contaminants of food and are the most acutely toxic of AFs (Park and. Pezzuto .... intake with 24â32% reduction in body weight gain particularly during the second phase of ...... enzyme activities and nutrient digestibility of cherry valley ducks.
EFFECT OF ADDING CURCUMA LONGA ON AFLATOXIN CONTAMINATED DIETS ON PRODUCTIVE AND PHYSIOLOGICAL ASPECTS OF BROILER CHICKS BY HAMADA ABDEL- HAMEID MEHANY ELWAN B.Sc. Agriculture sciences, 2003 M.Sc. Animal Production (Poultry Physiology), 2008 Faculty of Agric., Minia University THESIS Submitted to Animal Production Department, Faculty of Agriculture Minia University in Partial Fulfillment of the Requirement For The Ph.D. Degree in Poultry Physiology Under supervision of: Prof. Dr. Attiat, H. EL- Bogdady Prof. of Poultry Production Anim. Prod. Dept., Fac., of Agric. Minia Univ., Egypt Prof. Dr. Mahmoud, A. Toson Prof. of Poultry Production Anim. Prod. Dept., Fac., of Agric. Minia Univ., Egypt
Prof. Dr. Akrum, M.M. Hamdy Prof. of Poultry Physiology Anim. Prod. Dept., Fac., of Agric. Minia Univ., Egypt Prof. Dr. Shaker, A. Abd El-Latif Prof. of Poultry Production Nutrition Anim. Prod. Dept., Fac., of Agric. Minia Univ., Egypt
Minia University, Egypt (2013)
Curcuma longa Versus Aflatoxin Contamination
Acknowledgments All thanks, to Allah as it should accord with his Holiness and Glory. All thanks and praise go to him for all favors he bestowed upon me and for giving me the power to complete this work. My deep gratitude is direct to my supervisor professor. Attiat H. ELBogdady, professor of poultry production. Animal production department, Faculty of Agriculture, Minia University, whose favors upon me can never be deny. She facilitated all the difficulties that faced me during my research, my Allah assist me to do her some of the favors she did to me. I would like also to express my sincere appreciation to professor, Akrum M. M. Hamdy, professor of poultry physiology. Animal production department, Faculty of Agriculture, Minia University, for his keen supervision, genuine encouragement, and revising this thesis, Also, particular gratitude and sincere thanks to professor, Mahmoud, A. Toson, professor of poultry production. Animal production department, Faculty of Agriculture, Minia University, for his helps, advice and encouragement. In addition, I am greatly being holder to my supervisor professor, Shaker, A. Abd El-Latif, professor of poultry nutrition. Animal production department, Faculty of Agriculture, Minia University, for his valuable assistance and adequate support, I greatly needed during the accomplishment of this thesis. My deep thanks are also dedicate to all the department staff members whom I always appreciate.
Curcuma longa Versus Aflatoxin Contamination
Contents
Page
List of Tables
X
List of Figures
XII
List of Abbreviations
XXI
I. Introduction
1
II. Review of literature
2
1. Importance of studying aflatoxicosis
2
2. Aflatoxins in poultry feedstuffs and its toxicity
2
3. Metabolism of aflatoxins in poultry
4
4. Effect of aflatoxins on productive performance
5
4.1. Growth performance, efficiency of feed utilization and mortality rate
5
4.2. Effect of aflatoxins on digestibility of some nutrients
8
5. Effect of aflatoxins on physiological responses
9
5.1. Liver weight
9
5. 2. Kidney weight
11
5.3. Lymphoid organs weight
11
5.4. Blood physical characteristics
13
5.5. Blood serum constituents
14
5.5.1. Total protein, albumin and globulin
14
5.5.2. Liver enzymes
16
I
5.5.3. Kidney function
19
5.5.4. Lipid profiles
20
6. Immuno-responses
21
7. Histological and pathological changes
21
8. Methods applied to control aflatoxin contaminated feeds
25
9. Herbs and medical plants
27
10. Curcuma Longa as feed additives
29
10.1. Classification plant description
29
10.2 Chemical composition of Curcuma longa
30
10.3. Uses of Curcuma longa
31
10.4. Structure and chemical properties of curcumin
32
11. Effect of Curcuma longa on growth performance
33
12. Effect of Curcuma longa on nutrient metabolism
34
13. Effect of Curcuma longa on physiological response
35
13.1. Some organs weight
35
13. 2. Blood physical characteristics
35
13. 3. Blood biochemical
36
14. Effect of Curcuma longa on immuno-responses
39
15. Effect of Curcuma longa on histological structure of some organs
41
16. Protective effects of Curcuma Longa against aflatoxins
41
17. Curcuma longa and aflatoxins biotransformation
43
II
III. Materials and methods
44
1. Experimental birds
44
2. Experimental diets
44
3. Experimental design
45
4. Preparing of aflatoxins
46
5. Preparation of curcuma
47
6. Studied parameters
47
6.1. Productive performance
47
6.1.1. Live body weight and body weight gain
47
6.1. 2. Feed consumption
48
6.1.3. Feed conversion
48
6.1. 4. Mortality rates
48
6.2. Slaughter traits
48
6.3. Digestion trials
48
6.4. Proximate chemical analysis of basal diet and excreta
49
6.4.1. Moisture content
49
6.4.2. Crude protein
49
6.4.3. Ether extract
50
6.4.4. Crude fiber
50
6.4.5. Ash
50
6.4.6. Nitrogen free extract
51
III
6.4.7. Fecal nitrogen
51
6.5. Collection of blood samples
51
6.6. Hematological studies
52
6.6.1. Total erythrocytic count
52
6.6.2. Hemoglobin percentage 52
6.6.3. Microhaematocrit (Packed Cell Volume) 52
6.6. 4. Wintrobe erythrocyte indices
53
6.7. Serum biochemical determinations
53
6.7.1. Total protein concentration
53
6.7.2. Albumin concentration
53 54
6.7.3. Globulin concentration and Albumin/Globulin (A/G) ratio
54
6.7.4. Glucose
54
6.7.5. Some liver enzymes
54
6.7.5.1. Glutamic–Pyruvic Transminase
54
6.7.5.2. Glutamic –Oxaloacetic Transminase
55
6.7.5.3. Glutathione- S- Transferase
55
6.7.5.4. Lactic Dehydrogenase
55
6.7.6. Total lipids
55
6.7.7. Triglycerides
55
6.7.8. Cholesterol
56
6.7.9. High density lipoprotein cholesterol (HDL- cholesterol)
56
IV
6.7.10. Low density lipoprotein cholesterol (LDL- cholesterol)
56
6.7.11. Kidney function
56
6.7.11.1. Creatinine
56
6.7.11.2. Urea
57
6.7.11.3. Uric acid
57
6.8. Immunological responses
57
6.8.1. Total and differential white blood cells count
57
6.8.1.1. Total leucocytes count
57
6.8.1.2. Differential leucocytes count
57
6.8.2. Innate immune response
58
6.8. 2.1. Phagocytosis
58
6.8. 2.2. The micropore filter assay (chemokinetic assay)
58
6.8. 2.3. The agarose gel assay (chemotaxis movement assay)
59
6.8.3. Humeral immune response
60
6.8.3.1. Turbidity test for estimation of total immunoglobulin level
60
6.8.3.2. Precipitation test
62
6.8.3.3. Immunization and titration
64
6.9. Histopathological investigation
65
6.10. Economic efficiency
66
6.11. Statistical analysis
66
IV. Results and discussions
68
V
1. Effect of Treatments on productive performance
68
1.1. Feed intake
68
1.2. Feed conversion
73
1.3. Body weight
75
1.4. Body weight gain
79
1.5. Mortality rate
83
2. Effect of treatments on digestion trial
86
2.1. Dry matter
86
2.2. Organic matter
89
2.3. Crude protein
89
2.3. Total protein
90
2.4. Crude fiber
90
2.5. Ether extract
91
2.6. Nitrogen free extract
92
3. Effect of treatments on physiological studies
94
3.1. Absolute and relative weights of some organs
94
3.1.1. Some edible organs
94
3.1.1.1. Liver
95
3.1.1.2. Heart
100
3.1.1.3. Gizzard
101
3.1.1.4. Proventiculus
101
VI
3.1.2. Lymphoid organs
104
3.1.2.1. Spleen
104
3.1.2.2. Thymus
105
3.1.2.3. Bursa of fabricius
110
3.2. Effect of treatments on hematological studies
112
3.2.1. Red blood cells count
112
3.2.2. Hemoglobin
115
3.2.3. Paced cell volume
115
3.3. Wintrobe erythrocyte indices
116
3.3.1. Mean corpuscular volume
116
3.3.2. Mean corpuscular hemoglobin
119
3.3.3. Mean corpuscular hemoglobin concentration
119
4. Effect of treatments on blood serum biochemistry
122
4.1. Total protein
122
4.2. Albumin
125
4.3. Globulin
126
4.4. Albumin/Globulin ratio
126
4.5. Glucose
129
4.6. Liver enzymes
130
4.6.1. Transaminase enzymes (GOT and GPT)
130
4.6.2. The activity of Glutathione S-Transferees
134
VII
4.6.3. The activity of lactic dehydrogenase
135
4.6.4. Total lipid profiles
139
4.6.4.1. Total lipids
139
4.6.4.2. Triglycerides
139
4.6.4.3. Cholesterol
143
4.6.4.4. HDL-cholesterol
144
4.6.4.5. LDL-cholesterol
145
4.7. Kidney function
148
4.7.1. Creatinine
149
4.7.2. Urea
149
4.7.2. Uric acid
152
5. Effect of treatments on immunological studies
154
5.1. Total count of white blood cells
154
5.2. Differential count of white blood cells
155
5.2.1. Heterophile
155
5.2.2. Lymphocyte
158
5.2.3. H/ L ratio
158
5.2.4. Monocyte
159
5.2.5. Eosinophil
159
5.2.6. Basophil
162
5.3. Cell mediated reactions
163
VIII
5.3.1. Chemokinesis
163
5.3.2. Chemotaxis
166
5.3.3. Phagocytic activity
166
5.4. Humeral immune response
170
5.4.1. Total immunoglobulin
170
5.4.2. Hemagglutintion
172
5.4.3. Precipitation test
172
6. Histopathological studies
177
6.1. liver
177
6.2. kidney
180
6.3. Pathological changes of some lymphatic organs
183
6.3.1. Spleen
183
6.3.2. Thymus
186
6.3.3. Bursa of fabricius
190
7. Effect of dietary treatments on economic efficiency
194
5. Summary
199
6. REFERENCES
205
الملخص العربـــــــي
IX
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Curcuma longa Versus Aflatoxin Contamination
List of Tables Title Table (1): The proximate chemical analyses of the diets. Table (2): Standard calibration curve to estimate the total immunoglobulin’s (ZnSO4 turbidity test) using human serum with known level by RPPHS; (lot. No .97148 and Cat. No. 86100). Table (3): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on feed intake (gm) and feed conversion (gm feed/gm gain). Table (4): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on body weight (gm) and body weight gain (gm). Table (5): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on mortality rate. Table (6): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on digestibility of some nutrients. Table (7): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on absolute weight of some organs. Table (8): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on relative weight of some organs. Table (9): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on relative weight of some lymphoid organs. Table (10): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on relative weight of some lymphoid organs. Table (11): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on some hematological parameters. Table (12): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on Wintrobe erythrocyte indices. Table (13): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on liver function. Table (14): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on some liver enzymes (IU/L). X
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Table (15): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on some lipid profiles of serum. Table (16): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on kidney function. Table (17): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs.) during different experimental periods on white blood cells, heterophils %, lymphocyte % and H/L ratio. Table (18): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs.) during different experimental periods on monocyte %, esinophils % and basophils %. Table (19): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on Cell mediated immune responses. Table (20): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on humeral immune responses. Table (21): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on economic efficiency.
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Curcuma longa Versus Aflatoxin Contamination
List of Figures Title Fig. 1: Curcuma longa L. Fig. 2: Chemical structures of curcumin and its analogs. Fig. 3: Chemical structure of curcumin Fig. (4): Details of experimental groups during different periods. Fig. (5 and 6): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on feed intake (gm) and feed conversion (gm feed/gm gain). Fig. (7 and 8): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on body weight (gm) and body weight gain (gm). Fig. (9): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on digestibility of some nutrients. Fig. (10): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on absolute weight of some organs. Fig. (11): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on relative weight of some organs. Fig. (12 and 13): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on absolute weight of some lymphoid organs. Fig. (14): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on some hematological parameters. Fig. (15 and 16): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on wintrobe erythrocyte indices Fig. (17): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on liver function Fig. (18 and 19): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on liver function. Fig. (20 and 21): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on some liver enzymes. Fig. (22): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods XII
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Fig.
Fig.
Fig.
Fig.
Fig.
Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.
on some lipid profiles of serum. (23): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on some lipid profiles of serum. (24 and 25): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on kidney function. (26 and 27): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on white blood cells, heterophils % and lymphocyte %. (28 and 29): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs.) during different experimental periods on monocyte %, H/L ratio, eosinophils % and basophils %. (30 and 31): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs) during different experimental periods on Cell mediated immune and humeral immune-responses. (32): Precipitation of Antibodies of birds in T1. Note. The formation of sharp precipitating line (Arrow) (UV 360 nm wavelength). (33): Precipitation of Antibodies of birds in T2. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (34): Precipitation of Antibodies of birds in T3. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (35): Precipitation of Antibodies of birds in T4. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (36): Precipitation of Antibodies of birds in T5. Note. The absent of sharp precipitating line (UV 360nm wavelength). (37): Precipitation of Antibodies of birds in T6. Note. The absent of sharp precipitating line (UV 360 nm wavelength). (38): Precipitation of Antibodies of birds in T7. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (39): Precipitation of Antibodies of birds in T8. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (40): Precipitation of Antibodies of birds in T1. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (41): Precipitation of Antibodies of birds in T2. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (42): Precipitation of Antibodies of birds in T3. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (43): Precipitation of Antibodies of birds in T4. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (44): Precipitation of Antibodies of birds in T5. Note. The absent of sharp
XIII
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Fig. Fig. Fig. Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
Fig.
precipitating line (UV 360 nm wavelength). (45): Precipitation of Antibodies of birds in T6. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (46): Precipitation of Antibodies of birds in T7. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (47): Precipitation of Antibodies of birds in T8. Note. The formation of sharp precipitating line (UV 360 nm wavelength). (48): A photomicrograph of liver section of (T1) 3weeks of age, showing the normal hepatocytes (thick arrow), normal hepatocyte cell with two nuclei (zigzag arrow) and central vein (thin arrow) (H&E, x 500). (49): A photomicrograph of liver section of (T2) 3 week of age, showing the normal hepatocytes (thick arrow), normal hepatocyte cell with two nuclei (zigzag arrow) and central vein with blood (thin arrow) (H&E, x 500). (50): A photomicrograph of liver section of (T3) 3 week of age, showing the normal hepatocytes (thin arrow), normal hepatocyte cell with two nuclei (zigzag arrow) and central vein with blood (thick arrow) (H&E, x 500). (51): A photomicrograph of liver section of (T4) 3 week of age, showing the normal hepatocytes with two nuclei (zigzag arrow) and central vein with blood (thick arrow) (H&E, x 500). (52): A photomicrograph of liver section of (T5) group 3weeks of age, showing the central vein (thick arrow), damaged hepatocyte cell (thin arrow), hemorrhage (zigzag arrow), vacuolation (curved arrows) (H&E, x 500). (53): A photomicrograph of liver section of (T6) 3 week of age, showing the central vein (thick arrow), damaged hepatocyte cell pyknotic nuclei (zigzag arrow), central vein fibroblast and damaged cells (thin arrow) and vacuolation (curved arrows). (H&E, x 500). (54): A photomicrograph of liver section of (T7) 3 week of age, showing the normal hepatocytes (thick arrow), normal hepatocyte cell with two nuclei (zigzag arrow) and hemorrhage (thin arrow) (H&E, x 500). (55): A photomicrograph of liver section of (T8) 3 week of age, showing the normal hepatocytes (curved arrow), central vein with blood and hemorrhage (thick arrow) (H&E, x 500). (56): A photomicrograph of liver section of (T1) 6 weeks of age, showing the normal hepatocytes (thin arrow), hemorrhage (zigzag arrow) and central vein (thick arrow) (H&E, x 500). (57): A photomicrograph of liver section of (T2) 6 weeks of age, showing normal hepatocytes (thin arrow) and central vein (thick arrow) (H&E, x 500). (58): A photomicrograph of liver section of (T3) 6 weeks of age, showing
XIV
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normal hepatocytes (thin arrow), central vein (thick arrow) and hemorrhage (zigzag arrow) (H&E, x 500). Fig. (59): A photomicrograph of liver section of (T4) 6 weeks of age, showing normal hepatocytes, central vein (thick arrow), blood sinusoids (curved arrows) and hemorrhage (zigzag arrow). (H&E, x 500). Fig. (60): A photomicrograph of liver section of (T5) group 6 weeks of age, showing the portal vein (thick arrow), damaged hepatocyte with pyknotic nuclei (thin arrow), sever fibroblast around portal vein (zigzag arrow), vacuolation and sever damaged area (curved arrows) (H&E, x 500). Fig. (61): A photomicrograph of liver section of (T6) group 6 weeks of age, showing the portal vein (thick arrow), normal hepatocytes cell (zigzag arrow), vacuolation (head arrows) (H&E, x 500). Fig. (62): A photomicrograph of liver section of (T7) group 6 weeks of age, showing the congested blood vessels (thick arrow), hemorrhage (zigzag arrow) and congested blood sinusoids (curved arrows) (H&E, x 500). Fig. (63): A photomicrograph of liver section of (T8) group 6 weeks of age, showing the blood vessel (thick arrow), normal hepatocyte (H&E, x 500). Fig. (64): A photomicrograph in the cortex of kidney section for T1, illustrating the normal appearance of the proximal convoluted tubules (head arrow), distal convoluted tubules (thick arrow), Bowman's capsule (wavy arrow) glomerulus (curve arrow) and hemorrhage (pen arrow). (H&E, x 500). Fig. (65): A photomicrograph in the cortex of kidney section for T2, illustrating the normal appearance of the proximal convoluted tubules (wavy arrow), an increase in bowman's capsule space (thick arrow), glomerulus (pen arrow) and hemorrhage (curve arrow). (H&E, x 500). Fig. (66): A photomicrograph in the cortex of kidney section for T3, illustrating the normal appearance of the proximal convoluted tubules (curve arrow), an increase in bowman's capsule space (thick arrow) and glomerulus (head arrow). (H&E, x 500). Fig. (67): A photomicrograph in the cortex of kidney section for T4, illustrating the normal appearance of the proximal convoluted tubules (thick arrow) and glomerulus (head arrow). (H&E, x 500). Fig. (68): A photomicrograph in the cortex of kidney section for T5, illustrating the abnormal appearance of the proximal convoluted tubules (wavy arrow), distal convoluted tubules (curve arrow), congested blood vessel (thin arrow) and glomerulus (head arrow). (H&E, x 500). Fig. (69): A photomicrograph in the cortex of kidney section for T6, illustrating the abnormal appearance of the proximal convoluted tubules (wavy arrow), distal convoluted tubules (curve arrow), pyknotic nuclei (thin arrow), glomerulus with severs pyknotic nuclei (thick arrow) and XV
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hemorrhagic section appearance. (H&E, x 500). Fig. (70): A photomicrograph in the cortex of kidney section for T7, illustrating the normal appearance of the proximal convoluted tubules (curve arrow), an increase in bowman's capsule space (thin arrow) and glomerulus (head arrow) (H&E, x 500). Fig. (71): A photomicrograph in the cortex of kidney section for T8, illustrating the normal appearance of the proximal convoluted tubules, an increase in bowman's capsule space (zigzag arrow) and glomerulus (head arrow) (H&E, x 500). Fig. (72): A photomicrograph in the cortex of kidney section for T1, illustrating the normal appearance of the proximal convoluted tubules (thin arrow), distal convoluted tubules (head arrow), Bowman's capsule (wavy arrow) and glomerulus (thick arrows). (H&E, x 500). Fig. (73): A photomicrograph in the cortex of kidney section for T2, illustrating the normal appearance of the proximal convoluted tubules (thin arrow), distal convoluted tubules and dilated in urinary space (head arrow), and glomerulus (thick arrows). (H&E, x 500). Fig. (74): A photomicrograph in the cortex of kidney section for T3, illustrating the appearance of the proximal convoluted tubules (thin arrow), distal convoluted tubules and dilated in urinary space (head arrow), and glomerulus (thick arrows). (H&E, x 500). Fig. (75): A photomicrograph in the cortex of kidney section for T3, illustrating the appearance of the proximal convoluted tubules (head arrow), glomerulus (thick arrows) and dilated in urinary space (wavy arrow). (H&E, x 500). Fig. (76): A photomicrograph in the cortex of kidney section for T5, illustrating the abnormal appearance of the proximal convoluted tubules and distal convoluted tubules (curve arrow), glomerulus (thin arrow) and hemorrhage within glomerulus (zigzag arrow) section in general aberrance vacuolated, congested with blood and huge degenerated area. (H&E, x 500). Fig. (77): A photomicrograph in the cortex of kidney section for T6, illustrating the appearance of the proximal convoluted tubules and distal convoluted tubules. Glomerulus (thick arrow) and hemorrhage (zigzag arrow). (H&E, x 500). Fig. (78): A photomicrograph in the cortex of kidney section for T7, illustrating the appearance of the proximal convoluted tubules and distal convoluted tubules. Glomerulus (head and curve arrows) and glomerulus (thick arrows). (H&E, x 500). Fig. (79): A photomicrograph in the cortex of kidney section for T8, illustrating the appearance of the proximal convoluted tubules and distal convoluted tubules. Glomerulus (curve arrows) and glomerulus (thick arrows). (H&E, x 500). XVI
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Fig. (80): A photomicrograph in the spleen section for T1, showing red pulp (head arrow) white pulp (thick arrow) and trabeculae (wavy arrow) (H&E. x100). Fig. (81): A photomicrograph in the spleen section for T2, showing red pulp (head arrow) white pulp (thick arrow) and germinal zone (curve arrow) (H&E. x100). Fig. (82): A photomicrograph in the spleen section for T3, showing red pulp (head arrow) white pulp (thick arrow). (H&E. x100). Fig. (83): A photomicrograph in the spleen section for T4, showing red pulp (head arrow) white pulp (thick arrow) and blood vessels (curve arrow). (H&E. x100). Fig. (84): A photomicrograph in the spleen section for T5, showing red pulp and white pulp which have few lymphocytes and congested sinusoids and blood vessels with blood (wavy, curve and head arrows) (H&E. x100). Fig. (85): A photomicrograph in the spleen section for T6, showing red pulp (head arrow), white pulp (thick arrow) which have few lymphocytes and congested sinusoids and blood vessels with blood (wavy, curve head arrows) (H&E. x100). Fig. (86): A photomicrograph in the spleen section for T7, showing red pulp (head arrow), white pulp (thick arrow) and clear germinal zone (curve arrow). (H&E. x100). Fig. (87): A photomicrograph in the spleen section for T8, showing red pulp (head arrow), white pulp (thick arrow) and blood vessels (curve arrow). (H&E. x100). Fig. (88): A photomicrograph in the spleen section for T1, showing red pulp (head arrow) white pulp (thick arrow). (H&E. x100). Fig. (89): A photomicrograph in the spleen section for T2, showing red pulp (head arrow) white pulp (thick arrow). (H&E. x100). Fig. (90): A photomicrograph in the spleen section for T3, showing red pulp (head arrow) white pulp (thick arrow). (H&E. x100). Fig. (91): A photomicrograph in the spleen section for T4, showing red pulp (head arrow) white pulp (thick arrow) and blood vessel (curve arrow). (H&E. x100). Fig. (92): A photomicrograph in the spleen section for T5, showing red pulp and white pulp which have few lymphocytes and congested sinusoids and blood vessels with blood (thick, curve and head arrows) (H&E. x100). Fig. (93): A photomicrograph in the spleen section for T6, showing red pulp (head arrow) white pulp (thick arrow) and congested blood vessels with blood and trabeculae (wavy arrow) (H&E. x100). Fig. (94): A photomicrograph in the spleen section for T7, showing red pulp
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(head arrow) white pulp (thick arrow) and congested blood vessels with blood and trabeculae pink area and fibroblasts (zigzag arrow) (H&E. x100). Fig. (95): A photomicrograph in the spleen section for T8, showing red pulp (head arrow) white pulp (thick arrow) and few numbers of lymphocytes (H&E. x100). Fig. (96): A photomicrograph in the thymus section for T1, showing the cortex (thick arrow) and medulla (curve arrow). Note, Hassle's corpuscle (pink to red area). (H&E. x500). Fig. (97): A photomicrograph in the thymus section for T2, showing the cortex (thick arrow) and medulla (head arrow). Note, Hassle's corpuscle (pink to red area). (H&E. x500). Fig. (98): A photomicrograph in the thymus section for T3, showing the cortex (thick arrow) and medulla (head arrow). Note, Hassle's corpuscle (curve arrow). (H&E. x500). Fig. (99): A photomicrograph in the thymus section for T4, showing the cortex (thick arrow) and medulla (head arrow). Note, Hassle's corpuscle (pink to red area). (H&E. x500). Fig. (100): A photomicrograph in the thymus section for T5, showing the cortex (thick arrow) and medulla (head arrow). Note, Hassle's corpuscle (zigzag arrow), hemorrhages (curve arrow) and invasion of fibroblasts pink to rose area. (H&E. x500). Fig. (101): A photomicrograph in the thymus section for T6, showing the cortex (thick arrow) and medulla (head arrow). Note, Hassle's corpuscle (curve arrow), hemorrhages (red area) and invasion of fibroblasts pink to rose area. (H&E. x500). Fig. (102): A photomicrograph in the thymus section for T7, showing the cortex (thick arrow) and medulla (head arrow). And hemorrhages (red area). (H&E. x500). Fig. (103): A photomicrograph in the thymus section for T8, showing the cortex (thick arrow), medulla (head arrow), invasion of fibroblasts (curve area), Note, Hassle's corpuscle the highly curve arrow and connective tissues (wavy arrow). (H&E. x500). Fig. (104): A photomicrograph in the thymus section for T1, showing the cortex (thick arrow) and medulla (curve arrow). (H&E. x500). Fig. (105): A photomicrograph in the thymus section for T2, showing the cortex (thick arrow) and medulla (head arrow) and lymphocytic depletion (zigzag arrow). (H&E. x500). Fig. (106): A photomicrograph in the thymus section for T3, showing the cortex (thick arrow) and medulla (head arrow) and hemorrhage (wavy arrow). (H&E.x500). Fig. (107): A photomicrograph in the thymus section for T4, showing the cortex (thick arrow) and medulla (head arrow), hemorrhage (wavy arrow) and XVIII
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connective tissues (zigzag arrow). (H&E.x500). Fig. (108): A photomicrograph in the thymus section for T5, showing the cortex (thick arrow) and medulla (head arrow), focal areas of macrophage activity (thin arrow) and hemorrhage (curve arrow). (H&E. x500). Fig. (109): A photomicrograph in the thymus section for T6, showing the cortex (thick arrow) and medulla (head arrow), focal areas of macrophage activity (thin arrow) and hemorrhage (curve arrow). (H&E. x500). Fig. (110): A photomicrograph in the thymus section for T7, showing the cortex (thick arrow) and medulla (head arrow), focal areas of macrophage activity (thin arrow) and hemorrhage (curve arrow). (H&E. x500). Fig. (111): A photomicrograph in the thymus section for T8, showing the cortex (thick arrow) and medulla (head arrow) and focal areas of macrophage activity (wavy arrow). (H&E. x500). Fig. (112): A photomicrograph in the Bursa of Fabricius section for T1, showing the normal structure of epithelial cells and lymphocytes. (H&E. x500). Fig. (113): A photomicrograph in the Bursa of Fabricius section for T2, showing the normal structure of epithelial cells, lymphocytes and normal bursal lobules. (H&E. x500). Fig. (114): A photomicrograph in the Bursa of Fabricius section for T3, showing the normal structure of epithelial cells, lymphocytes and normal bursal lobules. (H&E. x500). Fig. (115): A photomicrograph in the Bursa of Fabricius section for T4, showing the normal structure of epithelial cells, lymphocytes and normal bursal lobules. (H&E. x500). Fig. (116): A photomicrograph in the Bursa of Fabricius section for T5, showing degeneration within the lobules, epithelial cells and losing lymphocytes. (H&E. x500). Fig. (117): A photomicrograph in the Bursa of Fabricius section for T6, showing degeneration within the lobules, epithelial cells and losing lymphocytes. (H&E. x500). Fig. (118): A photomicrograph in the Bursa of Fabricius section for T7, showing the normal structure of epithelial cells, lymphocytes and normal bursal lobules. (H&E. x500). Fig. (119): A photomicrograph in the Bursa of Fabricius section for T8, showing degeneration within the lobules, epithelial cells and little losing lymphocytes. (H&E. x500). Fig. (120): A photomicrograph in the Bursa of Fabricius section for T1, showing the normal structure of epithelial cells and lymphocytes in bursal lobules. (H&E. x500). Fig. (121): A photomicrograph in the Bursa of Fabricius section for T2, showing the normal structure of epithelial cells and lymphocytes in
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bursal lobules. (H&E. x500). (122): A photomicrograph in the Bursa of Fabricius section for T3, showing the normal structure of epithelial cells, lymphocytes in bursal lobules and normal connective tissues. (H&E. x500). (123): A photomicrograph in the Bursa of Fabricius section for T4, showing normal structure of epithelial cells, lymphocytes in bursal lobules and normal connective tissues. (H&E. x500). (124): A photomicrograph in the Bursa of Fabricius section for T5, showing severs degeneration within the lobules, epithelial cells and losing lymphocytes. (H&E. x500). (125): A photomicrograph in the Bursa of Fabricius section for T6, showing recovers of the degeneration within bursal lobules, epithelial cells and normal lymphocytes with some hemorrhage within connective tissues. (H&E. x500). (126): A photomicrograph in the Bursa of Fabricius section for T7, showing protection from the degeneration within bursal lobules, epithelial cells, normal lymphocytes with some hemorrhage and little losing of lymphocytes. (H&E. x500). (127): A photomicrograph in the Bursa of Fabricius section for T8, showing protection from the degeneration within bursal lobules, epithelial cells, normal lymphocytes with some hemorrhage and little losing of lymphocytes. (H&E. x500). (128): Effect of Curcuma longa (Cur.) addition to broiler diets inclusion with or without aflatoxins (AFs.) during different experimental periods on economic efficiency.
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Curcuma longa Versus Aflatoxin Contamination
LIST OF ABBREVIATIONS Alb
Albumin
A/G ratio
Albumin: Globulin ratio
ALT
Alanine Aminotransferase
ANOVA
Analysis Of Varian’s
AFs
Aflatoxins
AFB1
Aflatoxin B1
AST
Aspartate Aminotransferase
BW
Body Weight
BWG
Body Weight Gain
CF
Crude Fiber
CP
Crude Protein
CYP
Cytochrome P450
CYP1A1
Cytochrome P A1
CYP3A4
Cytochrome P450 3A4
Succinyl-CoA
Succinyl-Coenzyme A
DM
Dry Mater
DNA
Deoxyribonucleic acid
DTH
delayed-type hypersensitivity
E.E
Ether Extract
FCR
Feed Conversion Ratio
FI
Feed Intake
FL
Femtoliter
GIT
Gastro Intestinal Tract
GLM
General Liner Model
Glob
Globulin
GOT
Glotamic Oxaloacetic Transaminase XXI
GPT
Glotamic Pyruvate Transaminase
(GSTs)
Glutathione S Transferases
H/L ratio
Heterophil: Lymphocyte ratio
H2SO4
Sulfuric Acid
Hb.
Hemoglobin Percentage
Hct
Hematocrit
HDL
High Density Lipoprotein- cholesterol
HI
Hemagglutination
Ig
Immunoglobulin
IgA
Immunoglobulin A
IgG (IgY)
Immunoglobulin G
INF-γ
Interferon-γ
LDL
Low Density Lipoprotein - cholesterol
mRNA
Messenger Ribonucleic acid
MCH
Mean Corpuscular Hemoglobin
MCHC
Mean Corpuscular Hemoglobin Concentration
MCV
Mean Corpuscular Volume
ME
Metabolizable Energy
MO
Moisture content
MOS
Mannan- Oligosaccharide
Na Cl
Sodium Chloride
Na OH
Sodium Hydroxide
NDV
Newcastle Disease Virus
NFE
Nitrogen Free Extract
NKC’s
Natural Killer Cells
NRC
National Research Council
OM
Organic Mater
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OS
oxidative stress
PCV
Packed Cell Volume
Pg
Pictogram
pH
measure of the acidity
R.P.M
Round Per Minute
RBC's
Red Blood Cells
RID
Radial Immunodiffusion
ROS
Reactive Oxygen Species
RPPHS
Reference Preparation for Protein in Human Serum
SAS
Statistical Analysis Software
SE
Stander Error
SRBC's
Sheep Red Blood Cells
TP
Total Protein
TNF
Tumor Necrosis Factor
VLDL
Very Low Density Lipoprotein- cholesterol
WBC's
White Blood Cells
ZnSO4
Zinc Sulphate
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Curcuma longa Versus Aflatoxin Contamination
_________________________________________ I. INTRODUCTION I. INTRODUCTION
Aflatoxins
are concerning as part of mycotoxins. International
Agency for Research on Cancer categorized aflatoxins as class A1 human carcinogens. Commonly, maize is attributed mostly more than 60% of the chicken’s diet. Imported maize is susceptible of fungal contaminations. Shipping time, destination and storing conditions would be affected the contamination level. There are many ways to control and cope with aflatoxins. Physical, chemical, nutritional and microbial methods are familiar. However, there are modern roads, supplementing the chicken’s diet by medicinal herbs. Medicinal herbs well known to face many diseases and toxins, whether through prevention, treatment, or even the impact of antagonism. Since 1999, USA Ministry of Public Health listed Curcuma longa (turmeric rhizome powder) as Herbal Medicinal Products. Moreover, Curcuma longa are widely used in different purposes. Therefore, the present study conducted to speculate the effects of 0.5% Curcuma longa on productive performance, physiological, immunological reactions and histopathological alteration of broiler chicks fed diets contaminated with (100 µg AFs/kg diet) in focus on protection, therapeutic and antagonistic status.
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Curcuma longa Versus Aflatoxin Contamination
II. REVIEW OF LITERATURE
II. REVIEW OF LITERATURE Aflatoxins are a group of naturally occurring, extremely toxic and biologically active metabolites produced by the common molds Aspergillus flavous link and A. parasiticus. There are more than 20 isolated AFs, but only four, called AFB1, AFB2, AFG1 and AFG2 are the major significant contaminants of food and are the most acutely toxic of AFs (Park and Pezzuto, 2002). Aflatoxin B1 (AFB1) is the most toxic and a known carcinogenic. Acute or chronic aflatoxicosis in poultry leads to decreasing meat/egg production, immunosuppressant, and hepatotoxicosis (Khan et al., 2010). 1. Importance of studying aflatoxicosis: In the United States alone, the mean economic annual costs of farmer gate cereal crop losses due to AFs, fumonisins and trichothecenes, are estimated to be $932 million (Miller et al., 1998). Hegazy and Adachi, (2000) reported that 30.7 % of 1175 poultry feed samples collected from Egyptian farms were contaminated with AFs. Mycotoxin contamination of the food chain has a major economic impact. However, the insidious nature of many mycotoxicosis makes it difficult to estimate incidence and cost. In addition to crop losses and reduced animal productivity, costs are derived from the efforts made by producers and distributors to counteract their initial loss, the cost of improved technologies for production, storage and transport, the cost of analytical testing, especially as detection or regulations become more stringent, and the development of sampling plans (Whitaker, 2006). 2. Aflatoxins in poultry feedstuffs and its toxicity: There is no toxicity value for humans, but epidemiological, clinical, and experimental studies reveal, that exposure to large doses (>6.0 mg) of
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II. REVIEW OF LITERATURE
aflatoxin may cause acute toxicity with lethal effect (Eaton and Groopman, 1994). Chronic toxicity is due to long-term exposure of moderate to low levels of aflatoxin. The symptoms include, decrease in growth rate, lowered egg production, and immune-suppression. Sometimes, carcinogenicity is also observed. The liver is the main target organ and damage manifests itself as colour that is characteristic of jaundice. The gall bladder appears swollen. Immuno-suppression is due to the reactivity of AFs with T-cells, decrease in vitamin K activities, and a decrease in phagocytic activity in macrophages. These immuno-suppressive effects of AFs predispose the animals to many secondary infections due to other fungi, bacteria and viruses (McLean and Dutton, 1995). Aflatoxin is acutely toxic to most animal species at high levels. The acute medium lethal dose (LD50) value of AFB1 for most young animals (Ducklings, Rabbits, Turkeys and chickens) is about (0.5 mg/kg) of body weight. If this quantity is consumed, death of the animal will occur in about 72 h. If such animals are examined, it will be evident that they suffered from liver damage and hemorrhaging in the intestinal tract and peritoneal cavity (Marth, 1997). The acute medium lethal dose (LD 50) values for AFB1 was 0.30 in rabbits; 0.36 in duckling; 1.86 in turkeys and 6.50 in chickens (mg/kg body weight). The relative toxication of AFB1; G1; B2 and G2 were 100; 50; 25 and 12.5 % respectively (McKean, et al., 2006). Aflatoxins caused clinical illness and death when consumed in high quantity, but at lesser levels suppress immunity of young animals (Shehata, 2002). AFs have been detected in the pre-harvest, post-harvest, transport, storage and after processing and packing of grains (CAST, 2003).
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II. REVIEW OF LITERATURE
3. Metabolism of aflatoxins in poultry: Metabolism of xenobiotics including AFB1 can be divided into three phases, bioactivation (phase I), conjugation (phase II) and deconjugation (phase III), all of them can occur directly at the site of absorption, in the blood, after entering the liver as the main metabolizing organ, or in several extra hepatic tissues (Vermeulen, 1996). AFB1 itself is not a potent toxin, and phase I bioactivation is needed to exert toxic effects (Massey et al., 1995). Phase I reactions are mainly oxidation of AFB1 to hydroxylated metabolites such as aflatoxin M1, aflatoxin Q1 and aflatoxin P1 and to the highly reactive of AFB18, 9 epoxide (Eaton and Gallagher, 1994). The reduced form of nicotine amide adenine dinucleotide phosphate (NADPH) is required as a cofactor and oxygen is used as a substrate (Vermeulen, 1996). Although predominantly expressed within the liver, cytochrome P450 (CYPs) are additionally expressed extra hepatically within most tissues and especially in the respiratory and intestinal tract. Phase II metabolism includes conjugation of phase I metabolites with glutathione or glucuronic acid and is considered detoxification to enhance water solubility and excretion (Massey et al., 1995). Epoxide can be conjugated with glutathione with the help of Glutathione S transferase Cullen and Newberne, (1994), an enzyme essential in the reduction and prevention of AFB1induced carcinogenicity. Conjugates of epoxide and hydroxylated AFB1 metabolites are readily excreted via the bile in to the intestinal tract, where they might be subject to bacterial deconjugation as phase III reaction. The metabolism and toxicity of AFB1 have been studied in human cellular systems derived from liver
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II. REVIEW OF LITERATURE
(Van Vleet et al., 2001) or respiratory tract, but the impact of the intestinal metabolism is still to be investigated (Knasmüller et al., 2004). 4. Effect of aflatoxins on productive performance: 4.1. Growth performance, efficiency of feed utilization and mortality rate: Presence of AFs in poultry diets has been reported to be generally associated with growth retardation, decreased feed consumption and inefficient feed conversion. Aflatoxicosis in poultry is characterized by weakness, anorexia with lower growth rate, poor utilization, decreased weight gain, decreased egg production, increased susceptibility to environmental and microbial stresses, and increased mortality (Bailey et al., 1998). Quezada et al., (2000) observed that AFB1 (2 µg/g of feed) caused a decrease in body weight (10%) of 4-week-old chickens and a more severe reduction (20–30%) in 1-week-old chicks when compared with the control group. Results of Raju and Devegowda (2000) revealed that, body weight and feed intake were significantly reduced by feeding (0.3 mg/kg diet) of AFBl while feed conversion ratio was insignificantly affected. Main et al.,
(2001) reported that feeding one day-old broiler
chicks on aflatoxin B1-contaminated diet (200 ppb) for 8 weeks, significantly reduced body weight, feed efficiency and carcass yield. Elizabeth et al., (2003) showed that weight gain of broiler chicks was negatively affected (P < 0.01) by AF in diet (1000 ppb). Dersjant-Li et al., (2003) reported that the rate of body weight gain in broilers is reduced depending on the levels of aflatoxin in the diet.
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II. REVIEW OF LITERATURE
They suggested that with each mg/kg diet increase of aflatoxin in broiler diet would depress the growth rate by 5%. According to Verma et al., (2004a) broiler chicks received dietary aflatoxin at the levels of (0.5, 1.0 or 2.0 mg / kg diet) from 1 to 49 day of age, had significant depression in growth, reduced feed consumption and lowered feed conversion with the diets containing the greatest concentrations of AFs (1 and 2 mg / kg diet). Allameh et al., (2005) reported that chicks fed AFB1 containing diet (1 or 2 ppm AFB1) recorded a significant reduction (P < 0.05) in feed intake with 24–32% reduction in body weight gain particularly during the second phase of rearing (21-42 days) and increased feed conversion as compared to control. Azza, (2005) showed that basal diet contaminated with (100 or 200 µg AFB1/ kg diet) caused noticeable decrease in body weight by 6.7% and 10.52%, respectively. Qota et al., (2005) reported that one-day-old El-Salam chicks given a (500 ppb) AFB1 - contaminated diet for six weeks had lower body weight (25.12%) less feed intake (11.19%) and poorer feed conversion (21.40%) values than the control chicks The above results in good engagement with those of (Anong and Suparat, 2006; Pandey and Chauhan, 2007; Han et al., 2008 and Sultana and Hanif, 2009). Denli, et al., (2009) indicated that dietary AFB1 (1 mg/ kg) significantly (P < 0.05) decreased the body weight gain, feed intake, and impaired feed conversion rate of broiler chicks. Ananda et al., (2010) showed that at 2nd , 4th and 6th weeks of age broiler body weight, feed consumption and feed efficiency significantly (P0.05). Moreover, in the second phase (from 21 to 42 d) of the experiment, daily body weight gain and daily feed intake of the AFB1 treatments were decreased by 5.62% (P0.05), respectively, compared with the control, but the feed conversion was increased by 4.93% (P
