous fishes (e.g. tilapia, common carp, and milkfish), carnivorous fishes (e.g. salmon, trout, eel, seabass, seabream and tuna) and crustacean species (marine and brackish-water shrimps, freshwater prawns, crabs and lobsters) (FAO, 2012). According to FAO estimates, in 2008, about 31.7 million tonnes (46.1 % of the total global aquaculture production including aquatic plants) of fish and crustaceans were feed-dependent, either as farm-made aquafeeds or as

6


industrially manufactured compound aquafeeds. In 2008, feed aquaculture contributed to 81.2 %of the global farmed fish and crustacean production of 38.8 million tones and 60% of the global farmed aquatic animal production (FAO, 2013). Globally, 708 million tonnes of industrial compound animal feeds were produced in 2008, of which 29.2 million tonnes were aquafeeds (4.1 percent of all animal feeds). By volume, industrial compound aquafeeds used by major species and species groups are estimated to have been as follows in 2008: fed carps (9.1 million tonnes, 31.3 percent of the total), marine shrimps (17.3 percent), tilapias (13.5 percent), catfishes (10.1 percent), marine fishes (8.3 percent), salmons (7.0 percent), freshwater crustaceans (4.5 percent), trouts (3.0 percent), milkfish (2.0 percent), eels (1.4 percent), and miscellaneous freshwater fishes (1.6 percent) (Tacon et al., 2013). While there is no comprehensive information available on the global production of farm-made aquafeeds, the estimate is that it has been between 18.7 million and 30.7 million tonnes in 2006. Farm-made aquafeeds play an important role in the production of low-value freshwater fish species. More than 97 percent of carp feeds used by Indian farmers are farm-made aquafeeds (7.5 million tonnes in 2006/07), and they are the mainstay of feed inputs for low-value freshwater fishes in many other developing countries (FAO, 2012). Feed ingredients used for the production of aquafeeds are broadly categorized into three types depending upon their origin: animal, plant and microbial nutrient sources. Fish meal and fish oil derived from wildharvested whole fish and shellfish including by-catch currently constitute the major aquatic protein and lipid sources available for animal feed (Garcia,

7


2013). World reduction fisheries (marine capture fishery products converted to fish meal) were 18.2 million tonnes in 1976. This total rose progressively to 30.2 million tonnes in 1994, but then declined steadily to 17.9 million tonnes in 2009. As a result, fish meal and fish-oil production exhibited similar trends. Global fish meal production increased from 5.00 million tonnes in 1976 to 7.48 million tonnes in 1994 and then decreased steadily thereafter to 5.74 million tonnes in 2009. Similarly, global fish-oil production rose gradually from 1.02 million tonnes in 1976 to 1.50 million tonnes in 1994 (with the exception of production peaks of 1.67 million and 1.64 million tones recorded in 1986 and 1989, respectively), but then fell back steadily to 1.07 million tonnes in 2009. Hence, the analysis of the data for 15 years period (1994–2009) indicates that global fish meal and fish-oil production from marine capture fisheries have been decreasing at annual average rates of 1.7 and 2.6 percent, respectively. As a result, prices of fish meal at the same period raised by more than 267% from 393- 1050 US Dollars (Jackson and Shepherd, 2012). However, these prices raised more than double to about 2190 US dollars in 2013 (FAO Globefish, 2013). The amount of captured fish destined for non-food uses increased from 20.6 million tonnes in 1976 to 34.2 million tonnes in 1994 (a proportionate increase from 31.5 to 37.1 % of the total catch). Since 1995, this amount has been decreasing both in absolute terms and as a proportion of the total catch. In 1995, 31.3 million tonnes of global fish and shellfish landings were destined for non-food uses (33.9 percent of total catch), and, out of this total, 27.2 million tonnes (29.5 percent of total catch) were reduced into fish meal and fish oil. In 2009, the corresponding figure was 22.8 million tones (25.7 percent of total). Out of this total, 17.9 million tonnes (20.2 percent of total catch) were reduced into fish meal and fish oil. The amount of captured fish

8


destined for nonfood uses will probably decrease further in the near future (Ababouch, 2012). In recent years, increasing volumes of fish meal and fish oil have originated from fisheries by-products (capture fisheries and aquaculture). An estimated 6 million tonnes of trimmings and rejects from food fish are currently used for fish meal and fish oil production (Jackson and Shepherd, 2012). The International Fish meal and Fish Oil Organization estimates that about 25 percent of the fish meal production (1.23 million tonnes in 2008) comes from fisheries by-products. This amount will grow as its processing becomes increasingly viable. Accurate information on the proportion of byproduct fish meal and fish oil produced from aquaculture processing waste is not available, but it is probable that a significant volume of farmed fish wastes is contributed (OECD-FAO, 2013).

2.3. Fish Meal Replacement in Aquaculture

Fish meal is generally added to animal diets to increase feed efficiency and growth through better feed palatability; it enhances nutrient uptake, digestion, and absorption. Fish meal is valuable because of the quality of its protein; the amino acids that make up the protein are present in just the right balance for animal nutrition. The balanced amino acid composition of fish meal complements other animal and vegetable nutrients in the agricultural feed to provide synergistic effects that promote fast animal growth. The availability of the essential amino acids, phospholipids, and fatty acids (Docosahexaenoic acid and Eicosapentaenoic acid) are high in fish meal and promote optimum development, growth, and reproduction (Atanasoff, 2014).

9


Fish meal has high biological value as a feedstuff, as it has a high level of digestible essential amino acids, such as lysine, Methionine and Leucine, which are often deficient in plant feed ingredients, the typical base for most animal feeds (Lall and Anderson, 2005). Furthermore, fish meal has low fiber content and is also a valuable source of vitamins B1, B2, B6 and B12 in addition to calcium, phosphorous, magnesium, potassium, and trace elements including zinc, iodine, iron, copper, manganese, cobalt, selenium, and fluorine. The lack of nutritional inhibitors in fish meal also makes this meal more attractive than plant proteins for use in aquafeeds. Aquafeeds typically have a higher percentage of fish meal than feeds for other animal species (Ayoola, 2010). The level of fish meal in the feed formulation varies depending on whether the fish species is a carnivore or omnivore because of the variations in their biochemical utilization of nutrients. In a typical commercial feed for common carp, 30-35 % is protein ingredient (Mazurkiewicz, 2009), while this level is about 40-50 % in a typical salmon diet (Espe et al., 2012). The aquaculture feed industry consumes a considerable amount of the global fish meal production. About 46 % of the total annual fish meal produced is used to formulate aquafeeds. This figure is expected to rise because of the public attention that has been shifted to the consumption of the seafood products (FAO, 2012). However, the fluctuating supplies and increased prices of fish meal during the last two decades have encouraged researchers to find appropriate alternatives for fish meal in aquafeeds (Slawski, 2011). Because of their low price and their relatively consistent nutrient composition and supply, plant proteins, such as oil seed cakes, are often economically and nutritionally valuable sources of protein. However, potential problems associated with insufficient levels of indispensable amino acids (particularly Lysine and

10


Methionine), anti-nutritional factors and poor palatability are the main concerns for feed formulators. Among plant protein sources, soybean products are the most suitable sources to replace fish meal in aquatic feed because of its protein levels as well as amino acid profile that match the animal’s requirements except for the low Methionine level (Ayoola, 2010, NRC, 2011). However, Jovanovic et al. (2009) indicated that with modern processing technologies, properly processed plant ingredients containing high protein content with high digestibility of crude protein and low antinutritional components became potential protein sources for the replacement of fish meal in aquafeeds like extrusion which inactivate or destroy heatsensitive anti-nutritional factors commonly found in soybean meal and gelatinizing starch granules. Animal protein sources contain considerably high protein contents such as meat and bone meal, blood meal, hydrolyzed feather meal, fish waste meal shrimp silage and fish silage. Despite their good amino acid profile, meat and bone meal was banned in feed as a result of mad cow disease (Bovine Spongiform Encephalopathy, BSE) outbreaks (De Vos and Heres, 2009). Najim (2012) replaced fish meal partially by different ratios of fish waste meal in feeds for hybrid red tilapia Oreochromis mossambicus x O. niloticus fingerlings. Depending on growth performance and biochemical composition analysis, he concluded that up to 50% of fish meal could be replaced by fish waste meal without adverse effects on these parameters. Nogueira et al., (2012) incorporated only 10 % of blood meal and hydrolyzed feather meal in diets for gilthead seabream Sparus aurata indicating their inferior properties of imbalanced amino acid profile and low digestibility. Plascencia-Jatomea et al. (2002) investigated the feasibility of shrimp head silage as fish meal alternative in Nile tilapia Orechromis

11


niloticus feed. They concluded that shrimp head silage could be added to fish feeds with replacement ratios not exceeding 15% because of low digestibility due to the existence of considerable amounts of non-digestible chitin and mineral salts in shrimp shell. In contrast, Arruda et al. (2007) demonstrated that fish silage resembles fish meal more closely in nutrient content because it is produced from the same raw material. In addition, simple and cheap production and environmental considerations give it obvious superiority over other fish meal alternatives (Ramasubburayan, et al., 2013).

2.4. Applications of Fish Silage 2.4.1. Fish Silage as Protein Source in Aquafeeds

As stated by Arruda et al. (2007), silage production dates back to the 1920's with the use of sulphuric acid/hydrochloric acid to preserve green fodder. The same method was adopted in the 1930's to preserve fish waste for animal feed. Acid fish silage was produced commercially for 30 years in Denmark, where the annual production over the last years reached 60,000 tonnes. In Norway, fish silage is produced commercially from fish viscera and other wastes of fishing industry reaching about 140,000 tonnes/year, mainly from salmon aquaculture by- products (Rustad, 2003). Ramasubburayan et al. (2013) demonstrated that silage production acquires increased importance compared to fish meal with the following advantages: the process is virtually independent from the scale, the technology is simple and the investment is little. Due to the similarity of the protein source with the raw material and low cost, especially when

12


compared to fish meal, silage has a good potential use in aquaculture (Vidotti et al., 2003).

2.4.2. Other Potential Uses of Fish Silage From its very beginning, fish silage was used as protein ingredients in feeds intended for many animal groups. Species of livestock, poultry and even laboratory animals like rats were fed on diets containing different kinds and contents of fish silage (Hertrampf and Piedad-Pascual, 2003). The vast majority of studies on these animal species reported good results about feeding, growth efficiency and productivity of silage fed animals (Rahmi et al., 2008; Al-Marzooqi et al., 2010; Thuy et al., 2011). Ensiling process results in isolation of fish silage mixture into different separated layers. These are oil layer on the top, water and water soluble matters in the middle and finally the hydrolyzed protein layer on the bottom of ensiling container (Goosen et al., 2014). This was exploited to extract good quality fish oil from fish silage for different uses in human food, animal diet, pharmaceutical and industrial applications (Bower et al., 2009; Jayasinghe and Hawboldt, 2012). The hydrolyzed protein part of fish silage was also extracted and used as crude peptone preparation comparable to the standard preparations in microbiological media. Other potential uses of fish silage were to isolate active peptides and different enzymes for various applications (Aneiros and Garateix, 2004; Hsu, 2010; Vissesangua and Benjakul, 2012). Recently, some studies have examined the reutilization of biodegraded fisheries-waste products as liquid fertilizer (Kim, 2011). Waste fish biological silage has been found to contain compounds capable of promoting plant growth. Fish waste silage is known to be free of toxic or carcinogenic

13


materials, unlike other types of municipal and industrial effluents (Dao and Kim, 2011; Maynard and Lorenz, 2011). One of the most interesting modern applications of fish silages is the production of biogas and biodiesel (Mbatia, 2011). Kafle et al. (2013) ensiled fish industry wastes for biogas production at laboratory scale and evaluated the biochemical methane potential (BMP) and kinetics. Fish waste was found to be a potential substrate for biogas production with methane potential of fish waste silages in the range of 441–482 ml/g. Yahyaee et al. (2013) separated fish oil from fish wastes by the aid of an ensiling process. Fish waste silage produced about 11% of the extracted fish oil relative to the initial weight of fermented fish wastes. Biodiesel fuel was then produced from the extracted fish oil after the chemical reaction (transesterification, reaction between methanol, potassium hydroxide and oil from fish wastes). They reported that for each liter of extracted fish oil, 0.9 liter of biodiesel was produced.

2.5. Types of Fish Silage There are generally two types of fish silage which are widely made depending on the method of acidification or protein hydrolysis. Acid or chemical silage can be prepared by direct acidification with addition of organic or inorganic acids (Ramasubburayan, et al., 2013). Fermentative or biological fish silage (biosilage) is made by using bacterial inoculums and carbohydrate substrate as a sugar source (Dapkevicius, 2002). A third type, enzymatic fish silage is rarely applied using proteolytic enzyme preparations (Borghesi et al., 2008a)

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2.5.1. Chemical Fish Silage Chemical or acid fish silage is made by using both organic and inorganic acids. Sulphuric and hydrochloric acids are the mostly used inorganic acids for the preparation of chemical fish silage while formic and propionic acids are the mostly used organic acids. Inorganic acids are cheaper especially sulphuric acid in comparison with the organic acids but the latter are used with less quantity (Abd El-Hakim et al., 2007). In addition, chemical silage which is made by using inorganic acids reaches very low pH level of about 2 and needs neutralization before incorporation into feeds and this will add to the cost and affect silage quality (Hertrampf and Piedad-Pascual, 2003). Vedotti et al. (2003) recommended using a mixture of 20 ml/kg formic acid and 20 ml/kg sulfuric acid due to the high cost of other organic acids. Abd El-Hakim et al. (2007) used 1.5% formic acid with 1.5% sulphuric acid to ensilage Nile Tilapia wastes. Their examinations showed that this silage was of good nutritional quality and used it as fish meal replacer into feeds for Nile Tilapia Oreochromis niloticus.

Ramasubburayan, et al.(2013)

prepared acid silages using fishery wastes supplemented with three different concentrations (2, 2.5 and 3%) of formic acid which fermented for 30 days and concluded that 2% formic acid was adequate to produce good quality silage as tested in feeds for the common carp fingerlings.

2.5.2. Biological Fish Silage Concentrated organic and organic acids which are used to prepare chemical silage are hazardous materials imposing risks with handling and storage especially in the developing countries. This motivated researchers to evaluate various raw materials and fermentation methods to produce biological or fermentative fish silage (Moretro et al., 2010).

15


Biological silage can be produced through spontaneous acidification resulted by using different carbohydrate substrates like sugarcane molasses (Zahar et al., 2002a), tapioca and corn flours (Hertrampf et al., 2003), apple pomace (El-Ajnaf, 2009) and sugar beet pulp (Mousavi et al., 2013) by native or added lactic acid bacteria (LAB) like Lactobacillus plantarum (Dapkevicius, 2002; El-Ajnaf, 2009; Rao et al., 2009) , L. pentosus (Hasan, 2003) , L. curvatus (NRRL B-4562), L. casei (NRRL B-1922), Lactcoccus lactis (NRRL B-1821) and Pediococcus pentosaceus (NRRL B-14009) (Bower et al., 2011).

2.5.3. Enzymatic Fish Silage Different proteolytic enzymes were used to prepare enzymatic fish silage. Borghesi et al. (2008a) used 1g/kg protease type II from Aspergillus oryzae to produce enzymatic silage from Nile tilapia processing (filleting) wastes and whole fish (trash). They find that prepared enzymatic silage does not differ significantly from chemical and biological silages produced from the same raw materials. Ghaly et al. (2013) reviewed the utilization of several kinds of enzymes such as Alcalase, Neutrase, Carboxypeptidase, Chymotrypsin, Pepsin and Trypsin in the production of enzymatic fish silage. They mentioned that such products are especially appropriate for the extraction of valuable biological materials like proteins, amino acids and oils from enzymatic fish silage. Ramakrishnan (2013) prepared fish silage by using Alcalase enzyme at three enzyme concentrations (0.5, 1 or 2%) and four time intervals (1, 2, 3 and 4 h) to extract proteins and amino acids from the whole fish and fish wastes. He concluded that enzymatic hydrolysis of fish tissues is cost

16


effective method to extract many important biological materials with high quality.

2.6. Requisites for Fermentation of Biosilage

Sugar fermenting bacteria in mixtures intended for biosilage production such as LAB require suitable concentrations of carbohydrate substrates for use as essential energy source to issue fermentation. Fish and fishery wastes generally contain very low concentrations of carbohydrate substances as glycogen. This imposes the necessity to find appropriate carbohydrate materials and determining the optimum sugar levels which supports the fermentative activity in fish silage. Dapkevicius (2002) obtained high quality biological silage of blue whiting (Micromesistius poutassou Risso) by using 4-5% soluble sugars from sugarcane molasses which were added by 10-20% ratios to silage mixture. El- Ajnaf (2009) found that 5% fermentable sugar was the optimum level for ensiling sardine Sardina pilchardus using 15% apple pomace as a carbohydrate substrate at 35⁰C. Soltan and El-Laithy (2008) prepared fermented fish silage using tomato and potato by-products as carbohydrate substrates and evaluate it as a feed ingredient for Nile tilapia, Oreochromis niloticus. They concluded the possibility of replacing 30% of the dietary protein by silage in tilapia diets without adverse effects on growth or feed utilization parameters while improving the economics of feed costs. Bower et al. (2011) used potatoes as carbohydrate substrate for ensiling pink salmon Oncorhynchus gorbuscha processing wastes at mixing rates of 30-70% using 3 strains of LAB. They concluded that potatoes were adequate for short term ensiling, but stabilization of fish silage during extended storage requires supplemental

17


carbohydrates. Many other researchers determined the proper concentration of the carbohydrate substrate as a whole like 10-20% sugarcane molasses (Zahar et al., 2002a; Vidotti et al., 2003; Ramirez- Ramirez et al., 2008). Since fermentation process is carried out by sugar fermenting bacteria mostly LAB by the production of organic acids in fermentation medium, inoculum concentration is a very important factor which governs ensiling process. Very few studies were conducted on this aspect and their results demonstrated that inoculum concentrations of 3-4 log cfu/gm fish material of LAB were adequate to produce proper quantities of acid and lowering pH to levels required for suitable fermentation (Zahar et al., 2002a; Soltan and El-Laithy, 2008; Mousavi et al., 2013). Temperature is a crucial factor in bacterial growth. Successful fermentation processes require appropriate temperature to produce the needed quantities of acid, maintain the bacterial activity and stabilize the final product (El- Ajnaf, 2009). Many successful ensiling processes were reported within 20-40⁰C with a positive correlations between fermentation efficiency and temperature (Zahar et al., 2002b; Gildberg, 2004; Arruda et al., 2007). Zahar et al. (2002b) studied the effects of some parameters like temperature, anaerobiosis, stirring and salt addition on natural fermentation silage of sardine and sardine wastes in sugarcane molasses. They concluded that stirring was very important in addition to temperature. Anaeroboisis was also important to enhance LAB growth where it doubled acid production in comparison with aerobic conditions. Addition of salt showed an obvious inhibitory effect on ensiling process. However, Dapkevicius (2002) showed that salt addition with no more than 5% inhibited some spoilage and contaminating microbes while encouraged histamine degradation by some of

18


the most available LAB strains, regarding that as an innovative means of ensuring low levels of this toxic biogenic amine in the fermented product.

2.7. Storage Quality of Fish Silage

Fish silage can be used directly after preparation or can be stored as liquid or dried product for extended periods. However, there are many factors that govern the quality of the stored fish silage like chemical composition, acidity and storage conditions. The transport and storage of the liquid product are costly so it is desirable to produce dry silage in order to reduce transport costs and to optimize storage (Tanuja et al., 2014). Most studies of the use of co-dried mixtures of fish silage and cereals as aquafeed ingredients have been based on laboratory-scale manufacture and various drying methods have been reported. These include the use of drum dryers and ovens (Vidotti et al., 2002) or solar cabinets and sun-drying outdoors (Goddard et al., 2003). The dried products especially when mixed with cereals have a very good storage quality for extended periods without spoilage or significant alterations in its nutritional properties (Goddard and Perret, 2005). Dapkevicius et al. (1998) studied lipid and protein changes during the ensilage of blue whiting (Micromesistius poutassou Risso) by acid and biological methods and reported increases in protein and fat contents in fermented fish silage during storage for 15 days at ambient temperature. Espe and Lied (1999) monitored the chemical changes in fish silage prepared from different cooked and uncooked raw materials during storage at different temperatures (4-50⁰C) for 48 days. They reported that dry

19


matter, crude protein and total fat were not affected by the different storage temperatures or the length of the storage period nor was there any change in amino acid contents. Chemical composition in the four different silage types reflected the amounts in the raw materials used for silage production. Hydrolysis, on the other hand, varied with the type of raw material used for silage production as well as with the temperature under which the silages were stored. Cooked raw materials did not show any change in hydrolysis during storage. Geron et al. (2007) investigated the chemical characterization, dry matter and crude protein ruminal degradability and in vitro intestinal digestion of acid and fermented silages from tilapia filleting residues which were stored for up to 6 months. They concluded that the conservations processes (fermentation and acidification), of tilapia filleting residue altered the chemical composition, the amino acid and fatty acid profiles, without altering the nutritious value of the tilapia filleting residue silages while increasing the feed soluble protein. Diep (2009) used different raw materials from the local by-catch fish, lizardfish (Saurida undosquamis) and blue crab (Portunus pelagicus) to prepare 12 uncooked or cooked silages for feeding of cobia (Rachycentron canadum). He stored fish silages at ambient temperatures (30± 2⁰C) for 60 days with good general stability except silages with higher moisture content which tended to spoil after storage period. 2.8. Date Fruit Residues DFR as a Novel Ingredient in Fish Feeds Iraq is considered one of the main world producers of dates (Phoenix dactylifera) with annual production of 655500 tonnes from more than 15

20


million date palm trees in 2012 (Department of Agricultural Statistics, 2012). In addition to the direct human consumption of date fruits, date syrup (dibis) production, alcohol, vinegar, liquid sugar, bread yeast and citric acid are small examples of some of the products that result from processing dates (Zabar and Borway, 2012). Date fruits have good nutritional properties and energy content. Carbohydrates, essential amino acids and minerals exist in balanced quantities. Sugars represent more than 74% of dry weight, making dates one of richest fruits in energy content. It also contains high contents of some vitamins like vitamin A and B group especially Thiamine, Riboflavin and Niacin as well as Folic Acid. Calcium, phosphorus, potassium and sulfur also exist with good concentration in dates (El Hadrami and Al-Khayri, 2012). The chemical composition of dates and their residues varies widely depending on variety, maturity stage, soil type, storage period and analytical methods. As an average, date fruits contain 85-91% dry matter, 5-15% moisture, 1.9-7.9% protein, 0.7-11% lipid, 2-3% fiber, 2-3% ash and 6188% soluble sugars (Abbas and Ati, 2010; Hasnaoui et al., 2012). Date syrup is one of the primary products of date processing. This process produces large quantities of date fruit residues DFR as by-product which are discarded, disposed or used as animal feed. It is only recently that this by-product received the attention of researchers to produce high valued materials like biofuels, biopolymers, biosurfactants, organic acids, antibiotics, industrial enzymes and other possible industrial chemicals by using bioprocess technology (Chandrasekaran and Bahkali, 2013). The chemical composition of DFR is effected by the same factors as the fruit itself in addition to the syrup processing technique. Pressing and boiling are

21


the widest used techniques to produce date syrup. The latter is more efficient in extracting date sugars but with lower quality (Hussein, 2012). So, DFR from boiling contains less sugars and more moisture and therefore spoiled faster. In contrast, it was found from different studies that DFR contains 8085% dry matter, 15-20% moisture, 3-5% protein, 2-4% lipid, 10-15% fibers, 2-4% ash and 70-80% soluble sugars (Al-Farsi et al., 2007; Salah et al., 2010). Wasted or discarded dates, date pits and DFR were used widely in animal nutrition, but scarcely in fish feeding (Hussein, 2005; Taher et al. 2012). This may be due to its high content of simple sugars. Most fish species are known to tolerate very low levels of simple sugars in feed in comparison with complex carbohydrate sources and this imposed restricted limits on their inclusion ratios in fish feeds (Al-Dubaikel et al., 2009; Azaza et al. 2009). The peculiarities in the chemical composition of DFR promote its utilization as carbohydrate substrate for preparation of fish silage. Soluble sugar content is higher and crude fibers in DFR are lower than the previously used substrates like sugar beet pulp, sugarcane molasses and apple pomace. Higher content of soluble sugars ensures using low inclusion levels in fish silage which in turn is reflected in lower levels of insoluble fibers, a non-nutritive component known to decrease feed digestibility in contrast to its role in terrestrial vertebrates (Zahar et al., 2002a; Al-Amili et al., 2008; El-Ajnaf, 2009).

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2.9. Nutritional Quality of Fish Biosilage As a fish meal alternative aimed to replace different quantities of fish meal in aquafeeds, fish biosilage must have comparable nutritional properties as the replaced component (Da, 2012). There are many factors which can affect the nutritional quality of fish biosilage like raw material, ensiling conditions and feed processing method and parameters. These crucial aspects are studied and reviewed in details by many authors (Wassef, 2005; Arruda et al., 2007; Ramasubburayan et al., 2013). Fish biosilage, like other types of fish silage, contain good quantity of protein with high quality for fish feeding as experimented by in vitro and in vivo methods (Borghesi et al. 2008). Vidotti et al. (2003) observed that the protein content of fermentative fish silage is slightly less than the protein content of the raw fish and they attributed this to the dilution effect of the sugar and lactic acid bacteria culture added to the raw materials. Protein contents generally reported for fish silages lie between 40-60% on dry basis which is generally lower than 65-74% crude protein reported for fish meal (Abd El-Hakim et al., 2007; Ramirez-Ramirez et al., 2008; Ayoola, 2010; Ramasubburayan et al., 2013). However, the reported digestibility of fish silage protein is between 80-90% which is higher than 60-75% which reported for fish meal (Bezzera, 2002; Hertrampf and Piedad-Pascual, 2003; Al-Marzooqi et al., 2010; Wang et al., 2012 ). Vidotti et al. (2003) evaluated amino acid composition of fish silages produced from three raw materials, commercial marine fish waste, commercial freshwater fish waste, and tilapia filleting residue by acid digestion (20 ml/kg formic acid and 20 ml/kg sulfuric acid) and anaerobic fermentation (50 g/kg Lactobacillus plantarum, 150 g/kg sugar cane

23


molasses). Protein content and amino acid composition were determined for raw materials and silage. Marine fish waste has higher crude protein content (776.7 g/kg) compared to freshwater fish waste (496.2 g/kg) and tilapia filleting residue (429.9 g/kg). They indicated that all silages are lacked up to three amino acids for each product according to FAO standards for essential amino acids. However, being considered as the limiting factor is only the amino acids below the 30% of the minimum requirement for fish in general, they considered all products as satisfactory with respect to the essential amino acids and regarded it appropriate for use in balanced fish diets. The final composition of silage varies considerably according to the type of raw material used, especially as to the content of lipids, which varies in different fishing seasons (Arruda et al., 2007). The content of lipids is an important point for the investigation of fish silage quality, as fatty acids which are found in fish oil are highly unsaturated and can be easily oxidized, affecting the nutritional quality of the product, making proteins and amino acids unavailable or making the product inedible (Vidotti et al. 2003). In comparison with 5-10% lipid content in fish meal, fish silages normally contain higher percentages of lipid up to 30% depending on the raw material (Hertrampf and

Piedad-Pascual, 2003; Soltan and El-Laithy, 2008;

Ramasubburayan et al., 2013), but the reported quality and digestibility of fish silage lipids are higher than that of fish meal (Fagbenro and Jauncey, 1998; Wandzel and Medrzycka, 2013; Goosen et al., 2014). Fatty acid composition of fish silage oil varies considerably according to the raw material especially those produced from marine or freshwater resources. Borghesi et al. (2008a) studied the fatty acid composition of the acid silage (AS), biological silage (BS) and enzymatic silage (ES) produced

24


from discardings of the culture and from processing residues of the Nile tilapia Oreochromis niloticus. The values found for lipids (dry matter basis) have been: 12.45, 12.25 and 12.17 g/100 g for BS, AS, and ES, respectively. The fatty acids present in the lipid fraction of the silages are predominantly unsaturated. Oleic acid was present in larger amounts (30.49, 28.60 and 30.60 g/ 100 g of lipids for BS, AS and ES, respectively). Among saturated fatty acids, palmitic and stearic acids are present in larger amounts. Only traces of eicosapentaenoic (EPA) and docosahexaenoic (DHA) fatty acids are found. In contrast, Goosen et al. (2014) recovered silage oil from rainbow trout Oncorhynchus mykiss processing wastes and used it as an alternative for commercial pelagic fish oil in diets for Mozambique tilapia, and determined the effects on fillet fatty acid profile, production parameters, intestinal microflora and gut histology.

Their results indicated that silage oil

successfully substituted the commercial oil without adverse effects on production parameters, and improved cellular non-specific immunity by 33%. The silage oil is proved to be a good source of polyunsaturated fatty acids (36.9 g/100 g total fatty acids), exhibits antimicrobial properties in the feed and gastro-intestinal tract, and caused a significant shortening of intestinal folds (34.4%) in the mid-intestine of experimental fish. They concluded that rainbow trout silage oil is cost-effective alternative dietary oil for tilapia diets, with advantages over some conventional fish oils. Several studies have been carried out to evaluate fish silage as a partial replacement for fish meal in aquafeeds prepared for freshwater or marine species. All these studies report good results of feeding, growth performance and fish body composition with replacement ratios of 15-80% of fish meal

25


by different kinds of fish silages regarding it as a viable and cost effective alternative for fish meal, the main animal protein source in aquafeeds (Vidotti et al., 2003; Wassef et al., 2003; El-Ajnaf, 2009; Ramasubburayan et al., 2014). Ensiled fish can contain considerable amount of free amino acids that serve as precursors for the production of biogenic amines like histamine, cadaverine, putrescine and tyramine. Biogenic amines are produced from decarboxylation of the precursor amino acid either by endogenous amino acid decarboxylase activity or by the growth of decarboxylase positive microorganisms (Macan et al., 2006). The low pH (below 4.5) and the physical characteristics of fish silage that lead to lower oxygen concentration within ensiled fish are favorable for the action of amino acid decarboxylases. Thus, biogenic amines can represent potential risk in fish silage. These compounds, especially histamine, are very toxic to fish (Dapkevicius et al., 2000). Lumsden et al. (2002) observed some gastric abnormalities in rainbow trout and salmon fed histamine rich feed and considered histamine levels of 1–2 g/kg sufficient for development of toxic effects in fish. Dapkevicius et al., (2000) isolated 77 lactic acid bacteria cultures from natural fermented fish pastes at 15 and 22°C and examined selected combinations of these isolates for the production of histamine, tyramine, cadaverine and putrescine. Of the isolates tested, 17% are found to produce one or more of these biogenic amines and only five of these isolates can degrade as much as 20–56% of the histamine added to the medium within 30 hours, when used as pure cultures. Similarly, Zahar et al. (2002b) found that histamine levels increase during the first days of the ensiling process of

26


sardine at 25 and 35°C to reach a maximum then decline to stabilize during the rest of the experiment (39 days).

2.10. Effects of Fish Silage on Pelleted Feed Quality

The physical quality of feed pellets is important for a number of reasons. First of all, transportation and handling in both factory and on farm situation require pellets of certain integrity without fines produced by attrition stresses. Pellets of high physical quality must have properties which give a high nutritional quality for example in terms of higher feed intake and, perhaps, an improved nutritional value (Aarseth and Prestlokken, 2003; Salas-Bringas et al., 2007). Pellets also need to have a basic form of physical quality in terms of, e.g., hardness and durability to withstand the rigors of transportation. Hardness is the force necessary to crush a pellet or a series of pellets at a time; durability is the amount of fines returning from pellets after being subjected to mechanical or pneumatic agitation. Such quality parameters can also be used to evaluate the effects of diet formulation, conditioning, expander treatment, pellet binders, die selection and similar process parameters (Salas-Bringas, 2011). In addition to the nutritional adequacy and digestibility, fish feeds especially pelleted feeds must meet certain other conditions to be successful and productive because of the peculiarity of the aquatic environment which will be introduced into (Glencross et al., 2007; Sorensen et al., 2011). Water stability and related properties like bulk density and floatability depend on several factors such as chemical composition and process parameters.

27


Chemical composition includes the proportions of proteins, lipids, carbohydrates and added binding materials. Process parameters include cooking temperature, moisture content and machine type (Sorensen et al, 2010; Li, 2012). Fagbenro and Jauncey (1998) studied the physical and nutritional properties of moist fermented fish silage pellets as a protein supplement for tilapia (Oreochromis niloticus). They mixed the wet silage (2:1, w: w) with poultry by-product meal, soybean-hydrolyzed feather meal blend or menhaden fish meal; and each mixture was pelleted by cold extrusion method. They concluded that the moist pellets maintained a firm consistency during water immersion for 10 min and pellet stability has been similar among different diet formulations. Moreover, protein and lipid losses are low ( 0.05) in the major constituents where protein content ranges between 57.51 in FBS-11 and 58.88 in FBS-10, and lipid content from 22.26 in FBS-11 to 23.96 in FBS-9 (Table 6).

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CHAPTER FOUR: RESULTS & DISCUSSION

During the course of ensiling process, DM content decreased steadily in all the studied treatments (Figure 1). Starting from DM% of 24.41, 24.99 and 26.47% for FBS-9, FBS-10 and FBS-11, respectively, different decreasing rates are recorded during the ensiling period. Dry mater contents in FBS-9, FBS-10 and FBS-11 decrease by 7.82, 7.12 and 8.16%, respectively, at the end of ensiling period. Decreasing rates in DM content are relatively similar and approximately maintain the difference in initial DM between the three studied ensiling treatments. Table 6. Characteristics of upscaled successful ensiling treatments from preliminary experiment Parameter Moisture, % Protein , % DM Lipid % DM Ash, % DM Residual sugar, % DM Fibres, % DM pH Titratable acidity, % TVB-N, mg/100gm TBA, mg/kg FFA, % Histamine, mg/100 gm

Ensiling treatment FBS-10

FBS-9 a

6.55 ± 0.777 57.74 ± 1.158 a 23.96 ± 2.799 a 10.6 ± 0.612 a 0.00 1.39 ± 0.475 a 4.29 ± 0.199 a 3.94 ± 0.659 a

a

6.25 ± 0.692 58.88 ± 1.715 a 22.43 ± 1.490 a 10.42 ± 0.552 a 0.49 ± 0.191 a 1.53 ± 0.383 a 4.01± 0.175 b 4.77 ± 0.451 b

FBS-11 6.31 ± 0.850 a 57.51 ± 2.731 a 22.26 ± 1.897 a 10.65 ± 0.396 a 1.61 ± 0.468 b 1.66 ± 0.431 a 3.98 ± 0.193 b 4.79 ± 0.473 b

51.22 ± 4.398 a 44.17 ± 3.179 a 49.83 ± 5.720 a 14.36 ± 1.250 a 18.22 ± 1.024 a 21.82 ± 2.407 a 2.38 ± 0.545 a 1.93 ± 0.216 ab 1.55 ± 0.385 b 15.23 ± 2.606 a 8.69 ± 3.163 b 9.81 ± 2.823 b Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

The only significant differences in proximate composition (p ≤ 0.05) between the three biosilage products in this experiment are noticed in dry matter DM percentage (Figure 1), where all the three treatments differ significantly (p < 0.05) from each other. FBS-11 contain the higher

69


percentage (24.31 %) in comparison with the other two treatments (22.50 and 23.21 % for FBS-9 and FBS-10, respectively). 27

DM %

26 25

FBS-9 FBS-10

24

FBS-11

23 22 0

2

4

6

8

10

Days Figure 1. Time related variation in dry matter content during ensiling of the three studied biosilage treatments FBS-9, FBS-10 and FBS-11 within 10 days.

The second significant difference (p < 0.05) between ensiling products in this experiment is found in residual sugar contents (Table 6). Final biosilage products contain 0, 0.49 and 1.61 % of fermentable sugars from starting concentrations of 6.30, 7.01 and 7.72% in FBS-9, FBS-10 and FBS-11, respectively. This represent consumption ratios of fermentable sugars 100, 93 and 79.14% during fermentation course of 10 days at 35⁰C in FBS-9, FBS-10 and FBS-11, respectively. It can be estimated that, among other factors, this biosilage formulation requires between 6.3- 6.5% sugar content for successful fermentation. Also, a significant positive correlation (r =

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0.995, p < 0.05) is found between DM and residual sugar contents in biosilage. Differences in proximate composition of fish silage reflect the initial composition of raw materials in fermentation mixture (raw fish, carbohydrate source and additives) and the different hydrolysis rates of these components (Dapkevicius, 2002; Arruda et al., 2007). The DM contents in this study has been equal or close to the minimum level of 20-30% recorded by most of the previous studies (Fagbenro and Jauncey, 1998; Espe and Lied, 1999; Bezzera, 2002; Ramasubburayan et al., 2013). This decrease in DM yield may ascribed to the addition of vinegar which contains 96-98% water and contributed to 20% of product moisture. Most biosilage types are produced by using concentrated acids or inoculants and dried carbohydrate sources which considerably decrease moisture content. However, this high moisture content may have many advantages and some disadvantages. Zahar et al. (2002b) demonstrated that higher water content in ensiling environment can effectively dissolve and dilute solid matter like sugars and make it more homogenously distributed, easily accessible by acid producing bacteria and facilitate stirring of mixture. From the technical point of view, high water content in an oily product like silage renders it easier to incorporate into feed formulation with dry components without risk of clumping and heterogeneity problems if it is planned to be used directly after production as indicated by Fagbenro and Jauncey (1998). On the other hand, Oulavallickal (2010) mention that high moisture content in silage imposes the necessity of larger containers for processing, transport and storage which are common drawbacks with nearly all silage types. Diep (2009) demonstrated that high moisture content affects the stability of silages which

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tends to spoil during prolonged storage. However, this aspect needs further research efforts to modify this process or suggest viable and economical alternatives for vinegar addition into fish ensiling mixture in view of the known roles of vinegar as an available, safe and cheap source of acid and inoculants in biosilage as shown in the current study. The difference in residual sugars may be ascribed to the initial carbohydrate concentration, numbers and activity of different sugar consuming microorganisms in ensiling mixtures. Many researchers indicate that acid producing bacteria in fish biosilage require from 4 to 5% of fermentable matter in the mixture (Ramirez- Ramirez et al., 2008; Soltan and El-Laithy, 2008) which has been slightly lower than the level estimated in the current study. Zahar et al. (2002a) and Bower et al. (2011) show that the variation in required sugar concentrations for biosilage fermentation depends on important factors such as inoculating bacterial strains, carbohydrate source complexity, raw fish material and incubation temperature. 4.2.2.2. The pH and Titratable Acidity The three upscaled fish biosilage treatment show little or no differences as to pH level in comparison with the preliminary experiment. FBS-9, FBS10 and FBS-11 reach pH levels at this experiment of 4.29, 4.01 and 3.98 (table 6) in comparison with 4.21, 4.09 and 3.98 (Table 5), respectively, in the previous experiment. FBS-9 was significantly different (p< 0.05) from other two treatments (Table 6). A significant negative correlation (r = 0.791, p < 0.05) is found between sugar content and pH of ensiling medium.

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Figure (2) illustrates the time course of pH variation of the three examined fish biosilage treatments during fermentation period. FBS-9, FBS10 and FBS-11 start from initial pH levels of 6.36, 6.29 and 6.19 reaching final values of 4.29, 4.01 and 3.98, respectively, after 10 days. Relatively similar trends of pH variation can be seen where primary declines occur during the first 6 days and tend to slow or rise (as occurred in FBS-9) thereafter during the final 4 days. 6.5 6

pH

5.5 FBS-9

5

FBS-10

4.5

FBS-11

4 3.5 0

2

4

6

8

10

Days

Figure 2. The time course of pH variation during ensiling of the three studied biosilage treatments FBS-9, FBS-10 and FBS-11 within 10 days.

Titratable acidity values reach 3.94, 4.77 and 4.79 % in the three studied biosilage treatments FBS-9, FBS-10 and FBS-11, respectively. Similar to pH levels, FBS-9 was significantly different (p< 0.05) from the other two treatments (Table 6). A significant positive correlation (r = 0.876, p< 0.05) is found between sugar content and titratable acidity of ensiling medium while

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significant negative correlation (r = - 0.997, p < 0.05) is found between titratable acidity and pH level of ensiling medium. Production of acids and maintaining low pH of fish ensiling media are crucial factors for the success of fermentation and quality of the product. Biological fish silages are known to have mild pH values around 4.5 in comparison with 2-3 in acid silages. This may be due to the weak strength of organic acids which are used in biosilage in comparison with the strong inorganic acids that are normally used to produce acid silage. The dissociation factor pKa of sulphuric acid is less than 1 while pKa values of the most used acids in biosilage formic, citric, lactic and acetic acids are between 3.75- 4.75. The pH levels which fish biosilage reach in the current experiment are equal or lower than those recorded in other researches. Zahar et al. (2002b) report pH of 4.4 after 7 days of ensiling fish-sugarcane molasses at 35⁰C and emphasize that rapid fermentation is desired for economic reasons, to minimize the risk of spoilage and to avoid excessive protein hydrolysis in silage that depreciates its nutritive value. RamirezRamirez et al. (2008) record pH of 4.4 ± 0.02 in fish-molasses biosilage indicating that the decrease in pH and high lactic acid production prevents the growth of harmful microorganisms, which allowed the preservation of fish silage. Vazquez et al. (2011) reach pH of 4.39 with fish viscera wastes fermented biologically by adding glucose (~ 4%) at 30⁰C for 3 days concluding that the most important technological issue in the development of bio-silage with fish by-products is the ability of LAB strains to ferment the waste materials and thus to produce organic acids, basically lactic and acetic acids, in order to preserve and generate ingredients for animal feed.

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Titratable acidity values recorded in the current study (3.94- 4.79%) are lower or higher than 4.6% that recorded by Ennouali et al. (2006) in fish silage produced with 10% molasses and incubated at ambient temperature (30-33⁰C) for 15 days. They attributed this high acidity to the strong acidifying potential of LAB strains in the presence of molasses sugars. Total titratable acidity of 2.37 % is reported by Ndaw et al. (2008) in sardine fish ensiled with 4% glucose after 2 weeks of fermentation at 30⁰C. They explain that the safety of fermented product primarily depends on the rapid increase in medium acidity suggesting that a pH of 4.5 should be achieved as quickly as possible in order to inhibit the growth of spoilage microorganisms in the final product. El-Ajnaf (2009) reports titratable acidity of 3.77 in fish-apple pomace biological silage incubated at 30⁰C for 7 days. He considers this value suitable for conserving biosilage, ascribing most of this concentration to lactic and acetic acids which are produced by LAB during silage fermentation. 4.2.2.3. TVB-N Total volatile basis nitrogen TVB-N values are 51.22, 44.17 and 49.83 mg N /100 gm fish silage in FBS-9, FBS-10 and FBS-11, respectively (Table 6), with no significant difference (p > 0.05) between the three studied formulations. TVB-N values are widely used as a fish quality index because they include the measurement of trimethyl amine TMA, dimethyl amine DMA and ammonia resulted from the autolysis or bacterial spoilage of stored fish (Dapkevicius et al., 2007). TVB-N values recorded in the current study are lower or equal to those reported in other studies. Dapkevicius et al. (2007)

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record values between 50-80 mg/gm fish in fish- molasses biological silage incubated at 37⁰C for 15 days. They justify these high values by the extensive hydrolysis of proteins by autolytic enzymes and bacterial activity but not fish spoilage because fermented silage is free of any objectionable odours. Ndaw et al. (2008) reported TVB-N of 36.31 mg N/100 gm fish in sardine fish ensiled with 4% glucose at 30⁰C for two weeks. They conclude that it is generally difficult to establish the limits of acceptability especially for the TVB-N, because of the extensive variability between species and regions, particularity for oily fish. They also suggested that the TVB-N values can be influenced by fish species, age and sex, the catching season and the region of fishing. Besas and Dizon (2012) report TVB-N values between 76.41 and 226.8 mg N/100 gm in yellowfin tuna viscera ensiled by adding 10-25% salt for 7 days at ambient temperature. They find negative relationship between salt content and TVB-N values ascribing this effect to the preservative action of salt against the microorganism that exists in silage. 4.2.2.4. TBA and FFA The measured TBA values in the current study are 14.36, 18.22 and 21.82 mg/kg FBS-9, FBS-10 and FBS-11, respectively (Table 6). Although a positive correlation is observed between TBA values and increasing DFR ratios in silage from 9 to 11 mg/kg (r = 0.999, p < 0.05), there are no significant differences (p > 0.05) in TBA values between the means of different biosilage treatments. TBA values are good indicator of oxidative rancidity which occurs in the lipid part of fish flesh and silage (Ndaw et al., 2008). TBA values measured in the current study are comparable or lower than those reported by other

76


researchers. El-Ajnaf (2009) record high TBA values reaching 82.55 mg/kg in fish- molasses silage. He justifies these high values by the effect of the lyophilization process and the refrigerated storage of the samples before analysis which exerts higher rates of lipid oxidation during sample thawing. De Oliviera et al. (2013) also reports high TBA value of 45.22 mg/kg in biological silage prepared from filleting wastes from Nile tilapia processing industries which are fermented with cabbage, papaya, wheat flour and 8% vinegar and incubated at ambient temperature (± 30°C) for 6 days. They mention that the produced fish waste silage develops rancid flavor which indicated the extensive rate of oxidative rancidity and emphasizing the importance of adding an antioxidant agent to preserve lipid quality in silage. Khodanazary et al. (2013) report TBA value of 18 mg/kg after 8 days of ensiling anchovy fish with 25% wheat flour at 37⁰C. However, they indicate that this value decreased to 14 mg/kg after 14 days ascribing this to the production of various peptides from protein hydrolysis that have antioxidant properties during fermentation and preserved the fermented products from oxidation of lipids. Free fatty acid concentrations in FBS-9, FBS-10 and FBS-11 silages are 2.38, 1.93 and 1.55 %, respectively. There is significant difference (p < 0.05) between FBS-9 and FBS-11only, while FBS-10 does not differ significantly (p > 0.05) from the other ensiling treatments. A significant negative correlation (r = -0.999, p < 0.05) is observed between TBA and FFA values in different ensiling treatments (Table 6). Free fatty acid concentration is another indicator of lipid quality in fish and their products. The esterified lipid hydrolysis by lipolytic enzymes liberates FFA which in turn serves as substrates for TBA reactions. Most

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lipases have optimum pH close to 4.6 which is similar to pH level in most biological silages (Goosen et al., 2014). This can explain the inverse relationship between the two parameters in fish silage. Fagbenro (1994) records values of FFA up to 2.57% in biological fish silage prepared from minced tilapias (Oreochromis spp.) with different carbohydrate substrates (molasses, corn flour, tapioca flour) and Lactobacillus plantarum as inoculum which is incubated anaerobically for 30 days at 35°C. He used ginger extract as antioxidant and reported reduction in TBA and FFA values concluding that antioxidant addition could effectively preserve fish silage lipid quality. El-Ajnaf (2009) demonstrated that FFA content has increased with storage up to a maximum of 5.13% and 7.58% in apple pomace and molasses fish silages, respectively, after 30 days at 35⁰C as a result of lipolysis. He conclude that these high values illustrate the need for adding an antioxidant material to preserve lipid quality and elongate the storage life of fermented fish silage. Khodanazary et al. (2013) reported FFA values up to 5% in anchovy- 25% wheat flour silage which incubated at 37⁰C for 8 days. They indicate that these levels do not affect silage quality nor represent any risk of toxicity for fish when used as an ingredient in fish feeds because they possess ß-oxidative enzymes which facilitate FFA incorporation into energy metabolism. Finally, they suggest adding some antioxidant material to fish silage if it is intended for feeding fish after long storage periods to limit the expected deterioration in silage lipid quality. 4.2.2.5. Histamine Histamine measurements in the current study reveals different concentrations in the studied biosilage treatments. FBS-9, FBS-10 and FBS11 contained 15.23, 8.69 and 9.81 mg/100 gm fish silage (table 6). There are

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significant differences (p > 0.05) in histamine concentrations between FBS-9 and the two other treatments which have not differed significantly (p > 0.05) from each other. Significant correlations are found between histamine concentration and both pH and TVB-N values (r = 0.986 and 0.711, respectively, p > 0.05). Many strains of lactic acid bacteria are known to produce one or more of biogenic amines including histamine by using free amino acids as substrates especially the amino acid histidine supported by low pH and anaerobic conditions in fish silage (Dapkevicius et al., 2000). Zahar et al. (2002b) record increasing histamine levels from 11 to 38 mg/100 gm in 5 days and to 60 mg/ 100 gm in samples of silage prepared from sardine wastes and sugarcane molasses (60:40 w:w) which are incubated at 25 and 35⁰C, respectively, for up to 40 days. Final histamine concentrations are stabilized at 23 and 32 mg/100 gm in silages incubated at 25 and 35⁰C, respectively. They report that biogenic amines are formed from free amino acids by autolytic decarboxylation or microbial enzyme decarboxylation mainly from Enterobacteriacea that exists in the initial fish. Kuda et al. (2007) report histamine concentrations up to 30.5 ±2.6 mg/100g in different scombrid fish species fermented with rice bran at ambient temperatures for up to 6 months. They suggest that accumulation of histamine in fermented fish products may be affected by their histidine content and distribution of both histamine producing and degrading bacterial flora. Besas and Dizon (2012) report histamine concentrations up to 12 mg/ 100g in silages prepared from yellowfin tuna viscera which fermented for 7 days at ambient temperature. They indicate that adding salt at more than 17.5% to ensiling medium considerably reduces histamine content in fish silage. They explain this

79


effect by salt potential to retard microbial histidine decarboxylase activity, a phenomenon which they ascribed to retarding microbial growth by withdrawal of cellular soluble contents by the hyperosmotic medium. Histamine concentrations measured in the current study are well below toxicity level of ≥ 50 mg/100 gm regulated by USFDA but about the double of the defect action level of 5 mg/100 g (Patange et al., 2005). The European Economic Community (EEC) has recently established regulation for species of fish belonging to the Scombridae and Clupeidae families and determined the minimum and maximum allowable levels of histamine in fresh fish at 100-200 ppm (10-20 mg/100 gm) and enzymatically ripened fish products at 200-400 ppm (20-40 mg/100 gm) (Joshi and Bhoir, 2011). According to the results presented in Table 6, especially the significant differences (p < 0.05) between the three studied fish biosilage formulations FBS-1, FBS-2 and FBS-3 as to the contents of residual sugars, FFA and most importantly histamine; FBS-2 treatment has been chosen as a standard formulation of fish-DFR biosilage. This formulation is additionally improved by adding antimycotic agent (0.25% sorbic acid) to increase efficiency of acid producing bacteria and preserve silage microbial quality (Vidotti et al., 2011) and antioxidant (200 ppm BHT) to improve lipid quality in view of high values of TBA (Arruda et al., 2006). 4.3. Formulation of Fish Feeds Using Fish Biosilage as Fish Meal Alternative 4.3.1. Quality of Whole Fish, Fish Meal and Fish Biosilage Table 7 presents the proximate composition and different chemical quality indices of the whole fish, fish meal and fish biosilage which have been used in the current study. The proximate composition of the whole fish

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is explained in details in table 2 and it is repeated here for comparison purposes. This is also applied for fish biosilage where the results presented here are largely similar to those presented and discussed in the previous section for FBS-10 silage except few differences. Values TBA and histamine are significantly (p < 0.05) lower than those reported in Table 6. TBA values have been decreased by 56.28% from 21.82 to 9.54 mg/kg and histamine content is decreased by 40.97% from 8.69 to 5.13 mg/ 100 gm (Table 7).

Table 7. Proximate composition (% ± S.D.) and chemical quality of whole fish, fish meal and fish silage Composition

Whole fish

Fish meal

Fish silage

Moisture Protein Lipid Carbohydrates Fibres Ash Energy (Kcal./kg) Yield, % pH TVB-N TBA FFA Histamine

6.12 ± 0.544

6.90 ± 0.768

6.28 ± 1.01

65.73 ± 1.610

70.10 ± 0.907

59.03 ± 0.83

15.01 ± 0.840 1.64 ± 0.161

8.03 ± 0.840 1.10 ± 0.061

22.05 ± 0.38 0.41 ± 0.15

11.50 ± 0.951

13.87 ± 1.371

1.26 ± 0.21 10.17 ± 1.13

5048.30 ± 35.88 -

4730.01 ± 30.29 20.02 ± 1.622

4316.09 ± 56.35 24.14 ± 1.988

6.63 ± 0.931

6.15 ± 0.688

3.99 ± 0.201

12.11 ± 1.017

25.42 ± 2.101

48.25 ± 2.833

0.63 ± 0.102

1.31 ± 0.179

9.54 ± 1.387

1.32 ± 0.222

1.66 ± 0.214

1.99± 0.309

0.15 ± 0.017

2.03 ± 0.455

5.13 ± 0.711

The reasons, which stand behind these improvements, could be related to the addition of antimycotic and antioxidant agents. Vidotti et al. (2011) report that inhibition of fungal growth by antimycotic agents helps to improve LAB activity which could produce more acids and further lowering

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medium pH. This will have implications on both protein and lipid hydrolysis in addition to enhancing histamine degradation rates. Similarly, Arruda et al. (2006) and Tanuja et al. (2014) emphasize that adding BHT to ensiling medium lowers TBA values and significantly improves the quality of lipids in fish silage. Different chemical quality indices (pH, TVB-N, TBA, FFA and histamine) for the whole fish agreed well with the reported values for good quality fresh fish (Huss, 1995). This may be due to good preservation conditions on-board and during different handling steps, as indicated by Alfred (1998) and Chebet (2007). Fish meal produced from by-catch fish in the current study is also of good quality. It has a protein content of 70.10 and lipid content of 8.03% (Table 7) which agrees well with the reported values for fish meal produced from different fish species i.e. 65-75% protein and 5-10% lipid (Aryawansa, 2000; Windsor, 2001). However, fish meal yield in the current study (20.02%) has been near the minimum value of the range (20-25 %) reported for industrial fish meal. The presumed reason is the addition of stick water solubles to fish meal after concentration which is discarded in laboratory made fish meal (Windsor, 2001; Tacon and Metian, 2009). Chemical quality indices of fish meal (pH, TVB-N, TBA, FFA and histamine), presented in Table 7, agree well with the reported values for good quality fish meal (De Koning, 2002; Hertrampf and Piedad-Pascual, 2003). 4.3.2. Amino Acid Composition of Whole Fish, Fish Meal and Biosilage Amino acid profiles of the whole fish, fish meal and biosilage used as feed ingredients in this study are presented in Table 8. All essential and non-

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essential amino acids are detected with different concentrations. The higher essential amino acids in the whole fish are Leucine (56.507 mg/gm protein), Histidine in fish meal (79.237 mg/gm protein) and Phenylalanine in fish silage (59.693 mg/gm protein). Very low concentrations of the essential amino acid Isoleucine are detected in both fish meal and fish silage (3.533 and 2.764 mg/gm DM, respectively) in comparison with the whole fish content (26.374 mg/gm DM). Of the 8 detected non-essential amino acids, Glutamic acid scored higher in the whole fish (99.783 mg/gm DM) while Glycine was higher in both fish meal and fish silage (101.100 and 50.704 mg/gm DM). On the other hand, very low concentrations of the non-essential amino acid Cysteine are detected in both fish meal and fish silage (5.975 and 4.835 mg/gm DM, respectively) in comparison with the whole fish content (11.357 mg/gm DM). Essential amino acids in fish silage achieve 56.146% of the total amino acids which has been higher than the corresponding ratios in the whole fish and fish meal (49.628 and 51.247 %, respectively). Similarly, ensiling process elevated EAA/NEAA ratio in fish silage (128.028%) is comparison with that observed in the whole fish and fish meal (98.524 and 105.115 %). No significant differences (p > 0.05) are observed between the three tested materials as to EAA contents. According to the common carp requirements of essential amino acids illustrated by NRC (2011), fish meal suffer deficiencies in three essential amino acids, i.e. Isoleusine, Phenylalanine and Valine, while fish biosilage suffer similar deficiencies in Isoleusine, Methionine and Valine. However, Windsor (2001) indicate that fish meal is valuable not only for the quantity

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but also the quality of its protein namely the amino acids which make up the protein present in just the right balance for animal nutrition. Table 8. The specific and total essential amino acids (EAA and TEAA), non-essential amino acids (NEAA and TNEAA) in whole fish, fish meal and biosilage used in fish feeds in comparison with common carp requirements. Carp Whole Fish meal Fish silage Amino acid, requirement* fish mg/gm protein Essential amino acids 17 Arginine 41.462 31.540 30.660 8 Histidine 23.122 79.237 53.531 10 Isoleucine 26.374 3.533 2.764 13 Leucine 56.507 20.353 50.650 22 Lysine 55.373 78.274 41.457 8 Methionine 16.390 13.412 7.727 13 Phenylalanine 26.387 2.975 59.693 15 Threonine 31.976 59.547 27.089 3 Tryptophan 22.814 61.077 46.654 14 Valine 26.801 11.388 9.572 123 Total EAA 327.206 361.337 329.797 Non-essential amino acids

Alanine Aspartic acid Cysteine Glutamic acid Glycine Proline Serine Tyrosine Total NEAA TAA EAA/TAA TEAA/TNAA,%

41.366 29.104 50.540 87.871 50.892 27.249 11.357 5.975 4.835 99.783 52.325 34.645 34.538 101.100 50.704 11.713 64.666 46.983 24.718 6.466 31.463 20.762 33.226 11.180 332.107 343.754 257.598 659.313 705.091 587.396 49.628 51.247 56.146 98.524 105.115 128.028 * Essential amino acid requirements for carp (NRC, 2011); ND, not determined

ND ND ND ND ND ND ND ND ND ND ND ND

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In addition, Vidotti et al. (2003) suggest that fermented silages, in spite of minor deficiencies in certain essential amino acids, do not lose their nutritional value; considering this fact is even more important if silages are to be considered as an ingredient in balanced diets.

Li et al. (2008)

conclude that amino acids play important and versatile roles in fish nutrition and metabolism. These functions include cell signaling, appetite stimulation, growth and development regulation, energy utilization, immunity, osmoregulation,

ammonia

detoxification,

antioxidative

defense,

metamorphosis, pigmentation, gut development, neuronal development, stress responses, reproduction and suppression of aggressive behavior in aquatic animals. Differences in amino acid composition between different feed ingredients could reflect the species composition and type of raw materials as well as the effects of processing conditions (Windsor, 2001; Vidotti et al., 2003). Amino acid composition and ratios of essential and non- essential amino acids of fish meal and fish silage are comparable to those reported previously (Windsor, 2001; Vidotti et al., 2003; Limin, 2006; El-Ajnaf, 2009). This composition can meet the nutritional requirements of the common carp as determined by NRC (2011). The relative deficiencies in some amino acids in fish silage or fish meal, although not critical for fish health, could be compensated interchangeably by both ingredients or by soybean meal which have different amino acid profile and is widely used as a major ingredient in most fish feeds (Hertrampf and Piedad-Pascual, 2003; Stankovic et al., 2011).

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4.3.3. Fatty Acid Profiles of Whole Fish, Fish Meal and Fish Biosilage Fatty acid profiles of the whole fish, fish meal and fish silage consist of 8 saturated SFA, 10 monounsaturated MUFA and 15 polyunsaturated fatty acids PUFA (Table 9). There are notable, significant and insignificant differences in contents of SFA, MUFA and PUFA between the three examined materials. Contents of ω3 and ω6, ratios of ω3/ ω6 and unsaturated/ saturated fatty acids ratios are also significantly (p < 0.05) different between fish meal and both whole fish and fish silage. PUFA comprised 48.52, 37.02 and 47.43% of fatty acid content in the whole fish, fish meal and fish silage, respectively and again fish meal differed significantly (p < 0.05) from both whole fish and fish silage. Of PUFA, both EPA (C20:5 ω3) and DHA (C22:6 ω3) are detected in good quantities (Table 9). For both of these two essential fatty acids, i.e. EPA and DHA, higher quantity is detected in the whole fish (4.98 and 16.1%, respectively), followed by fish silage (4.75 and 15.67%, respectively) and lower values in fish meal (2.71 and 11.12 %, respectively). DHA is the most abundant fatty acid in the whole fish and fish silage, while the saturated fatty acid C16:0 (Palmitic acid) has been the most abundant fatty acid in fish meal (20.71%). The fatty acid profile of the whole fish, fish meal and fish silage examined in the present study is relatively similar to the corresponding profiles of these materials prepared from marine raw materials as reported previously (Turan et al., 2007; Borghesi et al., 2008a; Huang et al., 2012).

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Table 9. Fatty acid profiles (%) of whole fish, fish meal and fish biosilage used in fish feeds. Fatty acid C8:0 C10:0 C14:0 C15:0 C16:0 C17:0 C18:0 C20:0 ∑ SFA C14:1 ω7 C15:1 ω8 C16:1 ω7 C17:1 ω7 C18:1 ω7 C18:1 ω9 C20:1 ω9 C22:1 ω9 C22:1 ω11 C24:1 ω9 ∑ MUFA C16:3 ω3 C16:4 ω1 C18:2 ω6 C18:3 ω3 C18:3 ω6 C18:4 ω3 C20:2 ω6 C20:3 ω3 C20:3 ω6 C20:4 ω6 C20:5 ω3 C21:5 ω3 C22:4 ω6 C22:5 ω3 C22:6 ω3 ∑ PUFA ω3 ω6 ω3/ ω6 ∑ USFA/∑ SFA

Whole fish

Fish meal

Fish biosilage

0.13 0.11 4.81 0.22 14.27 0.33 3.13 1.11 24.11a 1.17 0.54 11.64 0.31 0.56 9.05 2.01 0.32 0.31 1.36 27.37a 3.20 0.99 10.50 2.01 1.91 4.82 0.22 0.25 0.31 3.15 4.98 2.67 0.33 2.29 16.1 48.52a 36.32a 16.42a 2.212 3.148

1.07 0.89 2.33 0.41 20.71 0.34 8.51 0.56 34.82b 1.23 0.38 6.99 0.32 0.61 12.27 4.71 0.26 0.25 1.14 28.16a 3.03 0.78 8.47 1.91 1.74 4.12 0.19 0.22 0.22 3.01 2.71 2.26 0.30 1.88 11.12 37.02b 27.25b 13.93b 1.956 1.872

0.98 1.44 4.48 0.33 15.23 0.40 4.01 0.75 27.62a 1.89 0.50 9.51 0.30 0.55 7.27 3.11 0.33 0.27 1.22 24.95b 3.04 1.79 10.01 2.11 2.02 4.51 0.20 0.25 0.28 3.09 4.75 2.63 0.22 2.01 15.67 47.43a 34.97a 15.82a 2.210 2.621

Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

87


However, there are some noteworthy observations about fatty acid profile of fish silage in the present study. EPA and DHA values recorded for fish silage in the present study are higher than those reported by Sudaryono (2005) from marine trash fish-formic acid silage (4.74 vs. 2.45 and 5.61 vs. 15.67 % for EPA and DHA, respectively). At the same time, SFA represents more than 40% of that silage in comparison with 27.62% in the present study. Borghesi et al. (2008a) detect only traces of EPA and DHA in biological fish silage prepared from Nile tilapia processing wastes concluding that the silages produced from discardings of the culture and processing residues of the Nile tilapia are not a good source of EPA and DHA for fish feeds. However, Goosen et al. (2014) obtain good quantities of DHA (maximum 11.4 %) but not EPA, after recovering oil from silage made of rainbow trout viscera and 2.5% formic acid. On the other hand, Windsor (2001) indicates that fish species and processing conditions could effectively influence fatty acid composition of fish meal. This coincides with the increase in SFA in fish meal from 24.11 to 34.82% in the whole fish (31 %) in comparison with 27.62% in fish silage (13 %). It is also well documented that marine fishes are richer in PUFA especially in ω3 fatty acids EPA and DHA in comparison with freshwater fish (Osman et al., 2001; Ugouala et al., 2008). Fish meal and fish silage produced in the present study have balanced fatty acid profiles rich in unsaturated fatty acids which adequately meet the requirements for the common carp feeding and ensiling process especially improves these contents (Takeuchi, 2002; NRC, 2011).

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4.3.4. Preparation and Examination of Fry Feeds Fry feeds were formulated to be isonitrogenous and isocaloric as shown in Table 10. Designed protein value is 42% (measured 42.03-43.09%) and caloric content 4600 Kcal/kg (calculated 4604-4634 Kcal/kg). Table 10. Fry feed formulations and proximate compositions using fish silage as a partial fish meal alternative Feedstuff, % Fishmeal Fish silage Soybean meal Corn meal Barley flour Wheat bran Corn oil Premix* Moisture Protein Lipid NFE Ash Energy, Kcal/kg P/E Ratio, mg/Kcal

Feed formulation FM1 FSa1 FSb1 43 32.25 21.5 0 12.75 25.5 17 17 17 15 15 15 10 10 10 7 7 7 6 4 2 2 2 2 Proximate composition, %

FSc1 10.75 38.25 17 15 10 7 0 2

6.51± 0.99 42.82± 3.68 12.71± 1.98 27.37± 3.22 10.59± 1.59 4634± 291.9

6.83± 1.55 43.09± 2.92 12.83± 1.66 26.40± 2.98 10.85± 1.87 4620± 313.9

6.87± 1.23 42.03± 3.13 12.91± 1.28 27.25± 3.09 10.94± 1.69 4604± 355.7

7.07± 1.41 42.77± 2.89 13.3± 2.02 25.88± 2.83 10.98± 1.44 4610± 366.6

92.41± 6.55

93.27± 7.31

91.30± 6.69

92.78± 7.81

FM1, 100% fish meal; FSa1, 75% fish meal+ 25% fish silage; FSb1, 50% fish meal+50% fish silage; FSc1, 25% fish meal+75% fish silage calculated as per protein content. *Vapcomix, VAPCO Veterinary and agricultural product manufacturing Co., Amman, Jordan.

These levels of protein and energy contents are selected according to Takeuchi et al. (2002) and Davies and Gouveia (2010) who determine 4042% protein and protein to energy ratio of 85-105 mg protein/Kcal as optimum ranges for maximum growth performance for the early stages of

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the common carp (P/E ratios 91.3-93.27 mg protein/Kcal in experimental feeds formulated for the current study). Feeds are formulated to replace 0, 25, 50 and 75% of fish meal content (43% in basal feed FM1) depending on protein contents in both ingredients (70.1 and 59.03% in fish meal and fish silage, respectively, Table 7). Owing to the higher lipid content in fish silage comparing to fish meal (22.05 vs. 8.03%, respectively), different quantities of corn oil (0-6%) have been added to equalize the caloric contents in different feed formulations, as suggested previously by Abbas (2007). 4.3.4.1. Physical Quality of Formulated Feeds The physical quality of different formulated feeds in this experiment is evaluated by using various parameters, i.e. bulk density, pellet durability index PDI, water stability, settling velocity and floatability (Table 11). The results presented in Table 11 show that bulk density increases with increasing fish silage percentage in feed from 0.808 in FS1 (0% fish silage) to 1.058 gm/cm3 in FSc1 (38.25% fish silage). There are different significant (p < 0.05) and insignificant differences (p > 0.05) in bulk density between the different treatments denoting that minimum silage addition ratio to bring a significant difference are 25.5 %. There is also a significant positive correlation between bulk density and fish silage percentage in feed (r = 0.999, p < 0.05). The bulk density of pelleted feed in the current study is relatively similar to the range of 0.882-0.955 gm/cm3 recorded by Al-Shameri and Ali (2010) using a formulation containing 10% animal protein concentrate. They conclude that grinding fineness and meal moisture affect significantly pellet

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bulk density. Al-Khshali (2012) reports higher pellet bulk density reached a maximum of 1.103 gm/cm3, by using a formulation containing 89% plant meals (corn, wheat and soybean). He confirms the role of meal moisture in determining pellet bulk density as well as temperature and plate die diameter which correlates inversely with bulk density. Table 11. Physical quality of experimental feeds with different substitution ratios of fish meal by fish silage. Feed type Parameter FM1 FSa1 FSb1 FSc1 Bulk density, gm/cm3 PDI, %

0.808 ± 0.029a

0.900± 0.018b

75.8± 2.98a

79.6± 3.13ab

0.982 ± 0.077bc 1.058 ± 0.043c 84.0± 3.30b

90.3± 1.55c

Water stability, % 66.08± 2.00a 74.38± 1.66b 79.05± 1.68c 85.14± 1.09d Settling velocity, 5.77± 0.58a 6.35± 0.49ab 7.01± 0.47b 9.10± 0.72c cm/sec 10.37 ± 2.13a 9.22 ± 0.51a 6.11 ± 1.68b 0.50 ± 0.66c Floatability, min FM1, 100% fish meal; FSa1, 75% fish meal+ 25% fish silage; FSb1, 50% fish meal+50% fish silage; FSc1, 25% fish meal+75% fish silage calculated as per protein content. Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

Fallahi et al. (2013) indicate that bulk density of pelleted feed reflects the density of different ingredients as well as different processing parameters, i.e. temperature, moisture and screw speed. Accordingly, since proportions of fish silage, fish meal and corn oil are the only different variables in feed formulation, differences in their bulk density seems responsible for the variation in feed bulk density. Tumuluru (2013) emphasizes that bulk density of aqua feed pellets must be designed to meet the intended type properties (floating or sinking) because it strongly affects other physical properties of pellets such as durability and water stability.

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Values of pellet durability index PDI increase steadily from 75.8 to 90.3% in FM1 and FSc1, respectively, with various significant (p < 0.05) and insignificant (p > 0.05) differences between the four studied feed formulations (Table 11). There are also significant positive correlations between PDI value and each of fish silage ratio in feed (r = 0.993, p < 0.05) and feed bulk density (r = 0.987, p < 0.05). In an agreement with the results of the current study, Fagbenro and Jauncey (1998) find an improvement in fish pelleted feed durability, hardness and water stability when fermented fish silage is added up to 66% of feed formulation. They explain this effect by the high pelletability of silage proteins in addition to the impact of residual molasses in fermented fish silage. Rosentrater et al. (2009) achieve important increases in aquafeed pellet PDI from 55.4 to 90.8% by varying several factors like ratios of soybean meal and corn starch in feed and initial feed moisture contents (2545%). They indicate that gelatinization of the starchy matter form a consistent network structure which encloses feed fines preventing it from easily dissociation upon water immersion. Similarly, Sorensen et al. (2009) show significant improvements in PDI values (from 40 to 94%) in extruded fish feeds when soybean meal was added up to 35.6% of feed formulation. They ascribed this effect to several factors like increasing pellet length, bulk density and lowering pellet expansion rate concluding that durability and breaking force of extruded fish feed is significantly improved by the inclusion of soy, regardless of previous heat treatment of this feed ingredient. Miladinovic et al. (2012) report PDI values of 72.77- 93.22 using different moisture removal techniques and pellet die hole diameters. They find negative relationships between die-hole diameter and each of bulk

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density and PDI values emphasizing that pellet durability is a very important factor which reflects pellet tolerance to handling, transportation and storage conditions with considerable implications on pellet quality and feeding economy. Water stability percentage of formulated fish feeds increases from 66.08 in FM1 without fish silage addition to 85.14% in FSc1 with higher silage addition rate of 38.25% (Table 11). There are significant differences (p < 0.05) between all the four examined feed types. There are also significant positive correlations between pellet water stability from one side and each of fish silage addition rate (r = 0.997, p < 0.05), bulk density (r = 0.996, p < 0.05) and PDI values (r = 0.979, p < 0.05). Misra et al. (2002) achieve significant improvements in water stability of pelleted feed from 47.51 to 71.41 % after water immersion for 12 hours by using extrusion processing instead of conventional steam pelleting. They conclude that extrusion process produced pellets of better water stability than steam cooked pellets ascribing the superior water stability of the extruded pellets to the gelatinization of dietary starch during extrusion under high temperature, high pressure and high shear. Oehme et al. (2012) record water stability ratios between 92.5 and 93% after 4 hours of water immersion in high energy extruded pellets of similar nutritional quality produced for Atlantic salmon Salmo salar by varying drying times to achieve moisture contents of 4.1-9.2%. They explain that through the effect of higher moisture content on water absorption rate which contributes considerably in pellet disintegration and determined its usefulness as a fish feed concluding that the better moisture content level for this type of extruded pellets is around 6%. Fallahi et al. (2013) obtain high stability rates

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of 83.84- 85.19% in extruded pellets produced by addition of distillers dried grains (DDGs), a non-fermentable co-product of ethanol plants, as a fish meal replacer. In agreement with the results of the current study, they also find a positive relationship between pellet bulk density and water stability. They explain these observations by the higher degrees of starch gelatinization and protein denaturation in the extruded blend which produced significant changes in the water stability of the extruded pellets. They conclude that water stability of pellets can be greatly improved through proper selection of feed ingredients, processing techniques and the use of proper processing equipment. Settling velocity of different examined pelleted feeds increases from minimum of 5.77 in FM1 to maximum of 9.10 cm/sec in FSc1 with significant differences (p < 0.05) between the different feed types except FSa1 and FSb1 which do not differ significantly (p > 0.05) as shown in Table 11. Positive significant correlations are found between pellet settling velocity and each of fish silage ratio (r = 0.964, p < 0.05) and pellet bulk density (r = 0.933, p < 0.05). Vassallo et al. (2006) determine the physical behaviour of feed pellets produced for Gilthead Sea Bream (Sparus aurata L.) and Sea Bass (Dicentrarchus labrax L.) in Mediterranean waters confirming that settled uneaten feed causes the most intense impact under sea cages, and settling velocity of the feed pellets represents a key parameter for waste dispersion models.. They record settling velocities of 8.7 to 14.4 cm/sec for pellets of different diameters between 3 to 6 mm with inverse relationship between pellet diameters and settling velocity. They relate this observation to the higher bulk density of smaller size pellets which increase settling velocity in

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comparison with the larger pellets of lower bulk density. This agrees well with the observed negative relationship between bulk density and settling velocity as shown in the current study. Piedecausa et al. (2009) investigate the settling velocity and total ammonia nitrogen leaching from commercial feed for gilthead seabream (Sparus aurata L. 1758) and seabass (Dicentrarchus labrax L. 1758). They record settling velocities of 6-14 cm/sec for pellets of varying diameters between 2 to 8 mm observing a significant negative correlation between pellet sizes and settling velocity. They indicate that pellet size especially diameter and weight are crucial factors influencing settling velocity. Therefore, they hypothesize that pellet weight increase is due to hydration and this causes a volume increase and shape change, causing a greater influence on settling velocity than weight because of greater friction produced, and a higher resistance to fall, so weight increment after soaking is higher in smaller pellets. Al-Khshali (2012) obtain improvements in settling velocity of fish feed pellets produced by steam pelletizer from 9.73 to 5.37 cm/sec by varying pellet moisture content from 5 to 6% and plate die holes from 2 to 6 mm. He find a positive correlation between pellet bulk density and settling velocity which agree with the results of the present study. He explain that through the effects of moisture content and plate die hole diameter on lowering bulk density of the produced pelleted feed from 1.103 to 0.873 which are reflected positively in slower settling velocity. Pellet floatability, as assessed by floatation time, decreases steadily from 10.37 in FM1 to .50 min in FSc1 with significant differences (p < 0.05) between the various examined feed types except FM1 and FSa1 which do not differed significantly (p > 0.05). Significant negative correlations are

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found between pellet floatability and each of fish silage ratio (r = - 0.956, p < 0.05) and pellet bulk density (r = - 0.943, p < 0.05). Jovanovic et al. (2009) examine the efficiency of cooking extruder and vacuum cooler for production of floated feeds for trout and carp. They achieve maximum floatation time for 6 hours when pellet bulk density reduces to 0.41 gm/cm3 which is considered as the minimum level for good pellet floatability. The most influencing factors on pellet bulk density, thus its floatability, in cooking extrusion technology are barrel temperature, screw speed, water vapour and pelletizer head pressure in addition to the initial ingredients of blend. Saalah et al. (2010) carry out a study to minimize the degree of swelling and mineral leaching and maximize pellet floatation time using several formulations of fish feeds. These formulations are based on common carbohydrate sources like corn flour, soy flour and tapioca flour. The recorded improved floatation times of pelleted feed from 10 to 40 minutes by adding palm oil stearin to give good floating characteristics for feed after increased inclusion of soy and tapioca flours. They explain this through the high bulk density of grain flours which reduces pellet floatability and using palm oil stearin resolves this by lowering bulk density and leaching out during drying to form a water resistant film coating pellets, delaying water absorption and elongate floatation time. The negative relationship which they found between pellet bulk density and floatability is consistent with the negative correlation between these two parameter of fish physical quality as indicated in the current study. Solomon et al. (2011) conduct a series of laboratory experiments to investigate water stability and floatation of fish feed bound with five different binding agents [Wheat Grain Starch (WGS), Corn Grain

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Starch (CGS), Millet Grain Starch (MGS), Guinea Corn Grain Starch (GGS) and Cassava Tuber Starch (CTS)] with yeast Saccharomyces cerevisae serving as the floating agent. They record pellet floatation times between 5 to 30 min with Cassava Tuber Starch (CTS) and Millet Grain Starch (MGS), respectively. They explain this through the different qualities of starch found in these ingredients which is reflected on formed gelatine network and expansion rates of pelleted feed and ultimately affects pellet bulk density and floatability. The inverse relationship that they find between pellet bulk density and floatation time agrees with that reported in the present study. Although the addition of fish silage to feed formulation has positive effects on some favourable properties of fish pelleted feed quality such as durability and water stability, it affects negatively other important pellet physical quality characteristics, i.e. settling velocity and floatability. It seems clearly from the results of the current study and the previous studies that feed bulk density is the main factor that governs these attributes (Fagbenro and Jauncey, 1995a). Therefore, if fish silage is intended to be used as an ingredient in fish feed, bulk density of feed must be improved through manipulation of feed ingredients, the addition of floatation aids and using modern technologies for the production of fish pelleted feeds (Piedecausa et al., 2009; Saalah et al., 2010; Fallahi et al., 2013). 4.3.4.2. Feeding and Growth Performance of Common Carp C. carpio Fry Feeding and growth performance parameters for common carp fry fed the four experimental diets with different fish silage ratios (0, 25, 50 and 75% of fish meal protein) are presented in Table 12.

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Table 12. Feeding and growth performance of common carp C. carpio fry fed different experimental feeds

Initial wt., gm Final wt., gm Weight gain, gm

FM1 0.664 ± 0.05 6.324± 0.75 5.66 ± 0.61ab

Experimental diet FSa1 FSb1 0.750 ± 0.06 0.691 ± 0.10 7.005± 1.01 6.390± 0.88 a 6.255 ± 0.94 5.699± 0.55ab

FSc1 0.611± 0.04 5.682 ± 0.85 5.071 ± 0.69b

SGR TGU

3.220 ± 0.35a 0.495± 0.05a

3.192 ± 0.44a 0.509 ± 0.06b

3.186 ± 0.32a 0.474 ± 0.04c

Parameter

3.178 ± 0.31a 0.492 ± 0.05a

2.101 ± 0.25a 2.133 ± 0.30 ab 2.148 ± 0.28b 2.151± 0.34b FCR 1.347± 0.14a 1.466± 0.11b 1.327± 0.17a 1.179± 0.12a PER 96.7 ± 1.0a 94.4 ± 0.58a 95.6 ± 0.58a 96.7 ± 1.0a Survival, % FM1, 100% fish meal; FSa1, 75% fish meal+ 25% fish silage; FSb1, 50% fish meal+50% fish silage; FSc1, 25% fish meal+75% fish silage calculated as per protein content. SGR, specific growth rate; TGC, Thermal growth coefficient; FCR, feed conversion ratio; PER, protein efficiency ratio. Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

Weight gain was very similar between the experimental diets except one significant difference (p < 0.05) between FSa1 and Fsc1 which can be attributed to the higher initial weight of fish (0.750 vs. 0.611 gm, respectively). This has been confirmed by specific growth rate SGR values which do not differ significantly (p > 0.05) between the four experimental feeds (3.178- 3.220). However, thermal growth coefficient TGC values show rather different trend and varied between 0.474- 0.509 with significant differences (p < 0.05) between both FM1 and FSb1 from one side and the other two experimental feeds. Feed conversion ratio FCR values vary between 2.101- 2.151 with significant differences (p < 0.05) between FM1 and treatments with higher fish silage contents FSb1 and FSc1. Values of protein efficiency ratio PER range between 1.179 in FSc1 to 1.466 in FSa1 which differed significantly (p < 0.05) from the other three treatments. Fish

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survival rates are within 94.4- 96.7 % with no significant differences (p > 0.05) between the four experimental diets. The four experimental feeds are performed in a very close manner with few significant differences in the studied growth parameters indicating the feasibility of using this fish biosilage as a partial replacement for fish meal in common carp fry diets. Several studies confirmed that fish silage could replace fish meal partially in fish feeds without any adverse effects on feeding, growth and survival of fish while reducing feed costs. They explained that through the close resemblance in major nutritional components between the two ingredients as they are produced from similar raw materials (Fagbenro and Jauncey, 1998; Soltan and El-Laithy, 2008; Ayoola, 2010). Growth parameters of fish in this experiment are compared similarly or favourably with some previous studies. Gumus et al. (2009) use tuna liver meal (TLM) to replace fish meal (FM) in diets for common carp fry. They feed fish with average weight of 0.32 gm on each of six isonitrogenous (42%), isolipidic (16%) and isoenergetic (18 KJ DE/g, 4302 Kcal/kg) diets prepared to include 0, 10, 20, 30, 40 and 50% of FM protein being substituted by TLM. The control diet contains fish meal (17.14%) and soybean meal (46.9%) as the main sources of dietary protein. After 13 weeks of feeding, they report SGR values of 1.49-2.24, FCR 1.53-3.52, PER 0.721.63 and survival rates 70-87.5% which are close or lower than those reported in the current study. They indicate that up to 20% of FM protein in fish diet can be replaced by TLM without adverse effects on fish growth, feed utilization and body composition. The differences in the results of the current study may be ascribed to the lower energy content in comparison with the current study (4302 vs. 4600 Kcal/kg, respectively) and lower

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temperature of rearing water (25 vs. 28⁰C, respectively). Takeuchi et al. (2002) demonstrate that energy content, thus P/E ratio, in common carp diets

are determinant factors that govern feeding and growth efficiency especially during the early life stages and diets must be designed carefully to satisfy the optimum levels of these components. Desai and Singh (2009) indicate that the common carp fry (Average weight 0.86 gm) show better feeding and growth performance when they are reared under temperature of 28⁰C in comparison with 32⁰C, considering it as an optimum for this life stage. Davies and Gouveia (2010) study the response of common carp fry fed diets containing a pea seed meal (Pisum sativum) subjected to different thermal processing methods. Starting from initial weight of 7.50 gm in a 7 week trial on 40% CP diets, they report SGR values of 2.51-2.84, FCR 1.58-1.82 and PER 1.38-1.58 which are relatively coincides with the results of the current study. They concluded that high grade protein pea concentrates (up to 60% CP after starch extraction) could replace about 20% of fish meal in common carp fry diets without significant alterations in feeding and growth performance. Rahman et al. (2012) rear mirror carp advanced fry (2.29 ± 0.08gm) in aquaria for seven weeks and fed it on different protein levels (2545%). At the end of their experiment, they record SGR values of 0.71-1.14, FCR values of 4.31-12.7 and survival rates of 70-93.33 %. These values are generally inferior in comparison with the results of the current study as shown in Table 12. There are several potential reasons for these differences such as fish strain (mirror vs. common carp), fish initial weight (2.29 vs. 0.68 gm in the current study), stocking density (1 fry/l. vs. 0.5 fry/l., in the present study) and feed protein source (10% from fish meal vs. about 30% from fish meal and fish silage in the current study). Many researchers indicate that younger fish have higher growth rates, animal protein sources

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perform better as feed ingredients than plant proteins and fish stocking density is inversely correlated with fish growth (Wilson, 2002; Jha and Barat, 2005; Dumas et al., 2010). As to the thermal growth unit coefficient TGC, three different formulae are observed in the literature based on the same concept of weight gain, water temperature and duration of study. The only difference is in multiplication factor. The first formula is that of Cho (1992) which has not contained any multiplication factor; the second is that of Bureau et al. (2000) which is multiplied by 100 and the last formula is reported by Jobling (2003) which is multiplied by 1000. The latter is chosen for the current study because it offers the best fit with fish growth data. Values from researches used other formulae were converted accordingly for comparison purposes. However, TGC values calculated in the current study (Table 12) are very similar to those reported previously for the common carp. Jafaryan et al. (2011) report TGC values of 0.319-0.472 when they investigate the enhancement of growth parameters in the common carp (Cyprinus carpio) larvae using probiotic in rearing tanks and feeding by various Artemia nauplii. They reared fish larvae (average weight 0.12 gm) at 24-26⁰C for 28 days and indicated that the addition of probiotic bacilli to rearing tanks has different effects on the growth parameters of common carp larvae when they are fed on different Artemia nauplii. Sahandi et al. (2012) report identical TGC values (0.319-0.472) to that of Jafaryan et al. (2011) when they investigate the effect of probiotic bacilli on the growth rate of common carp Cyprinus carpio and grass carp Ctenopharyngodon idella larvae (average weight 0.12 gm) reared at 30⁰C for 28 days and fed on Artemia sp. They conclude that inoculation of rearing water with probiotic bacteria could improve larval growth performance under the favorable conditions by

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enhancing digestive activities within the alimentary canal of fish larvae. AlDubaikel et al. (2012, 2013) report values of 0.40-0.49 at an average water temperature of 26⁰C and initial body weight 5.5-7.5 gm for common carp fingerlings fed either roquette oil (Eruca sativa) as an additive or different flavoring agents, respectively. Jobling (2003) emphasizes that the popularity of the TGC model for fish growth relates to its ease of use and flexibility; growth data collected for fish of given size at one temperature can be used to predict the growth of fish of a different size when held at other temperatures. The TGC has proven to be stable over a wide range of temperatures for several species and is also less affected by fish size and time interval between weightings than other growth rate estimates such as SGR, and thus offer a simple model for growth rate comparisons (Bureau et al., 2000). Water quality parameters of rearing water during feeding experiment of common carp fry are presented in Table 13. The values are within the suitable ranges for this species as described previously (Rahman et al. 2008; Markovic et al., 2009). Concentrations of inorganic nitrogenous wastes i.e. ammonia are of special importance in fish culture facilities. Ammonia is toxic to fish if allowed to accumulate in fish production systems. Ammonia is the main metabolite of protein catabolism in fish where it is excreted effectively through gills. Degradation of uneaten feed and faeces is another major source of ammonia in fish culture facilities. When ammonia accumulates to toxic levels, fish cannot extract energy from feed efficiently. If the ammonia concentration gets high enough, the fish will become lethargic and eventually fall into a coma and die. In properly managed rearing systems, ammonia seldom accumulates to lethal concentrations. However, ammonia can have sublethal effects such as reduced growth, poor

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feed conversion and reduced disease resistance (Hargreaves and Tucker, 2004). Gross et al. (2000) determine 1mg/l as a minimum lethal concentration for fish and sublethal concentrations down to 0.1 mg/l. In spite of the differences between feed treatments, the values of ammonia measured in the current study are well below risk levels. Suitable stocking density, good aeration and daily water exchange with removal of faeces and uneaten feed may have participated in lowering ammonia levels as suggested by Biswas et al. (2006).

Table 13. Water quality parameters of rearing water for common carp fry during feeding experiment Parameter

Feed type FM1

FSa1

FSb1

FSc1

Temperature, ⁰C

28.1 ± 0.382

28.2± 0.293

27.8± 0.366

27.7± 0.309

pH

7.47± 0.107

7.53± 0.097

7.75± 0.128

7.91± 0.112

Oxygen, mg/l

9.01± 0.188

8.95± 0.201

9.05± 0.199

8.88± 0.210

Salinity, ‰

1.77± 0.189

1.79± 0.227

1.81± 0.182

1.76± 0.237

Nitrate, mg/l

0.66± 0.082

0.69± 0.079

1.05± 0.088

1.49± 0.109

0.025± 0.003 0.028± 0.003 0.041± 0.005 0.048± 0.005 Ammonia, mg/l FM1, 100% fish meal; FSa1, 75% fish meal+ 25% fish silage; FSb1, 50% fish meal+50% fish silage; FSc1, 25% fish meal+75% fish silage calculated as per protein content.

4.3.5. Preparation and Examination of Fingerling Feeds Experimental feeds for common carp fingerlings are formulated to be isonitrogenous and isocaloric as shown in Table 14. Designed protein value is 35% (measured 34.42.-36.63%) and caloric content 4450 Kcal/kg (calculated 4402-4472 Kcal/kg). These levels of protein and energy contents are selected according to Takeuchi et al. (2002) and NRC (2011) who determine 30-35% protein and protein to energy ratio of 65-80 mg

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protein/Kcal as optimum ranges for maximum growth performance for common carp fingerlings at the beginning of production stage (P/E ratios 78.15-81.9 mg protein/Kcal in experimental feeds formulated for the current study). Table 14. Fingerling feed formulations and proximate compositions using fish silage as a partial fish meal alternative Feedstuff, % Fishmeal Fish silage Soybean meal Corn meal Barley flour Wheat bran Corn oil Premix* Moisture Protein Lipid NFE Ash Energy, Kcal/kg P/E Ratio, mg/Kcal

FM2 34 0 15 15 18 11 5 2

Feed formulation FSa2 FSb2 25.5 10.1 15 15 18 11 3.4 2

17 20.2 15 15 18 11 1.8 2

Proximate composition, % 6.50±0.52 6.88±0.64 6.91±0.49 36.63±1.17 35.41±1.79 35.24±1.58 10.90±0.91 11.28±0.85 11.15±1.04 35.76±2.69 34.84±1.71 35.58±2.15 11.21±0.83 11.59±1.21 11.12±1.50 4473 ± 33 4402 ± 45 4411 ± 38 81.90±1.54 80.43±1.71 79.88±1.66

FSc2 8.5 30.3 15 15 18 11.2 0 2

7.15±0.81 34.42±0.92 12.09±1.11 34.41±2.17 11.93±1.43 4404 ± 39 78.15±1.67

FM2, 100% fish meal; FSa2, 75% fish meal+ 25% fish silage; FSb2, 50% fish meal+50% fish silage; FSc2, 25% fish meal+75% fish silage calculated as per protein content. *Vapcomix, VAPCO Veterinary and agricultural product manufacturing Co., Amman, Jordan.

Feeds are formulated to replace 0, 25, 50 and 75% of fish meal content (34% in basal feed FM2) depending on protein contents in both ingredients (70.1 and 59.03% in fish meal and fish silage, respectively, Table 7). As previously performed in fry feed formulation, owing to the higher lipid

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content in fish silage comparing to fish meal (22.05 vs. 8.03%, respectively), different quantities of corn oil (0-5%) are added, as previously suggested by Abbas (2007) to equalize the caloric contents in different feed formulations. Table 15 presents feeding and growth performance parameters for common carp fingerlings that fed the four experimental diets with different replacement ratios of fish meal by fish silage (0, 25, 50 and 75% of fish meal protein). Weight gain at the end of experiment ranges from 20.42 gm in FSb2 to 22.30 gm in FSa2 with significant differences (p < 0.05) between the four feed treatments. Values of SGR range between 1.774 and 1.853 in FSc1 and FSa1, respectively. Significant differences (p < 0.05) are found between the various treatments except FSa2 and FSb2 (p > 0.05). Feed treatments show significantly different (p < 0.05) TGC values from 0.504 in FSc2 to 0.526 in FSa1. However, FM2 and FSa2 do not significantly differ as for calculated TGC values (0.520 and 0.526, p > 0.05). Feed conversion ratio FCR show values between 2.40 in FM2 and 2.51 in FSc2 which has been the only treatment that differed significantly (p < 0.05) from the others. Values of PER range between 1.096 and 1.197 in FSb2 and FSa2, respectively, with significant differences (p < 0.05) between the four treatments. The addition of fish silage has resulted in a gradual increase in feed intake from a minimum of 4.78% in FM2 to a maximum of 5.87% in FSc2. Feed intake values were differed significantly (p < 0.05) between the four feed treatments and correlated positively and significantly with fish silage addition rate (r = 0.998, p < 0.05). Fish survival rate differs significantly (p < 0.05) from 88.9% in FSc2 to 93.3% in both FM2 and FSb2.

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Table 15. Feeding and growth performance of common carp C. Carpio fingerlings fed different experimental feeds Parameter

Experimental diet FM2

FSa2

FSb2

FSc2

Initial wt., gm

5.98± 0.286

5.96± 0.309

5.46± 0.311

6.11± 0.374

Final wt., gm

27.71± 1.47

28.26± 1.80

25.88± 1.88

27.12± 2.03

Weight gain, gm

21.73± 1.12a

22.30± 1.07b

20.42± 1.30c

21.01± 1.65d

SGR

1.825± 0.105a

1.853± 0.151b

1.852± 0.148bc 1.774± 0.217d

TGC

0.520± 0.013a

0.526± 0.011a

0.514± 0.013b

0.504± 0.009c

FCR

2.40± 0.088a

2.42± 0.067a

2.49± 0.087a

2.51± 0.089b

PER

1.166± 0.440a

1.197± 0.476b

1.096± 0.411c

1.128± 0.433d

Feed intake, % BW

4.78± 0.479a

5.13± 0.481a

5.56± 0.445a

5.87± 0.505a

Survival, %

93.3± 2.11a

91.1± 3.17b

93.3± 2.11a

88.9± 4.33c

FM2, 100% fish meal; FSa2, 75% fish meal+ 25% fish silage; FSb2, 50% fish meal+50% fish silage; FSc2, 25% fish meal+75% fish silage calculated as per protein content. SGR, specific growth rate; TGC. Thermal growth coefficient; FCR, feed conversion ratio; PER, protein efficiency ratio. Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

Figure 3 illustrates feed intake of the common carp fingerlings as percentage of body wet weight during feeding on the different four experimental diets. Although being insignificantly different (p > 0.05), fish show relatively the same trend of feed intake partitioning during the course of the study. Feed intake decreases gradually during the first three hours and stops completely after that. Fish consume the main part of total feed intake during the first hour (60-65% of total intake); lower percentage during the second hour (26-29% of total intake) and the rest of feed intake (7-11% of total intake) has been consumed during the third hour.

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Feed intake, %BW

6

4 FM2 FSa2 2

FSb2 FSc2

0 1

2

3

4

Total

Time (h.) Figure 3. Feed intake by common carp fingerlings fed on different experimental feeds during 4 hours.

Al-Faraje (2000) report SGR values of 0.788-1.098, FCR 4.5-7.8 and PER 0.25-1.3, when he performed total replacement of animal protein by lactic acid fermented fish silage (Khishni Liza abu) in feeds for the common carp fingerlings (initial weight 4.5 gm). These values are lower than those reported in the current study. There are several potential reasons for these differences like lower protein (13.99- 28.86), lipid (3.86-6.92) and thus caloric (3800-3900 Kcal/kg) contents in feeds in comparison with the current study. Several previous studies indicated that replacement ratio of fish meal by fish silage in fish feed should not exceed 50% of dietary protein to

obtain

better

growth

(Fagbenro,

1994;

Dapkevicius,

2002).

Ramasubburayan et al. (2013) report SGR values of 1.06-1.49 for the common carp fingerlings (initial weight 2.5 gm) fed diets with different fish silage contents (0-3%). They conclude that the addition of fish silage has

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improved carp fingerling growth and that fish silage prepared from the processing wastes could effectively be utilized as fish feed stuff and indicating its potential means of minimizing fishmeal and reducing possible environmental pollution. These values are lower than those reported by the current study. This may be ascribed to the lower inclusion levels of fish silage (maximum 3% in comparison with 10-30% in the current study) and fish silage type (formic acid silage vs. biosilage in the present study). Values of TGC are close to those recorded in fry feeding experiments and compare favorably to those reported previously in other studies on the common carp fingerlings (Al-Dubaikel et al., 2012, 2013). Fagbenro (1994) and Dapkevicius (2002) indicated that fish silage may become more advantageous for fish feeding if it replace between 25-50% of dietary protein. They point out the superiority of biological fish silage over acid silage due to better digestibility of protein, higher quality of fish oil and the activity probiotic LAB bacteria with its metabolites that improve digestion, immunity and general health of fish. Atanasoff (2014) replace fish meal by ribotricin which is a fermented 60:40 mixture of waste non-standard fish and wheat bran in feeds for the common carp. He totally replaced fish meal (19% in the basal diet) by the alternative product (39% in experimental diet) and record SGR values of 2.07-2.15, FCR 2.20-2.36 and PER 1.31-1.40 which are very close to those reported in the present study. This rapprochement may be explained by using fish as a main component in fish meal replacing materials, i.e., biosilage and ribotricin, in both studies. Ayoola (2010) indicate that alternative protein resources made from fish (fish by-catch and processing wastes) perform better as fish meal replacer in comparison with other animal or plant materials for aquaculture feeds.

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Increases in feed intake with the elevated of inclusion rate of fish silage into feed formulations are previously indicated with its usage as one of the active attractants in fish feeds for increasing feed palatability and reducing feed wastes. Several mechanisms are proposed for this attractor effect like the roles of some fatty acids, amino acids and peptides resulted from fat and protein hydrolysis as feeding stimulants and gustatory attractants for different fish species (Xue and Cui, 2001; Yacoob and Browman, 2007). This agrees well with the gradual increase in feed intake by fingerlings in accordance with elevated fish silage ratio in experimental feeds during the current study. The improvements in feeding and growth performance which demonstrated in the current study with addition of up to 50% of fish silage to replace fish meal protein are in line with other studies (Fagbenro, 1994; Dapkevicius, 2002; Soltan and Al-Laithy, 2008). Ramasubburayan et al. (2013) indicated that high replacement ratios of fish meal by fish silage may require further improvement of feed physical quality. Fagbenro et al. (1998) explained that poor physical quality of silage containing feeds could contribute in considerable losses of nutrients through leaching which decrease its usefulness for fish. Another probable reason for poor growth with higher fish silage inclusions in fish feeds is the high content of free amino acids and hydrolyzed protein which could negatively affect protein stability in feed and its metabolism inside fish as suggested by Soltan and Al-Laithy (2008). Water quality parameters in culture system for the common carp fingerlings (Table 16) do not differ much from that previously mentioned (Table 13) for fry feeding because the same system has been used for both

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experiments. Therefore, the values lied again within the suitable ranges for culture of this species (Rahman et al. 2008; Markovic et al., 2009).

Table 16. Water quality parameters of rearing water for common carp fingerlings during feeding experiment Parameter

FM2

Temperature, ⁰C 27.7 ± 0.743 7.54± 0.131 pH 8.99± 0.203 Oxygen, mg/l 1.81± 0.215 Salinity, ‰ 0.94± 0.101 Nitrate, mg/l 0.038± 0.005 Ammonia, mg/l

Feed type FSa2 FSb2

FSc2

27.9± 0.889

27.7± 0.676

27.8± 0.512

7.66± 0.114

7.81± 0.101

7.99± 0.136

9.02± 0.219

9.01± 0.234

8.85± 0.242

1.80± 0.217

1.83± 0.221

1.79± 0.206

1.09± 0.121

1.34± 0.137

1.61± 0.099

0.039± 0.005 0.055± 0.009 0.067± 0.011 FM2, 100% fish meal; FSa2, 75% fish meal+ 25% fish silage; FSb2, 50% fish meal+50% fish silage; FSc2, 25% fish meal+75% fish silage calculated as per protein content.

However, it is noteworthy that ammonia concentrations are relatively higher than those measured in fry feeding experiment (maximum 0.067 vs. 0.048 mg/l., respectively). As was expected, larger fish size and higher metabolic rates could be the main reasons for this elevation. Although these concentrations do not reach the determined minimum of 0.1 mg/l for sublethal effect as indicated by Gross et al. (2000), cautionary measures should be taken to keep them under strict control when such high ratios of silage are intended for inclusion in fish feed. Frequent water exchange, effective aeration and balanced stocking density of fish as well as improving feed quality can contribute significantly in keeping ammonia levels within the accepted range (Hargreaves and Tucker, 2004; Biswas et al., 2006).

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4.3.6 Apparent Digestibility Coefficients ADCs of Different Nutrients in Common Carp Fingerling Feeds Table 17 presents different apparent digestibility coefficients for various nutrients in common carp fingerlings fed different fish silage ratios. Total ADC increased gradually from 68.62 in FM2 to 70.09% in FSc2 with insignificant difference (p > 0.05) between the basal feed FM2 and FSa2 only. Values of ADC for protein take the same trend as total ADC and increased gradually between 82.55-89.88% in FM2 and FSc2, respectively. All the four experimental treatments were differed significantly (p < 0.05) as for protein ADC. Lipid ADC values have also increased gradually with increasing fish silage addition ratio in feed. It differs significantly (p < 0.05) between the four experimental treatments (70.89-72.59% in FM2 and FSc2, respectively). Finally, ADC values for energy content are also differed significantly (p < 0.05) between the four feeds within 78.14-82.01% in FM2 and FSc2, respectively. Values of ADC for total, protein, lipid and energy are correlated positively and significantly with fish silage ratio in different feeds (r = 0.958, 0.999, 0.988 and 0.994, respectively, p < 0.05). It is obvious that the inclusion of increased ratios of fish biosilage in the common carp fingerling feeds have improved nutrient digestibility in a significant manner which agrees with many previous researches (Fagbenro, 1994; Al-Faraje, 2000; El-Ajnaf, 2009). Borghesi et al. (2008b) report value of 89.1 for protein ADC in Nile tilapia Oreochromis niloticus juveniles fed fermented fish silage which has been added to fish feed at a rate of 20%. They consider this result encouraging for using fermented fish silage as a partial alternative for fish meal in fish diets. El-Ajnaf (2009) report ADC values of 49.76, 83.36 and 73.41 for dry matter, protein and energy in European seabass (Dicentrarchus labrax) fed molasses-sardine fish silage at

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an inclusion rate of 36%. He demonstrate that this inclusion level which represents about 25% of fish meal protein does not result in any adverse effects on feeding and growth parameters of this fish species in comparison with complete fish meal diet. Table 17. Apparent digestibility coefficients ADCs of various nutrients for common carp fingerlings fed different fish silage ratios ADC

Feed type FM2

FSa2

FSb2

FSc2

Total

68.62 ± 1.77a

68.67 ± 1.38a

69.56± 1.57b

70.09± 2.22c

Protein

82.55 ± 3.14a

84.91 ± 3.90b

87.31 ± 4.01c

89.88± 5.36d

Lipid

70.89 ± 1.56a

71.66 ± 1.61b

72.21 ± 2.06c

72.59 ± 1.99d

78.14 ± 2.41a 79.11 ± 2.79b 80.88 ± 3.14c 82.01± 3.65d Energy FM2, 100% fish meal; FSa2, 75% fish meal+ 25% fish silage; FSb2, 50% fish meal+50% fish silage; FSc2, 25% fish meal+75% fish silage calculated as per protein content. Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

Hisano and de Pietro (2013) report ADC values of 63.42, 88.57, 81.6 and 58.7% for total, protein, lipid and energy ADCs, respectively when they add up to 12% of catfish viscera fermented silage in diets for juvenile Nile tilapia. They conclude that this silage can be included in diets for juvenile Nile tilapia up to 12% with no negative interference on growth performance and digestibility as well as reducing the cost of the diets. Several reasons can explain the improvement in nutrient digestibility with the inclusion of fish biosilage in fish feeds. The high quality of protein and lipid content may be the main reason for this improvement. Mild ensiling conditions preserve much of the indispensable amino and fatty acids in comparison with the more sever conditions for fish meal production (Vidotti et al., 2002, 2011; Ennouali et al., 2006). Other reasons are related to the

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organic acids and acid producing bacteria in fermented fish silage mainly LAB and their metabolites which have been proved to be beneficial for digestion especially in stomachless fish like carp and tilapia as well as intestinal microbe community which is reflected on the general health of fish (Arruda et al., 2007; Luckstadt, 2008; Vazquez et al., 2011). Marine biosilage has another peculiarity which is related to the existence of good quantities of the important ω3-fatty acids in comparison with freshwater fish silage (Abbas 2007; Goosen et al., 2014).

4.3.7. Proximate Composition of Common Carp Fingerlings Fed Different Fish Biosilage Ratios Proximate composition of common carp fingerlings based on weight wet before and after feeding on the different experimental feeds is presented in Table 18. Moisture content has decreased from initial 75.51 to 71.19 % in FSc2 with significant differences (p < 0.05) between the initial value and experimental feeds. Protein content show an opposite trend and increased form initial value of 12.89 to 14.76% in FSa2. Significant differences (p < 0.05) in protein content are found between the initial value and the other four experimental feed treatments. Lipid content shows a similar trend as protein and increases from initial 6.61 to 8.75 in FSc2 which both differ significantly (p < 0.05) from each other and from the other three experimental feeds. Ash content decreases from 2.69 to 2.06% in initial fish and FSa2. The initial value is differed significantly (p < 0.05) from the three biosilage treatments. Gross energy content of fish increased from initial 1405 to 1730 Kcal/kg in Fsc2 group. The initial value significantly (p < 0.05) differs from the rest of treatments. A significant positive correlation (r

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= 0.944, p < 0.05) is observed between fish biosilage ratio in feed and gross energy content in fish. Table 18. Proximate compositions (wet weight basis) of common carp fingerlings before and after feeding on different experimental feeds Experimental feeds Parameter, % Initial FM2 FSa2 FSb2 FSc2 a b b b 75.51±2.88 71.77±1.76 71.90±1.94 71.62±1.74 71.19±1.97b Moisture 12.89±1.04a 14.11±0.81b 14.76±0.73b 14.51±0.98b 14.24±1.18b Protein 6.61±1.17a 8.08±0.99b 8.11±1.22b 8.23±1.05b 8.75±1.32c Lipid 2.31±0.76a 3.49±0.66b 3.17±0.83b 3.33±0.63b 3.68±0.71b NFE 2.69±.46a 2.55±0.35a 2.06±0.26b 2.31±0.39b 2.14±0.30b Ash Gross energy, 1405±65.7a 1654±42.6b 1680±36.5b 1684±42.9b 1730±46.1c Kcal/kg FM2, 100% fish meal; FSa2, 75% fish meal+ 25% fish silage; FSb2, 50% fish meal+50% fish silage; FSc2, 25% fish meal+75% fish silage calculated as per protein content. Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

The results of the present study agree well with several previous studies which indicate improvements in biochemical composition of fish fed on diets containing fish silage as a partial replacement of fish meal. Fagbenro (1994) prepares 30 and 40% CP diets for juvenile tilapia O. niloticus and catfish Clarias gariepinus, respectively. Diets contained 0, 25, 50, 75 or 100% of fish silage as a major protein alternative for fish meal in 70 days feeding trial. He observes that 50% fish silage ratio attain higher protein content in comparison with the initial fish content in Nile tilapia (14.48 vs. 12.32%). However, body content of lipids is higher in 100% fish silage ratio in comparison with the initial fish content (5.50 vs. 4.06%). Catfish juveniles show slightly different trend where both protein and lipid contents are higher in 100% fish silage ratio treatment in comparison with the initial fish composition (16.40 vs. 13.87 and 6.23 vs. 5.11%, respectively). He

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concluded that addition of increased ratios of fermented fish silage have improved significantly the carcass proximate composition of the two tested fish species. El-Ajnaf (2009) observed slight increases in protein content (15.21-15.75%) and larger increases in lipid content (9.82-11.49%) when fermented apple pomace-sardine fish silage was added at 31.87% to diets for European sea bass Dicentrarchus labrax juveniles during a feeding experiment for 9 weeks. He conclude that fermented fish silage could be compared equally or favorably with fish meal when it used as a partial replacement in fish diets. Ramasubburayan et al. (2013) indicate that the addition of fish silage up to 3% in feeds for common carp fingerlings (average weight 2.5 gm) resulted in increasing protein and lipid contents (12.1- 14.2% and 2.88-3.62%, respectively) upon feeding on 40% CP diet for 30 days. They concluded that the addition of fish silage to the carp fingerling feeds improved both growth parameters and biochemical composition in fish. 4.3.8. Fatty Acid Profiles of Common Carp Fingerlings Fed Different Fish Biosilage Ratios Table 19 presents the fatty acid profiles of oils extracted from common carp fingerlings before and after feeding on different experimental diets containing various ratios of fish biosilage inclusion. Fatty acid profile of the common carp fingerlings from initial and different feeding group was composed of 30 fatty acids; 8 saturated SFA, 8 monounsaturated MUFA and 14 polyunsaturated fatty acids PUFA.

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Table 19. Fatty acid profiles of fish before and after feeding experiment Feed treatment Initial FM2 FSa2 FSb2 FSc2 1.85 1.15 1.11 1.09 1.08 C12:0 0.43 0.23 0.22 0.25 0.21 C13:0 2.79 3.39 3.38 3.44 3.42 C14:0 1.69 1.42 2.45 2.59 2.63 C15:0 19.91 17.91 16.4 16.41 16.37 C16:0 4.75 4.55 3.96 3.21 2.91 C17:0 6.94 7.31 7.34 6.97 6.31 C18:0 1.79 1.68 1.55 1.49 1.44 C20:0 a ab ab b 40.15 37.64 36.41 35.45 34.37b ∑ SFA 1.25 1.17 1.22 1.23 1.22 C14:1 ω7 0.23 0.22 0.22 0.23 0.23 C15:1 ω8 5.25 5.33 5.84 5.11 5.01 C16:1 ω7 2.11 1.84 1.31 1.49 1.74 C17:1 ω8 4.61 4.02 4.42 4.71 4.91 C18:1 ω7 17.22 18.22 18.76 18.82 18.86 C18:1 ω9 4.35 6.35 6.46 6.57 6.59 C20:1 ω9 0.37 0.44 0.48 0.57 0.37 C24:1 ω9 35.39a 37.59ab 38.71b 38.73b 38.93b ∑ MUFA 0.25 0.22 0.2 0.21 0.22 C16:2 ω4 10.38 10.44 10.67 10.93 11.02 C18:2 ω6 2.79 1.78 1.69 1.37 1.39 C18:3 ω3 0.67 0.75 1.02 1.29 1.31 C18:3 ω6 0.55 0.93 1.17 1.39 1.44 C18:4 ω3 0.45 0.69 0.71 0.76 0.76 C20:2ω6 0.39 0.85 0.85 0.94 0.99 C20:3 ω3 0.71 0.66 0.68 0.71 0.72 C20:3 ω6 2.78 2.47 1.93 1.89 1.95 C20:4 ω6 2.01 2.21 2.39 2.96 3.37 C20:5 ω3 0.27 0.48 0.49 0.51 0.61 C21:5 ω3 1.24 1.11 0.71 0.55 0.54 C22:4 ω6 1.33 1.47 1.65 1.59 1.63 C22:5 ω3 0.64 0.71 0.72 0.72 0.75 C22:6 ω3 24.46a 24.77a 24.88a 25.82ab 26.70b ∑ PUFA 7.98a 8.43a 8.96a 9.48ab 10.18b ∑ ω3 ab ab b ab 16.23 16.12 15.72 16.13 16.30ac ∑ ω6 0.492 0.523 0.570 0.588 0.625 ω3/ω6 1.491 1.657 1.746 1.821 1.910 USFA/SFA FM2, 100% fish meal; FSa2, 75% fish meal+ 25% fish silage; FSb2, 50% fish meal+50% fish silage; FSc2, 25% fish meal+75% fish silage calculated as per protein content. Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different. Fatty acid

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Palmitic acid (C16:0) is the major saturated fatty acid in carp oils (19.9116.37 in initial fish and FSc2 feed treatment) which decreases gradually with increasing fish biosilage ratio in feed. Oleic acid (C18:1 ω9) is the main monounsaturated fatty acid in carp oil which increases steadily with fish biosilage inclusion ratio from 17.22% in initial fish to a maximum of 18.86% in FSc2 feed treatment. Linoleic acid (C18:2 ω6) is the principal polyunsaturated fatty acid in carp oil which comprises a minimum of 10.38% in initial fish and increased gradually with fish biosilage inclusion ratio to reach a maximum of 11.02% in FSc2 feed treatment. EPA and DHA polyunsaturated fatty acids, which are of special importance for fish nutrition, comprise 2.01-3.37 and 0.64-0.75% of carp oil fatty acid composition, respectively. EPA shows greater increase rate than DHA (67.7 vs. 17.2%, respectively) with increasing inclusion ratio of fish biosilage in fish feeds. Many significant differences (p < 0.05) are detected in SFA, MUFA, PUFA and ω3 contents mostly between the corresponding levels in initial fish and the higher fish silage inclusion ratio in FSc2 feed treatment. Ratios of ω3/ ω6 and unsaturated USFA/ SFA fatty acids are also increased gradually with the increasing fish biosilage inclusion ratio (0.492-0.625 and 1.491-1.910, respectively). Fish depend on dietary fatty acids (FA) to support their physiological condition and health. Fatty acids (FA) play a major role in the nutrition of fish and humans. Omega-3 (ω-3) and omega-6 (ω-6) polyunsaturated FA (PUFA), including eicosapentaenoic (20:5 ω-3, EPA), docosahexaenoic (22:6 ω-3, DHA), and arachidonic acid (20:4 ω-6, ARA) are particularly important for somatic growth, reproduction, and general health of freshwater fish. As is the case for almost all animals, the ability of fish to bioconvert essential precursor PUFA is very limited and also depends on dietary supply

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of target PUFA. Moreover, from a human consumption perspective, the PUFA composition in fish strongly determines their nutritional quality since fish are very important in supplying particularly ω-3 PUFA for humans (Glencross, 2009; Manjappa et al., 2011; Bohm et al., 2014). Steffens and Wirth (2007) investigated the fatty acid composition of common carp (C. carpio) and tench (Tinca tinca) and showed that different methods of rearing and feeding cause substantial variations in the proportions of the n-6 and n-3 polyunsaturated fatty acids of these fish species. Carp reared on the basis of natural food in ponds exhibit high contents of n-6 and n-3 fatty acids in their muscle triacylglycerols TAG. On the other hand, carp fed supplementary wheat in ponds result in somewhat lower levels of these essential fatty acids. High amounts of ω-3 fatty acids can be found in carp fed high energy diets containing high levels of fish oil. They obtain analogous results in experiments with tench reared under different nutritional conditions. While rearing on the basis of only natural food in ponds as well as feeding supplementary wheat yield similar levels of ω-3 and ω-6 polyunsaturated fatty acids, they record higher contents of ω-3 fatty acids in tench fed pellets. Finally, they demonstrate that experiments with different cultured fish species prove that the fatty acid composition of the edible parts can be influenced by the diet. Therefore, a finishing diet with a suitable fatty acid profile can be used to improve the nutritional quality of fish products of farmed origin. Bohm et al. (2014) investigate diet effects on the composition of polar and neutral lipid fatty acids (PLFA and NLFA, respectively) in eight different tissues (dorsal and ventral muscle, heart, kidney, intestine, eyes, liver and adipose tissue) of the common carp. Two-year old carp are exposed to three diet sources (i.e., zooplankton, zooplankton plus supplementary

118


feeds containing vegetable, VO, or fish oil, FO) with different FA composition. The PLFA and NLFA response has been clearly tissue-specific after 210 days of feeding on different diets. PLFA are generally rich in omega-3 polyunsaturated FA and only marginally influences by dietary FA, whereas the NLFA composition strongly reflected dietary FA profiles. However, they indicate that NLFA composition in carp tissues varies considerably at low NLFA mass ratios, suggesting that carp is able to regulate the NLFA composition and thus FA quality in its tissues when NLFA contents are low. They conclude that FO were three times more retained than VO as NLFA particularly in muscle tissues, indicating that higher nutritional quality feeds are selectively allocated into tissues and thus available for human consumption. Goosen et al. (2014) conduct a study to evaluate fish silage oil recovered from rainbow trout processing waste as an alternative to conventional pelagic fish oil in formulated diets for Mozambique tilapia Oreochromis mossambicus and to determine the effects on fillet fatty acid profile. They carry out a feeding trial with the experimental treatment incorporating silage oil, and a control incorporating commercial pelagic marine fish oil. Silage oil successfully substitute the commercial oil with no negative effects on production parameters. The silage oil proves to be a good source of polyunsaturated fatty acids (36.9 g/100 g total fatty acids). They conclude that rainbow trout silage oil is cost-effective alternative dietary oil for tilapia diets, with advantages over some conventional fish oils. The results of the current study show clearly that biosilage of marine by-catch fish containing good fatty acid profile and proportions of mono- and polyunsaturated fatty acids especially ω-3 is reflected on fatty acid composition of fish fed biosilage feeds. These observations can make the basis for further

119


investigation on the enrichment of cultured freshwater fish with this group of nutritionally important fatty acids during the finishing stage of culture period by using marine fish biosilage which is a rich dietary source in these essential fatty acids as shown previously in section 4.3.2 and Table 9. This clearly coincides with many previous studies that reach the same conclusion about the effect of dietary lipid composition on common carp fatty acid profile (Mraz, 2011; Aprudu et al., 2012; Trbovic et al., 2013).

4.3.9. Lipid and Glycogen Contents in Experimental Fish Table 20 presents lipid and glycogen contents in muscle and liver of common carp fingerlings before and after feeding on different experimental feeds with the studied fish silage ratios. Lipid content in the muscle of fish increased by 45.7% from initial value of 4.33 to a maximum of 6.31% of wet weight in FSc2 feed treatment. The initial value is the only one differs significantly (p < 0.05) from the four feed treatments. Lipid content in fish liver shows lower increasing rate by 31% compared to muscle lipid with a similar trend increasing from the initial value 7.14 to 9.35% in FM2 feed treatment. The only significant difference (p < 0.05) was observed between the initial value and the other four feed treatments. Glycogen content in fish muscle has been more than doubled (119% increasing rate) from the initial value of 2.07 to 4.54 mg/gm in FM2 feed treatment. Initial value differed significantly (p < 0.05) from other four feed treatments which do not differ significantly (p > 0.05) from each other. Liver glycogen contents in fish increases with lower rate in comparison with muscle glycogen (45.3% increasing rate) from an initial value of 27.1 to 39.4 mg/gm in FSc2 feed treatment. Significant differences are observed only between the initial value and the other four experimental feeds.

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Table 20. Lipid and glycogen contents in muscle and liver of common carp fingerlings before and after feeding on experimental feeds Feed treatment Parameter

Organ Initial

Lipid, % wet wt. Glycogen, mg/gm

FM2

FSa2

Muscle 4.33± 0.84a 6.11± 0.94b 6.18± 1.07b Liver

FSb2

FSc2

6.22±0.89b

6.31± 1.33b

7.14± 0.97a 9.35± 1.32b 9.25± 1.46b 9.22± 1.25b 9.11± 1.33b

Muscle 2.07± 0.64a 4.54± 0.88b 4.32± 0.95b 4.28± 1.13b 4.42± 0.98b Liver

27.1± 4.23a 39.1± 5.35b 38.8± 6.01b 37.7± 5.83b 39.4± 5.74b

FM2, 100% fish meal; FSa2, 75% fish meal+ 25% fish silage; FSb2, 50% fish meal+50% fish silage; FSc2, 25% fish meal+75% fish silage calculated as per protein content. Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

Ahmad et al. (2012) conduct a study aiming to determine a feed formulation with best protein to energy ratio which would result in better liver composition and enzyme activity of Cyprinus carpio communis. Fingerlings (average weight 1.64 gm) are fed on four different formulated feeds and a control feed 6% of their body weight, three times a day, during 90 days. Feeds were formulated by using ground nut oil cake, mustard oil cake, rice bran, wheat bran, fish meal and soybean meal in order to suffice the balanced need of protein and energy of the common carp. They report a significant increase in liver lipid content with the increase in dietary carbohydrate level. They conclude that a diet containing 40% protein, 9.31% lipid and 10.08% carbohydrate is the best one for a more profitable and successful culture of the common carp. These results support the findings of the current study that the formulated isonitrogenous and isocaloric feeds did not significantly affect the proximate composition of liver in the common carp fingerlings.

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Lipid reserves in fish can be affected by many factors like age, sex, maturity stage, nutrition, physiological status, health condition and stressors (Silkina et al., 2007; Mraz, 2011). Lower lipid contents in muscle and liver of initial fish may partially reflect the smaller size of fish which could influence its biochemical composition, but the most probable explanation could be related to the nutritional status of fish. Fingerlings prepared for sale in outdoor stocking ponds usually do not receive adequate feed quantity for economical and water quality reasons. They depend largely on natural food in ponds which could fluctuate considerably in quantity and quality. However, fish will replenish its stores of biochemical components as soon as it receives suitable supplemental feeding (Sargent et al., 2002; Hardy, 2012). The same may be applied to glycogen contents, keeping in mind the limited capability of the common carp to metabolize carbohydrate ingredients (Takeuchi et al., 2002). Carp liver has a very limited ability to deal with high carbohydrate levels so it converts high proportions into lipids and remobilizes glycogen to other organs while keeping homeostatic levels (Ahmad et al., 2012; Diricx et al., 2013). Lipid and glycogen reserves in muscle and liver of fish could also reflect the swimming activity where the common carp is known as slow swimming fish, therefore high levels could not be expected in comparison with more active fish (Liew et al., 2012). It is noteworthy that the addition of fish silage does not significantly affect the fish reserves of lipid and glycogen in comparison with control feed (0% fish silage). This agrees well with many previous researchers who indicated that addition of fish silage in feeds for common carp and other species did not result in any adverse effects on the biochemical composition of fish (Fagbenro, 1994; El-Ajnaf, 2009; Ramasubburayan et al. 2013). The common carp has shown to accumulate more lipids with age if adequate

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feeding is supplied especially fish oil (Aprudu et al., 2012; Bohm et al., 2014). This agrees with the increased fish oil proportion in feeds with increasing inclusion level of fish biosilage as shown previously in section 4.3.4.1 and Table 14.

4.4. General Hematology and Plasma Biochemistry of Common Carp Fingerlings Fed on Experimental Feeds General hematological indices (RBC, WBC, Hb and Hct) of common carp fingerlings before and after feeding on experimental feeds which contain different inclusion ratios of fish biosilage are presented in table 21. RBC counts in initial fish averaged 2.251 x106 /mm3 and increased by 69.3% to a maximum of 3.812 x106 /mm3 in FSc2 feed treatment. WBC counts show similar, though lower, trend of increasing by only 11.3% from an initial value of 1.49 x104/mm3 to 1.66 x104 /mm3 in FM2 feed treatment as well. Hemoglobin Hb concentration increases from an initial value of 7.81 to 11.18 gm/dl in FSc2 feed treatment (43.1% increasing rate). Similar increases are observed in hematocrit values which increases from an initial value of 19.88 to 27.55% in FSc2 feed treatment (38.6% increasing rate). Initial values of all the investigated general hematological indices are significantly different (p < 0.05) from those of the four experimental diets which in turn do not differ significantly (p > 0.05) from each other. There are significant positive correlations between RBC counts and each of Hb concentration (r = 0.999, p < 0.05) and hematocrit Hct (r = 0.997, p < 0.05). Hb and Hct values are significantly and positively correlated also (r = 0.999, p < 0.05).

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Table 21. General haematology of common carp fingerlings before and after feeding on experimental feeds Feed treatment Parameter

Initial FM2

FSa2

FSb2

FSc2

RBC x 106/mm3 WBC x 104/mm3

2.251± 0.32a

3.808± 0.19b

3.791± 0.18b 3.799± 0.19b 3.812± 0.16b

1.492± 0.47a

1.661± 0.41b

1.632± 0.44b 1.599± 0.40b 1.658± 0.39b

Hb gm/dl

7.81± 0.91a

11.07± 0.65b

10.99± 0.62b 11.05± 0.71b 11.18± 0.69b

Hct, %

19.88± 2.79a

27.41± 2.28b

26.89± 2.31b 26.97± 3.05b 27.55± 2.99b


Abdelghany (2002) shows that replacement of fish meal partially or totally by Gambusia meal in diets for the common carp (average weight 5 gm) do not significantly affect RBC counts (3.08-3.17 x 106/mm3), but it significantly

improves

hematocrit

values

(27.53-35.07%).

These

improvements are close to the findings of the current study in fish groups fed different experimental diets. Moradi et al. (2013) conduct a study to evaluate the hematological and biochemical changes induced by replacing fish meal with plant protein in the common carp Cyprinus carpio which is fed for 8 weeks. They indicate that experimental diets show significant differences in hematocrit and hemoglobin, whereas no significant differences are found in white and red blood cell counts. They conclude that the maximum levels of fish meal replacement by corn gluten and sesame oil cake in diets of Cyprinus carpio could be 68 % of the total protein sources in diet without significant alterations in hematological indices in fish. Their values reported for the fish fed on fish meal diet are very close to the findings of the current study for the fish fed the four experimental diets where fish meal is replaced

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by various levels of fish biosilage. Nasir and Al-Sraji (2013) investigate the effects of different dietary protein and fats on some blood parameters in the common carp fingerlings (average weight 13 gm) which are reared in floating cages for 180 days. They indicate that the fish fed on high fish meal diet containing 23.68% dietary protein had hemoglobin levels, hematocrit, RBC and WBC counts higher than those fed on lower protein level of 13.82%. They conclude that in order to prevent and adverse effects on fish hematology, diets containing lower than 23% dietary protein should be avoided. This supports the findings of this study in that replacing fish meal by fish biosilage on protein content basis in isonitrogenous feeds (35% CP) does not significantly affect fish fed on the various experimental diets. Table 22 presents some blood plasma biochemistry indices of the common carp fingerlings before and after being fed on experimental diets which contain different inclusion ratios of fish biosilage. Total plasma protein shows significant (p < 0.05) increases from an initial value of 3.35 to 4.36 gm/dl in FSc2 feed treatment (30.1% increasing rate). Plasma albumen Alb concentration shows similar trend, increasing significantly (p < 0.05) by 73.2% (initial 1.68 to 2.91 gm/dl in FSc2 feed treatment) and represents the main contributor in rising levels of total plasma proteins. In contrast, plasma globulin Glob levels decreased significantly (p < 0.05) by 18% from an initial value of 1.67 to 1.37 gm/dl in FSa2 feed treatment. Ratio of Alb/Glob increased significantly (p < 0.05) by 164% (initial 1.01 to 2.09 in FM2 feed treatment). Plasma lipid indices show rather similar trends in comparison with plasma proteins. Total plasma cholesterol concentrations decrease significantly (p < 0.05) from an initial value of 255.3 to 178.2 mg/dl in FSc2 feed treatment (30.2% decreasing rate). Concentrations of plasma

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triglycerides decrease further by 41.5% from an initial value of 121.6 to 71.14 mg/dl in FSc2. HDL cholesterol levels show an opposite trend and increase significantly (p < 0.05) from an initial value of 79.15 to 118.2 mg/dl in FSc2 (49.3% increasing rate). LDL cholesterol levels decrease significantly (p < 0.05) by 69.9% from an initial value of 151.8 to 45.77 mg/dl in FSc2 feed treatment. As VLDL cholesterol is the result of dividing total cholesterol concentration by factor 5, its levels in different treatments follow the same trend of decrease in feed treatments as total cholesterol.

Table 22. Blood plasma biochemistry (proteins, gm/dl and lipids, mg/dl) of common carp fingerlings before and after feeding on experimental feeds Parameter

Experimental feed

Initial

FM2 a

4.26± 0.38

FSa2 b

4.20± 0.31

FSb2 b

4.27± 0.50

FSc2 b

4.36± 0.39b

Total protein

3.35± 0.49

Albumin

1.68± 0.47a

2.88± 0.54b

2.83± 0.48b

2.88± 0.51b

2.91± 0.46b

Globulin

1.67± 0.25a

1.38± 0.23b

1.37± 0.18b

1.39± 0.25b

1.45± 0.27b

Alb/Glob Total cholesterol Triglycerides

1.01± 0.31a

2.09± 0.37b

2.07± 0.36b

2.07± 0.39b

2.01± 0.29b

255.3± 33.2a

212.1± 29.3b

201.6± 23.7bc 191.9± 25.4bc 178.2± 27.7c

121.6± 25.1a

78.11± 23.1b

75.74± 22.6b

HDL

79.15± 17.9a

101.7± 15.2b

105.4± 15.7bc 111.2± 17.1bc 118.2± 14.3c

LDL

151.8± 11.5a

94.78± 10.4b

81.05± 9.9bc

73.54± 25.2b 65.99± 9.7bc

71.14± 20.9b 45.77± 8.8c

24.32± 5.22a 15.62± 4.17b 15.15± 4.26b 14.71± 5.06b 14.23± 4.89b VLDL FM2, 100% fish meal; FSa2, 75% fish meal+ 25% fish silage; FSb2, 50% fish meal+50% fish silage; FSc2, 25% fish meal+75% fish silage calculated as per protein content. Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

Abdelghany (2002) reports values of the total plasma protein between 2.71 to 4.37 gm/dl in the common carp fingerlings (average weight 5 gm)

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fed on diets containing different levels of Gambusia meal as partial or total replacements of fish meal. These values resemble well those reported in the current study. The study of Moradi et al. (2013) on the hematological and biochemical changes induced by replacing fish meal with plant protein in the common carp Cyprinus carpio fed for 8 weeks, indicate that experimental diets show significant differences in total protein levels while no significant differences are found in cholesterol, albumin and triglycerides. They determine the maximum levels of fish meal replacement by corn gluten and sesame oil cake in diets of Cyprinus carpio at 68 % of the total protein sources of diet without significant alterations in major biochemical indices in fish. Their values reported for fish meal feeding group are comparable to the findings of the current study for the fish fed on the four experimental diets where fish meal is replaced by various levels of fish biosilage. Significant differences in the plasma total protein, total cholesterol and triglycerides are reported in the study of Nasir and Al-Sraji (2013) who investigate the influence of different dietary protein and fats on some blood biochemistry parameters in the common carp fingerlings (average weight 13 gm) reared in floating cages for 180 days. They indicate that the fish fed on high fish meal diet containing 23.68% dietary protein had better blood biochemical profile than those fed on lower protein level of 13.82%. Based on these observations, they conclude that 23% dietary protein is more adequate for growth and health of this fish species under rearing conditions in floating cages. This agrees well with the results of this study that the using of isonitrogenous feeds (35% CP) by replacing fish meal with different levels of fish biosilage on protein content basis does not significantly affect the blood profile of the fish fed on the various experimental diets.

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In addition to the proved adequacy of fish biosilage as a fish meal replacer in the common carp fingerling feeds, it is important to note that low values of hematological and biochemical parameters in initial fish are lower than the normal levels reported for this species indicating some kind of anemia (Tripathi et al., 2004). This phenomenon was attributed to many factors like stress, disease and starvation (Hardy, 2012) which is the most likely reason as indicated previously in section 4.3.4.6. The significant improvements in all the studied blood profile indices of fish which supplemented with different experimental feeds in the current study could support this explanation since all feeds contains suitable quantity and quality of the major nutrients. Previous studies have shown that nutrition has a significant influence on blood profile of the common carp and other cultured species (Kumar et al., 2010; Slawski, 2011; Nasir and Al-Sraji, 2013).

4.5. Histological Study 4.5.1. General Histology of Intestine and Liver in Experimental Fish Figures 4, 5, 6, 7 and 8 illustrate the tissue and cellular components of intestine in fishes of different treatment groups (initial, FM2, Fsa2, Fsb2 and FSc2, respectively). Despite the size variation in some histological features as indicated previously, all histological components are represented in intestine tissue samples. Intestinal mucosa is the inner epithelial layer that covers the intestine lumen surface and serves as a barrier between lumen and intracellular media. It consists primarily of the apicocalyx portion of enterocytes and the mucus matter which is produced by goblet cells. The intestinal epithelial surface is expanded into finger-like villi. The attached basement membrane is the connective tissue layer that supports this layer and separates it from submucosal lamina propria which consists also of a

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connective tissue occupied mainly by lymphatic and blood vessels. The muscular layer consist of circular and longitudinal muscles that encircle the intestine and control its peristaltic movements. At the outer most part, the perimeter of the intestine is lined by the serosa; a thin connective tissue layer which is attached to the mesenteric tissue. The cellular components of intestinal villi are illustrated in figures 4-8 (B). It is composed of goblet cells, enterocytes and blood cells. Goblet cells look rounded from base with barley recognized nucleus. Their numbers are variable and they discrete along the mucosa layer. The enterocytes are more prominent with well stained nuclei. The outer cylindrical part is called the apicocalyx which makes the larger part of cell where their nucleus and nucleolus could be easily recognized in ordinary stained sections. The numbers of goblet cells are high normally and reflecting the feeding activity of fish and feed composition. It arranged in two or more adjacent layers. Blood cells are found normally since blood vessels exist inside the lamina propria layer. No infiltration of leucocytes is observed which is considered as a histopathological marker of inflammation. The results of the current study agree with many previous studies which indicate that the partial replacement of fish meal with fish silage does not alter the gut histology or function in various fish species (Fagbenro and Jauncey, 1995b; Fagbenro et al., 1997; Reyes-Becerril et al., 2012). Fagbenro (1994) uses fermented fish silage (0, 25, 50, 75 or 100%) to prepare 30 and 40% CP diets for juvenile tilapia O. niloticus and catfish C. gariepinus, respectively as a major protein alternative for fish meal in 70 days feeding trial. He indicates that this replacement does not result in any adverse effect or histopathological condition for fish based on examination of intestine and liver histology. El-Ajnaf (2009) also observe no adverse

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influence on gut histology of European seabass D. Labrax fed on different replacement levels of fermented fish silage prepared using molasses or apple pomace. Raskovic et al. (2011) indicate the importance of using histopathological methods to assess the effect of feed on common carp intestine and liver. They mention that fish fed on 30% soybean meal as fish meal replacer suffer from transient enteritis. They ascribe this adverse effect to the existence of anti-nutritional factors in soybean meal. However, they conclude that the common carp need a period of approximately one month to adapt to this component in diet then the enteritis is self-relieved. No inflammatory reaction is noticed in the current study since all experimental feeds contain 15% soybean meal in addition to fish meal or fish silage. Figures 9, 10 and 11 illustrate liver histology of fish in initial group and the four different feed treatment groups. As shown in Figure 9 (A), hepatocytes are polyhedral, globular or ovoid in shape with smaller size than other fish groups.. Hepatocytes are arranged in groups which are separated from each other by small sinusoids. Hepatic sinusoids are collect together to form hepatic veins. Hepatocyte nucleus is prominent and occupies large proportion of cellular cytoplasmic area. Small number of macrophage aggregates is observed in dark brown colour and discrete through the hepatic tissue. The gross histology of liver in initial fish group, although normal in general, reflect poor growth and the small cytoplasmic area of hepatocytes may indicate low cellular reserves of glycogen and lipid.

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Figure 4. (A). Cross section in fish intestine (initial group) basic structure of villi. L, intestine lumen; M, mucosa; Bm, basement membrane; Sm, submucosa; Ms, muscularis; Se, serosa (HE, 400X). (B). Villus cellular components. Gc, goblet cells; Lp, lamina propria; Ec, enterocytes; Er, erythrocytes (HE, 1000X).

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Figure 5. (A). Cross section in fish intestine (FM2 group, 0% fish silage) basic structure of villi. L, intestine lumen; M, mucosa; Bm, basement membrane; Sm, submucosa; Ms, muscularis; Se, serosa (HE, 400X). (B). Villus cellular components. Gc, goblet cells; Lp, lamina propria; Ec, enterocytes; Er, erythrocytes (HE, 1000X).

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Figure 6. (A). Cross section in fish intestine (FSa2 group, 25% fish silage) basic structure of villi. L, intestine lumen; M, mucosa; Bm, basement membrane; Sm, submucosa; Ms, muscularis; Se, serosa (HE, 400X). (B). Villus cellular components. Gc, goblet cells; Lp, lamina propria; Ec, enterocytes; Er, erythrocytes (HE, 1000X).

133


Figure 7. (A). Cross section in fish intestine (FSb2 group, 50% fish silage) basic structure of villi. L, intestine lumen; M, mucosa; Bm, basement membrane; Sm, submucosa; Ms, muscularis; Se, serosa (HE, 400X). (B). Villus cellular components. Gc, goblet cells; Lp, lamina propria; Ec, enterocytes; Er, erythrocytes (HE, 1000X).

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Figure 8. (A). Cross section in fish intestine (FSc2 group, 75% fish silage) basic structure of villi. L, intestine lumen; M, mucosa; Bm, basement membrane; Sm, submucosa; Ms, muscularis; Se, serosa (HE, 400X). (B). Villus cellular components. Gc, goblet cells; Lp, lamina propria; Ec, enterocytes; Er, erythrocytes (HE, 1000X).

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In contrast, histological features in livers of fish in experimental feed treatments (Figures 9B-11) represent more developed structures. Large hepatocytes are prevailed with larger cytoplasmic area denoting better function and storage capacity. Nucleolus can be observed inside many hepatocyte nuclei while some hepatocytes show nuclei in mitotic state (FSb2, Figure 10B). All fish fed experimental diets show 1-3 well developed hepatopancreata with prominent pyramidal cells and well defined nuclei which is elongated in shape and protrude into the hepatopancreatic lumen (Figure 11B). These distinctive structures indicate good exocrine and endocrine functions of liver. No differences in liver histology are observed between the four experimental feed treatments. No histopathological changes like degeneration, necrosis, leukocyte infiltration, haemorrhage, steatosis, tissue or cellular abnormalities are observed in the examined sections even with replacement ratio up to 75% of fish meal by biosilage. Liver histopathological examination is one of the powerful tools that can be used effectively to monitor fish health in general. As liver is the main gland associated with the digestive system in fish, liver histopathological methods can reveal any adverse effects of feed components especially the novel or innovated ingredients (Raskovic et al., 2011, 2013). Many previous studies show that replacing fish meal with fish silage does not lead to any adverse effects on liver histology in several cultured fish species (Fagbenro and Jauncey, 1995b; El-Ajnaf, 2009; Majumdar et al., 2014). This agrees with the results of the current study. Dietary lipids are the major feed constituents reported to influence liver structure and function. Replacement of fish oil completely with different vegetable oils in feeds for different fish species leads to some kind of fatty

136


liver disease known as steatosis (Benedito-Palos et al., 2008; Bell et al., 2010; Szabo et al., 2011). However, this disease is rarely detected in the common carp because it does not store large quantity of lipids in the liver but instead uses visceral lipids as a primary storage site (Yilmaz and Genc, 2006; Steffens and Wirth, 2007; Bohme et al., 2014). On the other hand, the nutritional adequacy of fish biosilage, as indicated by the histological examination of fish intestine and liver in the current study, may be partially ascribed to its content of fish oil which represent about 22% of dry matter with fatty acid profile rich in polyunsaturated fatty acids that are reported to enhance general metabolism, tissue structural integrity, nutritional and health status of fish (Turchini et al., 2009; Torstensen et al., 2010; Fard et al., 2014).

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Figure 9. Fish liver histology. (A). Initial group. (B). FM2 feed group (0% fish biosilage). C, capsule; Hc, hepatocyte; Hp, hepatopancreas; Mm, melanomacrophage; Ss, sinusoid; V, vein. (HE, 400X)

138


Figure 10. Fish liver histology. (A). FSa2 feed group, 25% fish biosilage. (B). FSb2 feed group, 50% fish biosilage. Hc, hepatocyte; Hp, hepatopancreas; Mc, mitotic cell; Mm, melanomacrophage; Ss, sinusoid; V, vein. (HE, 400X)

139


Figure 11. Fish liver histology. (A). FSc2 feed group, 75% fish biosilage. (B). Enlarged hepatopancreas of the same section. Er, erythrocyte; Hc, hepatocyte; Hp, hepatopancreas; Mc, mitotic cell; Mm, melanomacrophage; N, nucleus; Ss, sinusoid; V, vein. (HE; A,400X; B, 1000X)

140


4.5.2. Histological Measurements of Fish Intestine and Liver Components Various measurements of intestinal villi length, width, enterocyte height and goblet cell numbers are presented in Table 23. Villus length increases from an initial value of 42.8 to 142.1 µm in Fsb2 feed treatment representing 232% increasing rate. Villus width also increased, but with lower rate (32%) from an initial value of 37.2 to 49.1 µm in FM2 feed treatment. The ratio between villus length and width was increased from 1.151 to 2.932 in FSc2. Intestinal enterocyte height was increased from an initial value of 2.19 to 4.69 µm in FSb2 feed treatment (114% increasing rate). The average number of goblet cells also increases by 80.4% from an initial value of 3.12 to 5.63 cells/ 100 µm of villus length in FM2 feed treatment. Significant differences (p < 0.05) are observed, for all the measured intestine histological features, between fish in initial group and various experimental feed treatments which do not differ significantly (p > 0.05) from each other. Intestinal villi are folds or envaginations that distribute along the length of fish intestine as an adaptation to increase the surface area for absorption. Its main cellular components are goblet cells which secret the mucus material enveloping intestine luminal surface and enterocytes which are responsible for secretion and uptake processes (Genten et al., 2009). Epler et al. (2009) indicate that enterocyte height and goblet cell count decrease when carp is fed on different vegetable oils (sunflower, rapeseed and linseed oils) instead of fish oil. They explain that the absorptive function of intestine is influenced adversely by the abnormal metabolism of lipids which is reflected on the main cellular components of intestinal lumen and conclude that fish oil is superior in maintaining normal functions on intestine in comparison with vegetable oils. This conclusion seems applicable for the

141


current study where all experimental feeds contain various ratios of fish oil in addition to corn oil and fish do not show any abnormality in intestine histological features except for initial fish group which has been deprived of adequate feeding.

Table 23. Measurements of some intestine tissue and cellular properties of common carp fingerlings before and after feeding on experimental feeds Feed treatment Parameter Initial

FM2

FSa2

FSb2

FSc2

Villus length, µm

42.8± 4.5a

137.6± 8.8b 139.7± 8.1b 142.1± 9.9b 138.1± 7.3b

Villus width, µm

37.2±3.9a

49.1± 5.1b

Villus L/W ratio

47.9± 4.9b

48.7± 5.25b 47.1± 5.02b

1.151± 0.87a 2.802± 1.07b 2.916± 0.99b 2.918± 1.11b 2.932± 1.23b

Enterocyte height, 2.19± 0.91a 4.37± 1.37b 4.41± 1.38b 4.69± 1.69b 4.44± 1.41b µm No. goblet cells 3.12± 1.05a 5.63± 2.19b 5.37± 1.97b 5.22± 2.05b 5.52± 2.22b per 100 µm FM2, 100% fish meal; FSa2, 75% fish meal+ 25% fish silage; FSb2, 50% fish meal+50% fish silage; FSc2, 25% fish meal+75% fish silage calculated as per protein content. Values in the same raw which carry different superscript letters are significantly (p ≤ 0.05) different.

Omar et al. (2012) test a novel yeast co-product obtained from a bioethanol process in which wheat is the predominant feedstock in a series of isonitrogenous (38% crude protein) and isolipidic (8%) diets for juvenile mirror carp (Cyprinus carpio). The fishmeal protein component of a basal diet (control treatment) is effectively replaced by a yeast protein concentrate (YPC) at 7.5, 15, 20, and 50% of total dietary protein. The preliminary histological assessment of intestinal tissues gives no indication of impairment to health, but high YPC inclusion (≥15%) elevate the number of

142


goblet cells present in the posterior intestine after 8 weeks of feeding. They demonstrate that the probable cause may be due to some components (i.e. ßglucans or Mannan oligosaccharide, MOS) within the cell wall of the yeast which has immunostimulatory properties. However, they conclude that up to half the fishmeal protein component within experimental diets for mirror carp can be effectively replaced with a YPC without any obvious adverse effects on health as measured by several physiological and biochemical indices and general tissue morphology. This coincides with the results of the current study where replacing fish meal by different proportions of fish biosilage does not lead to any adverse alterations in intestine histology. Poleksic et al. (2014) find that enterocytes height and intestinal folds length decrease with increasing ratios of soybean meal and soy oil in the common carp fingerling diets. They mention that growth, starvation and feed components are the major factors that affect the development of intestine histological features in fish. This agrees well with the results of the current study as initial fish which presumably suffered from starvation as indicated in the previous sections and have lower values of all measured parameters of intestine histology. These values have significantly improve when fish are fed on experimental feeds in spite of feed type. In general, the results of the current study suggest that fish meal can be partially replaced with fish biosilage without any adverse influence on intestine tissue and cellular dimensions. Table 24 presents the measurements of diameter and area of hepatocytes and their nuclei. Hepatocyte diameter increases from an initial value of 5.91 to 10.07 µm in FSb2 feed treatment which represents an increasing rate of 70.3%. Nucleus diameter is increased by lower rate (38.6%) from an initial

143


value of 2.33 to 3.23 µm in FSc2 feed treatment. Corresponding to hepatocyte diameter, hepatocyte area is increased from an initial value of 110 to 318 µm2 in FSb2 feed treatment (189% increasing rate). Nuclear area followed analogous ascending trend and increased by 94% from an initial value of 17 to 33 µm2 in FSc2 feed treatment. The nuclear area represents 15.5% of hepatocyte area in initial fish group to a minimum of 10% in FSb2 feed treatment which represents a decrease by 35.5%. Similarly to intestine histological measurements, hepatocyte measurements differ significantly (p 0.05) differ from each other.

Table 24. Measurements of hepatocytes in livers of common carp fingerlings before and after feeding on experimental feeds Feed treatment Parameter Initial

FM2

FSa2

FSb2

FSc2

Hepatocyte diameter, µm

5.91± 0.97a

9.39± 1.8b

9.46± 2.0b

10.07± 2.2b

9.95± 1.9b

Nucleus diameter, µm

2.33± 0.3a

3.11± 0.4b

3.09± 0.4b

3.19± 0.5b

3.23± 0.3b

Hepatocyte area, µm2

110± 12a

277± 35b

281± 37b

318± 45b

311± 43b

Nuclear area, µm2

17± 0.4a

30± 0.9b

30± 0.9b

32± 0.9b

33± 0.9b

15.5± 0.6a

11± 0.4b

10.7± 0.5b

10± 0.4

10.6± 0.5b

N/H area , %


Hepatocytes are the basic structural units of fish liver which serves functions similar to those in mammals. Its functions include assimilation of

144


nutrients, production of bile, detoxification and maintenance of the body metabolic homeostasis that includes processing of carbohydrates, proteins, lipids and vitamins. The liver also plays a key role in the synthesis of plasma proteins, like albumin, fibrinogen, and complement factors (Genten et al., 2009). Epler et al. (2009) show that hepatocyte area of carp liver decreases significantly when fish oil is replaced by different mixtures of vegetable oils in fish feeds. They ascribe this observation to an abnormal lipid metabolism which decrease lipid reserves inside hepatocytes confirming the role of fish oil in normal structure and function of hepatocytes. This agree well with the results of the present study where hepatocyte dimensions increase significantly after fish are fed on experimental feeds containing various proportions of fish oil. Omar et al. (2012) replace the fishmeal protein component of a basal diet (control treatment) by a yeast protein concentrate (YPC) at 7.5, 15, 20, and 50% of total dietary protein in diets for juvenile mirror carp (Cyprinus carpio). The examination of liver ultrastructure shows no obvious detrimental changes associated with increased dietary inclusion of the novel yeast product. Light microscopy examination of hepatic tissue shows consistent hepatocyte density, size and nuclear to cytoplasm ratio. They conclude that replacement of fish meal with YPC has no influence on liver cell synthesis in terms of hyperplasia or altered nutritional state. Poleksic et al. (2014) find that hepatocyte cytoplasm area decreases with increasing ratios of soybean meal and soy oil in the common carp fingerling diets. They ascribe this effect to the sensitivity of liver toward vegetable oil sources which may be ameliorated through the inclusion of fish oil. These researches agree with the results of the present study where no significant changes in hepatocyte dimensions are observed after fish are fed on different experimental diets containing fish biosilage. This can give an indication on

145


the suitability of fish silage as a partial replacement of fish meal without any adverse alterations in liver hepatocyte morphometry.

4.5.3. Histochemical Detection of Glycogen and Lipids in Fish Hepatic Tissue. Figures 12, 13 and 14 illustrate glycogen accumulations and some other features in hepatic tissue sections from initial fish group and the other four experimental feed groups. It can be observed from Figure 12A that glycogen content in hepatic tissue from initial group is lower than the other experimental groups depending on the prevailed magenta and pink color intensity and density of dark brown glycogen accumulations. Glycogen accumulations are discrete irregularly through cells and tissue in general. They take different shapes from globular to ovoid or even amorphous. In contrast, color intensity, glycogen accumulation density and distribution patterns were clearly different in experimental feed groups in comparison with initial group (Figures 12B-14). Glycogen accumulations are larger in size probably due to merging of granules from adjacent cells and distributed mainly parallel along sinusoid sides. It can be also observed that cells around hepatopancreata have more pale magenta color denoting poor staining with PAS. Sinusoids are poorly stained with PAS also due to the lack of cellular components. Small numbers of melanomacrophages can be observed distributing discretely through hepatocytes and containing minute glycogen granules. Liver capsule is stained with pale color in comparison with the other tissue components indicating lower reactivity with PAS stain thus lower glycogen content in the fibroconnective tissue.

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Figure 12. Glycogen detection in hepatic tissue. (A) Initial group. (B). FM2 feed group (0% fish biosilage). G, glycogen; Ga, glycogen accumulation; Hc, hepatocyte; Hp, hepatopancreas; Mm, melanomacrophage; Ss, sinusoid; V, vein. (PAS, 400X)

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Figure 13. Glycogen detection in hepatic tissue. (A) FSa2 feed group, 25% fish biosilage. (B). FSb2 feed group, 50% fish biosilage. G, glycogen; Ga, glycogen accumulation; Hc, hepatocyte; Hp, hepatopancreas; Mm, melanomacrophage; Ss, sinusoid; V, vein. (PAS, 400X)

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Figure 14. Glycogen detection in hepatic tissue. FSc2 feed group, 25% fish biosilage. C, capsule; Ga, glycogen accumulation; Hc, hepatocyte; Hp, hepatopancreas; Mm, melanomacrophage; Ss, sinusoid; V, vein (PAS, 400X). Hepatocyte appeared normal in shape so as hepatopancreatic cells while their color intensity varies depending on their position; more peripheral cells have paler color in comparison with more central cells indicating lower glycogen content. The PAS technique is without question the most versatile and widely used technique for the demonstration of carbohydrates or glycoconjugates. The first histochemical use of this technique is for the demonstration of mucin. Subsequently other studies have demonstrated the utility of the PAS technique for demonstration of other carbohydrate-containing molecules, such as glycogen and certain glycoproteins. The technique is based upon the oxidation of carbohydrate by a mild oxidant (periodic acid) to form free aldehyde groups and the reactivity of these groups within carbohydrates with

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the Schiff reagent to form a bright red/magenta end product in which its intensity depends on carbohydrate type and concentration (Layton and Bancroft, 2013). In fish, liver is considered as the main storage site for carbohydrates and thus serves as the biggest source of blood glucose. The capability of fish to utilize carbohydrates varies according to their feeding habits. Generally, the common carp cannot utilize high carbohydrate levels efficiently. The basic energy reserves of glycogen form only 1% of the total body weight. This source is sufficient to provide the energy need for a short time, but not for a long time. The glycogen amount stored in liver depends on the physical, chemical and biological factors faced by the fish. Rapid movement, stress factors or starvation cause glycogen reserves to diminish in liver and subsequently in muscles. It has also been determined by various studies that the hormonal changes in fish affect the conversion of liver glycogen into blood glucose (Al-Dubaikel et al., 2009; Coban and Sen, 2011; Liew et al., 2012). Lower glycogen reserves in initial fish are agreed with low glycogen concentrations due to inadequate nutrition as previously indicated in the current study. However, fish replenish their liver glycogen stores after resuming feeding on various experimental feeds. Fish meal partial replacement with fish biosilage do not influence carbohydrate metabolism and subsequently glycogen stores in hepatic tissue. This seems in accordance with biochemical analysis (Table 20). Carbohydrate do not constitute an important proportion of fish biosilage composition in contrast to protein and lipid. Therefore, no direct effect of fish silage on carbohydrate metabolism can be expected although different feed components can interact in many ways. This agrees with previous studies showing normal liver glycogen and

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function in different fish species that is fed on fish silage as a protein alternative for fish meal (Fagbenro, 1994; El-Ajnaf, 2009; Ayoola, 2010). Fish liver is the central site for the storage of lipids, carbohydrates, lipidsoluble vitamins and iron. It contributes to digestion, reproduction, immune defense and detoxification. Therefore, any disturbance in the structure or function of liver will influence these vital activities including lipid metabolism. This is very indicative to select fish liver as a preferred organ for histopathological examinations especially to assess the effects of different feed ingredients (Raskovic et al., 2011, 2013). For the detection of lipid distribution in hepatic tissue from initial fish group and the four experimental feed groups, two staining techniques are used i.e. osmium tetraoxide, OsO4 and Sudan black B, SBB (Figures 15-18). Initial fish show smaller hepatocyte volume and high nuclear area as previously indicated. Nuclei are stained in black as a result of lipid content in the nuclear membrane. Vacuolation is abundant among hepatocytes. Veins and sinusoids are stained in black because of blood lipids (Figures 15A and 16A). In another hepatic tissue section from the same fish group which is stained with lipid-soluble SBB stain, lipids appear to deposit inside branched minute fissures of irregular shape. Vacuolation is also visible with very poor staining while nuclei appear darker again owing to nuclear membrane lipids (Figure 15B). In FM2 feed group, hepatocytes and their cytoplasmic area are larger and the nuclei occupy the lower proportion of the cellular area. Veins and sinusoids are wider and more widespread through the hepatic tissue. Fewer vacuolations and more lipid depositions are observed inside hepatocytes in comparison with the initial fish (Figure 16A). With higher magnification

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power, lipids are deposited also in teeny branched fissures which look more filled with darker stain than the previous group. Vacuolation is still visible and darker stained nuclei are observable also through the tissue section (Figure 16B). Feed groups FSa2 and FSb2 fed on 25 and 50% fish biosilage, respectively, are very similar in lipid distribution through their hepatic tissue. Veins and sinusoids are larger and more numerous. No obvious vacuolation can be detected and lipid deposits are more densely stained and homogenously distributed through hepatocytes (Figure 17A and B, respectively). The last feed group FSC2 fed on 75% fish biosilage shows a rather denser lipid depositions into hepatocytes in comparison with the other fish groups. The dark lipid depositions are distributing evenly inside hepatocytes through the hepatic tissue with no obvious vacuolation or heavy lipid accumulations suggestible for steatosis. Veins and sinusoids are wide and numerous. The shape of hepatocytes and their nuclei show no abnormality in comparison with the other fish groups (Figure 18A). In SBB stained section (figure 18B), lipid appear also to distribute more evenly and darkly. Numerous minute fissures filled with dark lipid material can be observed through the hepatic tissue section. Hepatocytes and their nuclei are more evidently stained than previous similar sections. Melanomacrophages and macrophage centers are observable staining deeply with SBB stain, suggesting effective immunocompetence of fish. Osmium tetraoxide is water soluble oxidant which reacts specifically with the unsaturated bonds within lipids and phospholipids. This reaction produces stable non-dissolvable black complexes which cannot be lost from tissue during section preparation steps. In contrast, Sudan Black B is a non-

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fluorescent, relatively thermostable lysochrome (fat-soluble dye) diazo dye used for staining of unsaturated esters, neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections. This method is not true histochemical technique because it depends only on stain solubility in fat giving it easily recognizable dark blue color. Therefore, SBB stained sections should be mounted in aqueous media like glycerin jelly instead of ordinary solvent containing media like Canada balsam and DPX (Bancroft and Stonard, 2013). Lipid is very important macronutrient in fish nutrition. In addition to its major role as an energy producing material, it serves as a precursor for many biologically active molecules like prostaglandin hormones. Essential fatty acids especially of ω6 and ω3 groups have vital roles in lipid metabolism like pre-oxidation of microsomal fats in the liver (Sargent et al., 2002). The initial fish group shows a deficiency of lipid depositions in hepatocytes. In addition to previously-examined physiological and biochemical indicators, this suggests that fish do not receive adequate feeding before the beginning of feeding experiment. Several authors indicate the effect of starvation on lipid stores in fish liver which decrease slowly but not depleted in live fish due to metabolic regulation regarded as a survival strategy. However, with adequate feeding , the liver resumes its reserves from nutrients and normal function while hepatocytes grow to their normal number and volume (Fontagne et al. 2000; Yee, 2005; Mraz, 2011). This agrees well with the findings of the current study where fish in the experimental feed groups differed in their lipid deposition and lower vacuolation in comparison with the initial group.

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Figure 15. Lipid detection in hepatic tissue. Initial fish group. (A) Osmium tetraoxide stain (B) Sudan black B stain. Hc, hepatocyte; L, lipid; N, nucleus; Ss, sinusoid; V, vein; Va, vacuolation. (1000X).

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Figure 16. Lipid detection in hepatic tissue. FM2 group (0% fish biosilage). (A) Osmium tetraoxide stain, 400X (B) Sudan black B stain, 1000X. Hc, hepatocyte; L, lipid; N, nucleus; Ss, sinusoid; V, vein; Va, vacuolation. .

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Figure 17. Lipid detection in hepatic tissue. (A) FSa2 group (25% fish biosilage). (B) FSb2 group (50% fish biosilage). Hc, hepatocyte; N, nucleus; Ss, sinusoid; V, vein. Osmium tetraoxide stain, 400X.

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Figure 18. Lipid detection in hepatic tissue. FSc2 group (75% fish biosilage). (A) Osmium tetraoxide stain, 400X (B) Sudan black B stain, 1000X. Hc, hepatocyte;L, lipid; Mc, macrophage centre; Mm, melanomacrophage; N, nucleus; Ss, sinusoid; V, vein.

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Fish silage is reported as a good alternative for fish meal in diets for several fish species depending on liver histology examination. It performs better than the other fish meal alternatives especially those of plant origin. Lipidosis or hepatic steatosis is frequently associated with fish fed high dietary vegetable oils but not fish oil (Yilmaz and Genc, 2006; Kumar et al., 2010). The good marine fish oil content and quality in fish meal and biosilage used in feed formulation and the fact that the common carp depends on visceral tissues as a lipid storage site instead of the liver, support our finding that no hepatic steatosis is observed in the examined liver sections in the current study (Steffense and Wirth, 2007; Ramasubburayan et al., 2013; Goosen et al., 2014). Additionally, higher fish biosilage inclusion level (75%) and thus higher marine fish oil proportion enhance cellular immunity as observed through melanomacrophages and macrophage centers in hepatic tissue in the current study. Similar observations are reported by Bohme et al. (2014), Fard et al. (2014) and Poleksic et al. (2014) in the common carp and other fish species fed diets containing different ratios of fish oil and various vegetable oils. In conclusion, it can be indicated from the previously reviewed researches in this section and the results of the histological examination of intestine and liver tissues of common carp fingerlings which fed on different inclusion levels of fish biosilage as an alternative to fish meal protein in the current study that this replacement does not cause any adverse alterations in these tissues on the microscopic level. Glycogen and lipid detection demonstrate normal distribution of these nutrients in hepatic tissue similarly to fish fed fish meal feeds which further support this conclusion.

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CHAPTER FIVE: CONCLUSIONS & RECOMMENDATIONS

CHAPTER FIVE: CONCLUSIONS AND RECOMMENDATIONS

5.1. Conclusions From the results of the present study, it can be concluded that: 1- Marine by-catch fish is a valuable source of nutrients like protein and oil which can be recovered through fish meal production or ensiling process to serve as fish feed ingredients. 2- Date fruit residues are very suitable carbohydrate substrate for fermenting fish biosilage when added at 10% level because of its high sugar, low fiber, availability and cheap price in comparison with similarly used substrates. 3- Domestic vinegar is an adequate inoculation and acidification medium for biosilage fermentation despite its high water content which elevates its effective addition level to 20%. 4- Citric acid is very cheap, available and non-harmful organic acid which can be used effectively as a main starting acidifier in biosilage fermentations at 2% level. 5- The addition of antioxidants and antimycotic agents considerably improves fish biosilage quality especially conserving lipid quality and lowering histamine levels. 6- Marine fish biosilage has a very suitable amino acid profile for use as fish feed ingredient containing good quantities of many essential amino acids as revealed by HPLC analysis. 7- Fatty acid profile of fish biosilage, as demonstrated by GC-MS analysis, is very rich in unsaturated fatty acids especially PUFAs (EPA and DHA) which play a vital roles in fish feeding.

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8- Marine fish biosilage performs equally to fish meal in feeds formulated for common carp advanced fry at up to 75% fish meal replacement ratio as evidenced from feeding and growth results. 9- Replacement of fish meal by up to 75% of marine biosilage in feeds formulated for the common carp fingerlings does not cause significant influences on feeding and growth parameters nor on the examined biochemical and hematological indicators. However, some important physical quality parameters of feed such as settling velocity and floatability are adversely affected by silage addition. 10-Histological evidence supports the other indicators of the adequacy of marine biosilage as fish meal alternative in feeds for the common carp fingerlings. Gross histological examination of intestine and liver, histochemical detection of glycogen and lipid in hepatic tissue show no abnormal alterations in comparison with fish meal feeding fish.

5.2. Recommendations The current study recommends the following: 1- The availability of good quantities of date fruit residues and its cheap price make an economic incentive to exploit this material in fish feeding in Iraq and may be some neighboring countries, indirectly this time, through serving as a carbohydrate source in fish ensiling process. This will attain double advantage by its sound contribution to the valorization of this agricultural waste while resolving an environmental issue represented by disposal problem. 2- Research on fish biosilage should be expanded to include other fishery materials like fish by-catch from freshwater fisheries, fish and shrimp

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wastes to be used as protein sources in animal feeding especially for aquafeeds. 3- Further research is needed to address some problems which arouse from the current study like using domestic vinegar with its high water content as an inoculant. Available and economically viable alternative inoculants should be explored. Feed extrusion technology should be adopted instead of conventional steam pelleting for feeds which include high fish silage contents to improve floatability that adversely affected by fish silage addition. 4- This research should extend to feeding fish at farm level in earthen ponds or floating cages to assess the practical efficiency of fish biosilage as feed ingredient for the common carp and other cultured species. 5- Marine fish biosilage can be used as a fish oil enrichment agent to improve freshwater fish lipid profile during the final stages of culture process especially its content of PUFA fatty acids which is proved to be very beneficial for human health. 6- Complete replacement of fish meal by fish silage is suggested to be an option in further detailed studies keeping in mind the high oil level and histamine content in fish biosilage which must be effectively controlled. Measuring histamine levels in fish blood plasma can be one targeted biomarker if the suitable technology is provided in future.

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CHAPTER SIX: REFERENCES

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‫اﻟﺨﻼﺻﺔ‬

‫اﻟﺨﻼﺻﺔ‬ ‫أﺟﺮﯾﺖ اﻟﺪراﺳﺔ اﻟﺤﺎﻟﯿﺔ ﻟﺘﻘﯿﯿﻢ اﺳﺘﺨﺪام ﺳﯿﻼج ﺳﻤﻚ اﻟﺼﯿﺪ اﻟﺒﺤﺮي اﻟﻌﺮﺿﻲ اﻟﻤﺘﺨﻤﺮ ﺑﺎﺳﺘﻌﻤﺎل‬ ‫ﺛﻔﻞ اﻟﺘﻤﺮ ﻛﻮﺳﻂ ﻛﺮﺑﻮھﯿﺪراﺗﻲ ﻓﻲ ﺗﻐﺬﯾﺔ زرﯾﻌﺔ وإﺻﺒﻌﯿﺎت أﺳﻤﺎك اﻟﻜﺎرب اﻟﺸﺎﺋﻊ ‪Cyprinus‬‬ ‫‪ .carpio L.‬وھﺪﻓﺖ اﻟﺪراﺳﺔ أﯾﻀﺎ ﻟﺘﻘﯿﯿﻢ ﺑﻌﺾ اﻟﺘﺄﺛﯿﺮات اﻟﻔﺴﻠﺠﯿﺔ واﻟﻨﺴﺠﯿﺔ ﻹﺿﺎﻓﺔ ھﺬا اﻟﺴﯿﻼج‬ ‫اﻟﺴﻤﻜﻲ اﻟﺤﯿﺎﺗﻲ ﻓﻲ أﻋﻼف اﻷﺳﻤﺎك‪ .‬ﺗﺄﻟﻔﺖ ﻋﯿﻨﺔ ﺳﻤﻚ اﻟﺼﯿﺪ اﻟﺒﺤﺮي اﻟﻌﺮﺿﻲ ﻣﻦ ‪ 23‬ﻧﻮﻋﺎ ﻣﻦ‬ ‫اﻷﺳﻤﺎك اﻟﺒﺤﺮﯾﺔ اﻟﺘﻲ ﺗﻨﺘﻤﻲ إﻟﻰ ‪ 18‬ﻋﺎﺋﻠﺔ‪ .‬أﺟﺮﯾﺖ ﺳﻠﺴﻠﺔ ﻣﻦ اﻟﺘﺠﺎرب ﻟﺘﺤﺪﯾﺪ اﻟﻈﺮوف اﻟﻤﺜﻠﻰ‬ ‫ﻹﻧﺘﺎج اﻟﺴﯿﻼج اﻟﺴﻤﻜﻲ اﻟﻤﺘﺨﻤﺮ‪ ،‬وأﺳﺘﻨﺪ اﻟﺘﻘﯿﯿﻢ ﻋﻠﻰ ﻋﺪد ﻣﻦ اﻟﺘﺤﻠﯿﻼت اﻟﻜﯿﻤﻮﺣﯿﻮﯾﺔ‪ .‬أن ﺛﻔﻞ اﻟﺘﻤﺮ‬ ‫ﻣﻦ اﻟﻤﺨﻠﻔﺎت اﻟﺰراﻋﯿﺔ اﻟﻤﺘﺎﺣﺔ ﺑﻜﺜﺮة وﻗﺪ أﺿﯿﻒ ﺑﻨﺠﺎح إﻟﻰ وﺳﻂ اﻟﺘﺨﻤﯿﺮ ﺑﻨﺴﺒﺔ ‪ %10‬وﻋﻤﻞ اﻟﺨﻞ‬ ‫اﻟﻤﻨﺰﻟﻲ ﻛﻠﻘﺎح ﻣﯿﻜﺮوﺑﻲ ﺣﯿﻦ أﺿﯿﻒ ﻟﻮﺳﻂ اﻟﺘﺨﻤﯿﺮ ﺑﻨﺴﺒﺔ ‪%20‬ﻓﯿﻤﺎ وﻓﺮ ﺣﺎﻣﺾ اﻟﺴﺘﺮﯾﻚ ﺑﺘﺮﻛﯿﺰ‬ ‫‪ %2‬اﻟﺤﻤﻮﺿﺔ اﻟﻜﺎﻓﯿﺔ ﻟﺒﺪء ﻋﻤﻠﯿﺔ اﻟﺴﯿﻠﺠﺔ‪ .‬ﺗﺤﺴﻦ اﻟﺴﯿﻼج اﻟﻤﻨﺘﺞ أﻛﺜﺮ ﺑﺈﺿﺎﻓﺔ ﻋﻮاﻣﻞ ﻣﻀﺎدة‬ ‫ﻟﻸﻛﺴﺪة واﻟﻔﻄﺮﯾﺎت ﻟﻠﺤﻔﺎظ ﻋﻠﻰ ﺟﻮدة اﻟﺪھﻦ وﺗﺨﻔﯿﺾ ﻣﺤﺘﻮى اﻟﮭﺴﺘﺎﻣﯿﻦ‪ .‬ﺣﻀﺮ ﻣﺴﺤﻮق اﻟﺴﻤﻚ‬ ‫ﺑﺎﻟﻄﺮﯾﻘﺔ اﻟﻘﯿﺎﺳﯿﺔ ﻣﻦ ﻋﯿﻨﺔ اﻷﺳﻤﺎك ﻧﻔﺴﮭﺎ ﻷﻏﺮاض اﻟﻤﻘﺎرﻧﺔ‪ .‬ﻛﺎن ﺗﺮﻛﯿﺐ اﻷﺣﻤﺎض اﻷﻣﯿﻨﯿﺔ ﻟﻠﺴﯿﻼج‬ ‫اﻟﺴﻤﻜﻲ أﻗﺮب ﻟﻸﺳﻤﺎك اﻟﺨﺎم ﻣﻦ ﻣﺴﺤﻮق اﻟﺴﻤﻚ ﺧﺼﻮﺻﺎ اﻷﺣﻤﺎض اﻷﻣﯿﻨﯿﺔ اﻷﺳﺎﺳﯿﺔ وأﻋﺘﺒﺮ‬ ‫ﻣﻘﺒﻮﻻ ﻟﺘﻐﺬﯾﺔ اﻷﺳﻤﺎك وﻓﻘﺎ ﻟﻠﻤﻌﺎﯾﯿﺮ اﻟﻤﻮﺛﻘﺔ‪ .‬وﻛﺎن زﯾﺖ اﻟﺴﯿﻼج اﻟﺴﻤﻜﻲ ﻏﻨﯿﺎ ﺟﺪا ﺑﺎﻷﺣﻤﺎض اﻟﺪھﻨﯿﺔ‬ ‫ﻏﯿﺮ اﻟﻤﺸﺒﻌﺔ ﺧﺼﻮﺻﺎ ﻣﺘﻌﺪدة ﻋﺪم اﻟﺘﺸﺒﻊ ‪ PUFA‬اﻟﺘﻲ ﺷﻜﻠﺖ ‪ %47‬ﻣﻦ اﻷﺣﻤﺎض اﻟﺪھﻨﯿﺔ اﻟﻜﻠﯿﺔ‬ ‫ﻓﻲ اﻟﺴﯿﻼج اﻟﺴﻤﻜﻲ ﺑﺎﻟﻤﻘﺎرﻧﺔ ﻣﻊ ‪ %37‬ﻓﻲ ﻣﺴﺤﻮق اﻟﺴﻤﻚ وھﺬه ﺟﻌﻠﺘﮫ ﻣﺼﺪرا ﻗﯿﻤﺎ ﻟﺰﯾﺖ اﻟﺴﻤﻚ‬ ‫اﻟﺒﺤﺮي ﻓﻲ ﺗﻐﺬﯾﺔ اﻷﺳﻤﺎك‪.‬‬ ‫وﻟﺘﻘﯿﯿﻢ اﻟﺴﯿﻼج اﻟﺴﻤﻜﻲ اﻟﺤﯿﺎﺗﻲ ﻛﺒﺪﯾﻞ ﻟﻤﺴﺤﻮق اﻟﺴﻤﻚ ﻓﻲ أﻋﻼف زرﯾﻌﺔ اﻟﻜﺎرب أﺿﯿﻒ‬ ‫ﻻﺳﺘﺒﺪال ‪ 0‬و ‪ 25‬و ‪ 50‬و ‪ %75‬ﻣﻦ ﺑﺮوﺗﯿﻦ ﻣﺴﺤﻮق اﻟﺴﻤﻚ ﻓﻲ أﻋﻼف ﻣﺘﻤﺎﺛﻠﺔ اﻟﻨﺘﺮوﺟﯿﻦ )ﺑﺮوﺗﯿﻦ‬ ‫ﺧﺎم ‪ (%42‬واﻟﻄﺎﻗﺔ )‪ 4600‬ﻛﯿﻠﻮ ﺳﻌﺮة‪/‬ﻛﻐﻢ(‪ .‬أﺧﺘﺒﺮت ﺧﺼﺎﺋﺺ اﻟﺠﻮدة اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻟﻸﻋﻼف‬ ‫اﻟﻤﺤﻀﺮة ﻟﺘﺤﺪﯾﺪ ﺗﺄﺛﯿﺮ إﺿﺎﻓﺔ اﻟﺴﯿﻼج ﻋﻠﯿﮭﺎ‪ .‬ﺗﺤﺴﻨﺖ اﻟﻜﺜﺎﻓﺔ اﻟﺤﺠﻤﯿﺔ وﻣﺘﺎﻧﺔ اﻷﻗﺮاص اﻟﻌﻠﻔﯿﺔ‬ ‫وﺛﺒﺎﺗﯿﺘﮭﺎ اﻟﻤﺎﺋﯿﺔ ﺑﺎرﺗﻔﺎع إﺿﺎﻓﺔ اﻟﺴﯿﻼج ﻓﻲ ﺣﯿﻦ أظﮭﺮت ﺳﺮﻋﺔ اﻟﻐﻄﺲ وﻗﺎﺑﻠﯿﺔ اﻟﻄﻔﻮ إﺗﺠﺎھﺎ ﻣﻌﺎﻛﺴﺎ‬ ‫ﺑﺎﻟﻤﻘﺎرﻧﺔ ﻣﻊ ﻋﻠﻒ ﻣﺴﺤﻮق اﻟﺴﻤﻚ‪ .‬أﺷﺎرت ﻧﺘﺎﺋﺞ دراﺳﺔ ﻛﻔﺎءة اﻟﺘﻐﺬﯾﺔ واﻟﻨﻤﻮ اﻟﺘﻲ أﺟﺮﯾﺖ ﻋﻠﻰ‬ ‫زرﯾﻌﺔ اﻟﻜﺎرب )ﻣﺘﻮﺳﻂ وزن ‪ 0.86‬ﻏﻢ( إﻟﻰ ﺗﺸﺎﺑﮫ ﻛﺒﯿﺮ ﺑﯿﻦ اﻷﻋﻼف اﻷرﺑﻌﺔ اﻟﻤﺨﺘﻠﻔﺔ ﻛﻤﺎ ﻗﯿﻤﺖ ﻣﻦ‬ ‫اﻟﺰﯾﺎدة اﻟﻮزﻧﯿﺔ وﻣﻌﺎﻣﻞ اﻟﻨﻤﻮ اﻟﻨﻮﻋﻲ وﻣﻌﺎﻣﻞ اﻟﻨﻤﻮ اﻟﺤﺮاري وﻣﻌﺎﻣﻞ اﻟﺘﺤﻮﯾﻞ اﻟﻐﺬاﺋﻲ وﻣﻌﺎﻣﻞ ﻛﻔﺎءة‬ ‫اﻟﺒﺮوﺗﯿﻦ وﻣﻌﺪل اﻟﺒﻘﺎء‪ .‬أﺟﺮﯾﺖ ﺗﺠﺮﺑﺔ ﺗﻐﺬﯾﺔ أﺧﺮى ﻋﻠﻰ إﺻﺒﻌﯿﺎت اﻟﻜﺎرب )ﻣﺘﻮﺳﻂ وزن ‪ 5.81‬ﻏﻢ(‬

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‫اﻟﺘﻲ ﻏﺬﯾﺖ أﻋﻼﻓﺎ ﻣﺘﻤﺎﺛﻠﺔ اﻟﻨﺘﺮوﺟﯿﻦ )ﺑﺮوﺗﯿﻦ ﺧﺎم ‪ (%35‬واﻟﻄﺎﻗﺔ )‪ 4400‬ﻛﯿﻠﻮ ﺳﻌﺮة‪/‬ﻛﻐﻢ(‬ ‫وﺑﻤﻌﺪﻻت إﺿﺎﻓﺔ ﺳﯿﻼج ﻣﺸﺎﺑﮭﺔ ﻟﻌﻠﻒ اﻟﺰرﯾﻌﺔ‪ .‬وﻛﺎﻧﺖ إﺿﺎﻓﺔ اﻟﺴﯿﻼج ﺑﻤﻌﺪل ‪ %50‬ﻣﺸﺎﺑﮭﺔ ﺟﺪا‬ ‫ﻣﻦ ﺣﯿﺚ ﻣﻘﺎﯾﯿﺲ ﻛﻔﺎءة اﻟﺘﻐﺬﯾﺔ واﻟﻨﻤﻮ ﻟﻌﻠﻒ ﻣﺴﺤﻮق اﻟﺴﻤﻚ اﻷﺳﺎس‪ .‬وﺧﻔﺾ ﻣﻌﺪل اﻹﺿﺎﻓﺔ اﻷﻋﻠﻰ‬ ‫اﻟﻌﺪﯾﺪ ﻣﻦ اﻟﻤﻘﺎﯾﯿﺲ ﺑﺎﺳﺘﺜﻨﺎء ﺗﻨﺎول اﻟﻌﻠﻒ اﻟﺬي ارﺗﻔﻊ ﺑﺰﯾﺎدة ﻣﻌﺪل اﻹﺿﺎﻓﺔ‪ .‬ﻟﻢ ﺗﺘﺄﺛﺮ ﻣﻘﺎﯾﯿﺲ ﺟﻮدة‬ ‫اﻟﻤﯿﺎه ﺳﻠﺒﺎ ﺑﺈﺿﺎﻓﺔ اﻟﺴﯿﻼج ﻓﻲ ﻛﻼ ﺗﺠﺮﺑﺘﻲ ﺗﻐﺬﯾﺔ اﻟﺰرﯾﻌﺔ واﻻﺻﺒﻌﯿﺎت ‪ ،‬ﻓﯿﻤﺎ ﺗﺤﺴﻨﺖ ﻣﻌﺎﻣﻼت‬ ‫اﻟﮭﻀﻢ اﻟﻈﺎھﺮﯾﺔ ﻟﻠﻤﻐﺬﯾﺎت اﻟﻌﻠﻔﯿﺔ اﻟﺮﺋﯿﺴﺔ ﺑﺈﺿﺎﻓﺔ اﻟﺴﯿﻼج ﺧﺼﻮﺻﺎ ﻣﻌﺎﻣﻞ اﻟﮭﻀﻢ اﻟﻈﺎھﺮي‬ ‫ﻟﻠﺒﺮوﺗﯿﻦ اﻟﺬي وﺻﻞ إﻟﻰ ‪ %98.88‬ﻓﻲ ﻋﻠﻒ ‪ %75‬ﺳﯿﻼج ﻣﻘﺎرﻧﺔ ﻣﻊ ‪ %82.55‬ﻓﻲ ﻋﻠﻒ ﻣﺴﺤﻮق‬ ‫اﻟﺴﻤﻚ‪ .‬ﻟﻢ ﺗﻈﮭﺮ ﻓﺮوﻗﺎت ﻣﻌﻨﻮﯾﺔ ﻓﻲ اﻟﺘﺮﻛﯿﺐ اﻟﺘﻘﺮﯾﺒﻲ ﻟﻺﺻﺒﻌﯿﺎت اﻟﺘﻲ ﻏﺬﯾﺖ اﻷﻋﻼف اﻟﻤﺨﺘﻠﻔﺔ‪.‬‬ ‫وﻣﻊ ذﻟﻚ‪ ،‬ﺗﺤﺴﻦ ﺗﺮﻛﯿﺐ اﻷﺣﻤﺎض اﻟﺪھﻨﯿﺔ ﺑﺎرﺗﻔﺎع إﺿﺎﻓﺔ اﻟﺴﯿﻼج ﺧﺎﺻﺔ ﻣﺘﻌﺪدة ﻋﺪم اﻟﺘﺸﺒﻊ ﻣﻦ‬ ‫ﻣﺠﻤﻮﻋﺔ أوﻣﯿﺠﺎ‪ 3 -‬اﻟﺘﻲ ارﺗﻔﻌﺖ ﻣﻌﻨﻮﯾﺎ ﻣﻦ ‪ 7.98‬إﻟﻰ ‪ %10.18‬ﻓﻲ ﻣﺠﻤﻮﻋﺘﻲ ﻣﺴﺤﻮق اﻟﺴﻤﻚ‬ ‫وﻋﻠﻒ ‪ %75‬ﺳﯿﻼج‪ ،‬ﻋﻠﻰ اﻟﺘﻮاﻟﻲ‪ .‬ﻛﻤﺎ ﻟﻢ ﺗﺨﺘﻠﻒ ﻣﺤﺘﻮﯾﺎت اﻟﺪھﻦ واﻟﺠﻠﯿﻜﻮﺟﯿﻦ ﻣﻌﻨﻮﯾﺎ ﻓﻲ ﻛﺒﺪ‬ ‫وﻋﻀﻼت اﻷﺳﻤﺎك ﺑﺈﺿﺎﻓﺔ اﻟﺴﯿﻼج ﻟﻠﻌﻠﻒ‪.‬‬ ‫ﻟﻢ ﺗﻈﮭﺮ اﻟﻤﻘﺎﯾﯿﺲ اﻟﺪﻣﯿﺔ اﻟﻌﺎﻣﺔ اﺧﺘﻼﻓﺎت ﻣﻌﻨﻮﯾﺔ ﺑﯿﻦ ﻣﺠﻤﻮﻋﺎت اﻟﻌﻠﻒ اﻷرﺑﻌﺔ ﺑﻐﺾ اﻟﻨﻈﺮ ﻋﻦ‬ ‫ﻣﻌﺪل إﺿﺎﻓﺔ اﻟﺴﯿﻼج وﻛﺬﻟﻚ اﻟﺤﺎل ﺑﺎﻟﻨﺴﺒﺔ ﻟﺒﺮوﺗﯿﻨﺎت اﻟﺒﻼزﻣﺎ ‪ .‬وﻣﻊ ذﻟﻚ‪ ،‬ﺗﺤﺴﻦ ﺗﺮﻛﯿﺐ دھﻮن‬ ‫اﻟﺒﻼزﻣﺎ ﺑﺰﯾﺎدة إﺿﺎﻓﺔ اﻟﺴﯿﻼج ﻟﻤﺤﺘﻮى اﻟﺴﯿﻼج ﻣﻦ زﯾﺖ اﻟﺴﻤﻚ ﺧﺎﺻﺔ ﻣﺴﺘﻮﯾﺎت اﻟﻜﻮﻟﺴﺘﺮول اﻟﻜﻠﻲ‬ ‫اﻟﺘﻲ اﻧﺨﻔﻀﺖ ﻣﻦ ‪ 212‬اﻟﻰ‪ 178‬ﻣﻠﻐﻢ‪100/‬ﻣﻞ ﻓﻲ ﻣﺠﻤﻮﻋﺘﻲ ﻣﺴﺤﻮق اﻟﺴﻤﻚ و‪ %75‬ﺳﯿﻼج ﺳﻤﻜﻲ‪،‬‬ ‫ﻋﻠﻰ اﻟﺘﻮاﻟﻲ‪.‬‬ ‫ﺗﻀﻤﻨﺖ اﻟﺪراﺳﺔ اﻟﻨﺴﺠﯿﺔ ﻗﯿﺎس ﺑﻌﺾ اﻟﻤﻜﻮﻧﺎت اﻟﻨﺴﺠﯿﺔ واﻟﺨﻠﻮﯾﺔ ﻷﻣﻌﺎء اﻷﺳﻤﺎك وﻛﺒﺪھﺎ‪ ،‬وﻟﻢ‬ ‫ﺗﻈﮭﺮ ﻧﺘﺎﺋﺞ ھﺬه اﻟﺪراﺳﺔ أي ﻓﺮوﻗﺎت ﻣﻌﻨﻮﯾﺔ ﺑﯿﻦ ﻣﺠﻤﻮﻋﺎت ﻋﻠﻒ ﻣﺴﺤﻮق اﻷﺳﻤﺎك اﻷﺳﺎس وأﻋﻼف‬ ‫اﻟﺴﯿﻼج اﻟﺴﻤﻜﻲ‪ .‬وﺗﺄﻛﺪت ھﺬه اﻟﻨﺘﯿﺠﺔ ﺑﻔﺤﺺ اﻟﺘﺮﻛﯿﺐ اﻟﻨﺴﺠﻲ اﻟﻌﺎم ﻟﻤﻘﺎطﻊ اﻷﻣﻌﺎء واﻟﻜﺒﺪ اﻟﺘﻲ ﻟﻢ‬ ‫ﺗﻈﮭﺮ أي ﻋﻼﻣﺎت ﺷﺎذة‪ .‬أوﺿﺢ ﻛﺸﻒ اﻟﺠﻠﯿﻜﻮﺟﯿﻦ ﻓﻲ ﻧﺴﯿﺞ اﻟﻜﺒﺪ ﺑﺼﺒﻐﺔ ‪ PAS‬وﺟﻮد ﻧﻤﻂ ﻣﺘﺸﺎﺑﮫ‬ ‫ﻓﻲ اﻟﺘﻮزﯾﻊ إﻟﻰ ﺣﺪ ﻣﺎ ﺑﯿﻦ ﻣﺠﻤﻮﻋﺎت اﻟﻌﻠﻒ اﻟﻤﺨﺘﻠﻔﺔ‪ .‬وﻣﻊ ذﻟﻚ‪ ،‬أوﺿﺢ ﻛﺸﻒ اﻟﺪھﻮن ﺑﺼﺒﻐﺘﻲ راﺑﻊ‬ ‫أوﻛﺴﯿﺪ اﻻوزﻣﯿﻮم وأﺳﻮد اﻟﺴﻮدان ب وﺟﻮد ﺗﺤﺴﻦ ﺗﺪرﯾﺠﻲ ﻓﻲ ﻣﺨﺰوﻧﺎت دھﻮن اﻟﻜﺒﺪ ﻣﻊ ارﺗﻔﺎع‬ ‫إﺿﺎﻓﺔ اﻟﺴﯿﻼج ﻓﻲ اﻟﻌﻠﻒ رﻏﻢ أن ﻛﺒﺪ اﻟﻜﺎرب‪ ،‬ﻛﻤﺎ ﺛﺒﺖ‪ ،‬ﻟﯿﺴﺖ ﻣﻮﻗﻊ اﻟﺨﺰن اﻟﺮﺋﯿﺲ ﻟﻠﺪھﻦ ﻓﯿﻤﺎ ﻟﻢ‬ ‫ﺗﻼﺣﻆ أي ﻋﻼﻣﺎت اﺿﻄﺮاب أو ﺷﺬوذ ﻓﻲ أﯾﺾ اﻟﺪھﻦ ﻓﻲ ﻣﻘﺎطﻊ ﻧﺴﯿﺞ اﻟﻜﺒﺪ اﻟﻤﻔﺤﻮﺻﺔ‪.‬‬ ‫إﺳﺘﻨﺘﺠﺖ اﻟﺪراﺳﺔ إﻣﻜﺎﻧﯿﺔ ﺗﺤﻀﯿﺮ اﻟﺴﯿﻼج اﻟﺴﻤﻜﻲ اﻟﺤﯿﻮي ﺑﺴﮭﻮﻟﺔ ﺑﺎﺳﺘﻌﻤﺎل ﻣﻮاد ﺧﺎم ﻣﺤﻠﯿﺔ‬ ‫ﻣﺜﻞ اﺳﻤﺎك اﻟﺼﯿﺪ اﻟﻌﺮﺿﻲ وﺛﻔﻞ اﻟﺘﻤﺮ واﻟﺨﻞ‪ .‬وھﻮ أﯾﻀﺎ ﺑﺪﯾﻞ ﺟﯿﺪ ﻟﻤﺴﺤﻮق اﻟﺴﻤﻚ ﻓﻲ أﻋﻼف‬

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‫زرﯾﻌﺔ وإﺻﺒﻌﯿﺎت اﻟﻜﺎرب اﻟﺸﺎﺋﻊ ﻟﺨﻮاﺻﮫ اﻟﺘﻐﺬوﯾﺔ اﻟﻤﻼﺋﻤﺔ‪ .‬وﻣﻊ ذﻟﻚ‪ ،‬ﺗﻮﺟﺪ ﺣﺎﺟﺔ ﻹﺟﺮاء اﻟﻤﺰﯾﺪ‬ ‫ﻣﻦ اﻟﺘﺤﺴﯿﻨﺎت ﻋﻠﻰ ھﺬا اﻟﺴﯿﻼج ﻗﺒﻞ إﺳﺘﺜﻤﺎره ﻓﻲ اﻷﻋﻼف اﻟﺘﻄﺒﯿﻘﯿﺔ ﺧﺼﻮﺻﺎ ﺑﻌﺾ ﻣﻘﺎﯾﯿﺲ اﻟﺠﻮدة‬ ‫اﻟﻔﯿﺰﯾﺎﺋﯿﺔ ﻟﻠﻌﻠﻒ‪ .‬وأوﺻﺖ اﻟﺪراﺳﺔ ﺑﺒﻌﺾ اﻟﺠﻮاﻧﺐ ﻟﻠﻤﺰﯾﺪ ﻣﻦ اﻟﺒﺤﺚ ﺣﻮل اﻟﺴﯿﻼج اﻟﺴﻤﻜﻲ‬ ‫وﺗﻄﺒﯿﻘﺎﺗﮫ ﻓﻲ أﻋﻼف اﻷﺣﯿﺎء اﻟﻤﺎﺋﯿﺔ‪.‬‬

‫ج‬

‫ﺇﺳﺘﺨﺪﺍﻡ ﺍﻟﺴﻴﻼﺝ ﺍﻟﺴﻤﻜﻲ ﺍﳌﺘﺨﻤﺮ ﺑﺈﺿﺎﻓﺔ ﺛﻔﻞ ﺍﻟﺘﻤﺮ‬

‫ﰲ ﺗﻐﺬﻳﺔ ﺃﲰﺎﻙ ﺍﻟﻜﺎﺭﺏ ﺍﻟﺸﺎﺋﻊ ‪Cyprinus carpio L.‬‬ ‫ﻭﺗﺄﺛﲑﺍﺗﻪ ﺍﻟﻔﺴﻠﺠﻴﺔ ﻭﺍﻟﻨﺴﺠﻴﺔ‬

‫أطﺮوﺣﺔ ﻣﻘﺪﻣﺔ اﻟﻰ‬ ‫ﻣﺠﻠﺲ ﻛﻠﯿﺔ اﻟﺰراﻋﺔ – ﺟﺎﻣﻌﺔ اﻟﺒﺼﺮة‬ ‫ﻛﺠﺰء ﻣﻦ ﻣﺘﻄﻠﺒﺎت ﻧﯿﻞ درﺟﺔ دﻛﺘﻮراه ﻓﻠﺴﻔﺔ ﻓﻲ اﻟﻌﻠﻮم اﻟﺰراﻋﯿﺔ‬ ‫"اﻷﺳﻤﺎك واﻟﺜﺮوة اﻟﺒﺤﺮﯾﺔ"‬ ‫)ﺗﺮﺑﯿﺔ وﺗﻐﺬﯾﺔ اﻷﺳﻤﺎك(‬ ‫ﻣﻦ ﻗﺒﻞ‬

‫ﺻﻼﺡ ﻣﻬﺪﻱ ﳒﻢ ﺍﻟﻜﻨﻌﺎﻧﻲ‬ ‫ﺑﻜﺎﻟﻮرﯾﻮس ﻋﻠﻮم زراﻋﯿﺔ ﻓﻲ اﻷﺳﻤﺎك واﻟﺜﺮوة اﻟﺒﺤﺮﯾﺔ )‪(1984‬‬ ‫ﻣﺎﺟﺴﺘﯿﺮ ﻋﻠﻮم زراﻋﯿﺔ ﻓﻲ اﻷﺳﻤﺎك واﻟﺜﺮوة اﻟﺒﺤﺮﯾﺔ )‪(1989‬‬

‫ﺇﺷﺮﺍﻑ‬ ‫ﺃ‪.‬ﺩ‪ .‬ﺳﺎﺟﺪ ﺳﻌﺪ ﺍﻟﻨﻮﺭ‬

‫ﺫﻭ ﺍﻟﻘﻌﺪﺓ ‪ 1435‬ﻫـ‬

‫ﺃ‪.‬ﻡ‪.‬ﺩ‪ .‬ﺑﺎﺳﻢ ﳏﻤﺪ ﺟﺎﺳﻢ‬

‫ﺃﻳﻠﻮﻝ ‪ 2014‬ﻡ‬