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ANTIBIOTIC RESIDUE IN POULTRY PRODUCTS
BY
FARHEEN ASAD (M.Phill Biochemistry)
2000-ag-1548
A thesis submitted in partial fulfillment Of the requirements for the
Degree of
DOCTOR OF PHILOSOPHY
IN
BIOCHEMISTRY
DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY, FACULTY OF SCIENCES,
UNIVERSITY OF AGRICULTURE, FAISALABAD, PAKISTAN
2012
DECLARATION
I hereby declare that the contents of the thesis, “Antibiotic Residues in
Poultry Products” are product of my own research and no part has been copied from
any published source (except the references, standard mathematical or genetic
models/equations/formulae /protocols etc.). I further declare that this work has not
been submitted for award of any other diploma/degree. The university may take
action if the information provided is found inaccurate at any stage.
Farheen Asad (2000-ag-1548)
Ph. D Biochemistry
To The Controller of Examinations, University of Agriculture,
Faisalabad.
“We, the supervisory committee, certify that the contents and form of
the thesis submitted by Miss Farheen Asad, Regd. No. 2000-ag-1548 have been
found satisfactory and recommend that it be processed for evaluation, by External
Examiner(s) for the award of degree”.
SUPERVISORY COMMITTEE: Supervisor : ------------------------------------------------ (Prof. Dr. Munir Ahmad Sheikh) Member : ------------------------------------------------ (Dr. Amer Jamil) Member : ------------------------------------------------ (Prof. Dr. Zia-ur-Rahman)
ACKNOWLEDGMENT
In the foremost, I present my earnest thank givings to “Almighty Allah” for enabling me to
carry on and complete this work. I proffer my humble wishes to Holy Prophet (PBUH) and Hazrat
Ali (A.S.) whose valuable and great teachings ever served as a source of inspiration for me.
We are highly obliaged for the grant from Higher Education Commission, Islamabad
(HEC) to do this research.
My evermore and sincere gratitude goes to my supervisor, Prof. Dr. Munir Ahmad
Sheikh who was abundantly helpful and offered invaluable assistance and guidance. I am
beholden to him for his rousing guidance, his trusted supervision and his hearty approval and
support. I am indebted to him more than he knows.
It will be a miss on my part if I fail to acknowledge the much needed cooperation of
Prof. Dr. Amer Jamil, Department of Chemistry and Biochemistry, UAF, who had been a
constant source of inspiration for me during this research work.
This thesis would not have been possible without the absolute support of
Prof. Dr. Zia-ur-Rahman Department of Physiology and Pharmacology, University of
Agriculture Faisalabad. Above all and the most needed, he provided me unflinching
encouragement and support in various ways. His generous contributions in providing
resources whenever I needed, his undemanding accessibility in the times of disappointment
make me stand indebted to him.
I am greatly thankful to my parents, my dear sisters, and brothers, and especially my
dear friend Sadia Batool whose admirable feelings are always with me.
I extend my thanks to all those who have helped me in my research work in the
Department of Physiology and Pharmacology University of Agriculture, Faisalabad.
FARHEEN ASAD
Dedication
To,
Those who are who are
Nearest and dearest to me
Whose hands for prayers
Are always with me
TABLE OF CONTENTS
Chapter No.
Content Page No.
LIST OF TABLES i
LIST OF FIGURES viii
ABSTRACT ix
1 INTRODUCTION 01
2 REVIEW OF LITERATURE 04
2.1 Scope of Antibiotic Drugs in Livestock 04
2.2 Prevalence of Antibiotic drug Residues In Poultry Products 05
2.3 Fluoroquinolone Residue in Poultry products 07
2.4 Resistance Developed by Antibiotic Residue 12
2.5 Detection Methods for Antibiotic Residues 13
2.6 Seasonal effect of antibiotic residue persistence in poultry tissues and eggs
28
2.7 Cooking effect on depletion and removal of antibiotic residue from poultry products
28
2.8 Occurrence of antibiotic residue in poultry products in Pakistan 29
3 MATERIALS AND METHODS 31
3.1 Selection of birds 31
3.2 Sampling 31
3.3 Swab Test on Animal Food (STAF) 32
3.4 Experimental Protocol 36
3.4 Cooking Operation 38
3.5 Health Biomarkers 43
3.5.1 Total Oxidant Status (TOS; mMol/L) 44
3.5.2 Total Antioxidant Capacity (TAC; mmol/L) 46
3.5.3 Paraoxonase Activity (PON1; Unit/L) 48
3.5.4 Arylesterase Activity (K Unit/L) 48
3.5.5 Catalase Activity 49
3.6 Statistical Analysis 50
TABLE OF CONTENTS … CONTINUED
Chapter
No. Content Page
No.
4 RESULTS 51
PHASE I: (Surveillance) 51
4.1 Survey of Broilers 51
4.2 Survey of Layers 59
PHASE II: 71
4.3 Withdrawal Time in Broilers 71
4.4 Health Biomarkers in Broilers 75
4.4.1 Total Oxidant Status (TOS; µmol/L±SE) in Broilers 75
4.4.2. Total Antioxidant Capacity (TAC; mmol/L±SE) in Broilers
80
4.4.3 Arylesterase concentration (KU/L±SE) in Broilers 85
4.4.4 Paraoxonase concentration (PON1; U/L±SE) in Broilers 90
4.4.5 Catalase concentration (KU/L±SE) in Broilers 94
4.6 Concentration of fluoroquinolones Before and After Cooking
99
4.7 Concentration of fluoroquinolones after cooking in Electric and Microwave Ovens
101
PHASE II: (withdrawal time of layer birds) 103
4.8 Layers 103
4.9 Health Biomarkers in Layers 108
4.9.1 Total oxidant status (TOS; µmol/L ± SE) in layers 108
4.9.2 Total antioxidant capacity (TAC; mmol/L±SE) in layers
113
4.9.3 Arylesterase concentration (KU/L±SE) in layers 118
4.9.4 Paraoxonase concentration (PON1; U/L±SE) in layers 123
4.9.5 Catalase concentration (KU/L±SE) in layers 128
4.10 Concentration of Fluoroquinolones in Layers Before and after Cooking
133
4.11 Concentration of Fluoroquinolones After Cooking in Electric and Microwave Ovens
135
TABLE OF CONTENTS … CONTINUED
Chapter
No. Content Page
No.
5 DISCUSSION 137
6 SUMMARY 144
REFERENCES 146
APPENDIX 172
i
LIST OF TABLES Table No.
Title Page No.
2.1 A maximum residue limits of fluoroquinolones in poultry products 11 2.2 Summary of different techniques applied for detection of drug residues 14 2.3 Extraction of flouroquinolone with water immiscible organic solvent 19 2.4 Extraction of flouroquinolones with water miscible organic solvent 20 2.5 Extraction of flouroquinolone with acidic solution 22 2.6 Extraction of flouroquinolones with basic or neutral buffer solution 26 3.1 Number of samples of layer birds collected from different farm houses in and
around Faisalabad 31
3.2 Number of samples of broiler birds collected from different farm houses in and round Faisalabad
32
3.3 Fluoroquinolone antibiotics (1ml/4L) added in layers and broiler drinking water (ad libitum).
36
3.4 Accuracy (%) and precision cv (%) of different quinalones during intra and inter-day assay
43
4.1 Survey of antibiotic residue from leg meat of broilers from different farm houses located in and around Faisalabad district
51
4.2 Survey of antibiotic residue from breast meat of broilers from different farm houses located in and around Faisalabad district
52
4.3 Survey of antibiotic residue from liver of broilers from different farm houses located in and around Faisalabad district
53
4.4 Survey of antibiotic residue from lungs of broilers from different farm houses located in and around Faisalabad district
54
4.5 Survey of antibiotic residue from heart of broilers from in and around Faisalabad district
55
4.6 Mean inhibition zones (mm) of different tissues of broilers from different areas of Faisalabad
56
4.7 Surveys of antibiotic residue in different tissue and organ of broiler during different months of experimental period
57
4.8 Surveys of antibiotic residue in different tissue and organs of broilers during different seasons
58
4.9 Survey of antibiotic residue in the leg meat of layer from different towns of Faisalabad
59
4.10 Survey of antibiotic residue in the breast meat of layer from different towns of Faisalabad
60
4.11 Survey of antibiotic residue in the liver of layer from different farms located in and around Faisalabad district
61
4.12 Survey of antibiotic residue in the lungs of layer from different farms located in and around Faisalabad district
62
4.13 Survey of antibiotic residue in the heart of layers from different farms located in and around Faisalabad district
63
4.14 Mean inhibition zones (mm) of different tissues of layers from different areas of Faisalabad
64
4.15 Survey of antibiotic residue in various organs of laying hens during various months of experimental conditions
65
ii
LIST OF TABLES … CONTINUED Table
No. Title Page
No. 4.16 Surveys of antibiotic residue in different tissue and organs of layer during three
different seasons 66
4.17 Survey of antibiotic residue in the egg yolk of layer at different days from different farms in and around Faisalabad district
67
4.18 Survey of antibiotic residue in the egg white of layer at different days from different farms in and around Faisalabad district
68
4.19 Mean inhibition zones (mm) of egg white and yolk of layers from different areas of Faisalabad
69
4.20 Survey of antibiotic residue of egg white and egg yolk of layers during different months of a year
70
4.21 Survey of antibiotic residue of egg white and egg yolk of layers during three different season
70
4.22 Analysis of variance of concentration of different fluoroquinolones at different groups on different days
71
4.23 Mean concentration (ppm±SE) of different fluoroquinolones in serum of broilers at different days after therapy
71
4.24 Analysis of variance of concentration of fluoroquinolones in muscle of broiler at different days
72
4.25 Mean fluoroquinolones concentration (ppm ± SE) of muscle from broiler at different days after therapy
72
4.26 Analysis of variance of concentration of fluoroquinolones in the liver of broilers at different days after therapy
73
4.27 Mean fluoroquinolone (ppm ± SE) concentration of liver from broilers at different days after therapy
73
4.28 Analysis of variance of concentration of fluoroquinolones in the kidney of broiler at different days of experimental period
74
4.29 Mean fluoroquinolones concentration (ppm±SE) in the kidney of broilers in different groups at various time intervals
74
4.30 Analysis of variance of total oxidant status in serum of broiler birds fed with different fluoroquinolones
75
4.31 Mean total oxidant status (TOS; µmol/L ± SE) in serum of broiler birds fed with different fluoroquinolones
75
4.32 Analysis of variance of total oxidant status in broiler muscles fed with different fluoroquinolones
76
4.33 Mean total oxidant status (TOS; µmol/L ± SE) of liver from broilers fed different fluoroquinolones
76
4.34 Analysis of variance of total oxidant status in the liver of broilers fed different fluoroquinolones
77
4.35 Mean total oxidant status (TOS; µmol/L ± SE) of liver from broilers fed different fluoroquinolones
77
4.36 Analysis of variance of total oxidant status in broiler kidney fed different fluoroquinolones
78
4.37 Mean total oxidant status (TOS; µmol/L ± SE) in kidney of broilers fed different fluoroquinolones.
78
4.38 Analysis of variance of total oxidant status in heart of broiler fed different fluoroquinolones
79
iii
LIST OF TABLES … CONTINUED Table No.
Title Page No.
4.39 Mean of total oxidant status (TOS; µmol/L ± SE) in heart of broiler fed different fluoroquinolones
79
4.40 Analysis of variance of serum concentration of total antioxidant capacity in broiler at different days of experimental period
80
4.41 Mean serum concentration of total antioxidant capacity (TAC; mmol/L±SE) in broiler showing its effects on different days of experimental period
80
4.42 Analysis of variance of total antioxidant capacity in muscles of broiler fed fluoroquinolones in different groups at different days of experimental period
81
4.43 Mean total antioxidant capacity (TAC; mmol/L±SE) of broiler muscles fed different fluoroquinolones during different days
81
4.44 Analysis of variance of total antioxidant capacity (TAC) of broiler liver obtained from different groups fed fluoroquinolons at different days
82
4.45 Mean total antioxidants capacity (TAC; mmol/L±SE) of liver from broiler fed fluoroquinolons during different days
82
4.46 Analysis of variance OF TAC (mmol/L±SE) in broiler kidney 83 4.47 Mean total antioxidants capacity (TAC; mmol/L±SE) of kidney from broiler
fed fluoroquinolons during different days 83
4.48 Analysis of variance of total antioxidant capacity in broiler heart fed different fluoroquinolones
84
4.49 Mean total antioxidants capacity (TAC; mmol/L±SE) of heart from broiler fed fluoroquinolons during different days
84
4.50 Analysis of variance of serum arylesterase concentration of broiler fed different fluoroquinolones
85
4.51 Mean serum arylesterase (KU/L±SE) concentration of broiler fed different fluoroquinolones
85
4.52 Analysis of variance of muscle arylesterase concentration of broiler fed different fluoroquinolones
86
4.53 Mean muscle arylesterase concentration (KU/L±SE) of broiler fed fluoroquinolones at different days of therapeutic dose.
86
4.54 Analysis of variance of liver arylesterase concentration of broiler fed different fluoroquinolones
87
4.55 Mean liver arylesterase concentration (KU/L±SE) of broiler fed different fluoroquinolones at different days of therapeutic dose
87
4.56 Analysis of variance of kidney arylesterase concentration of broiler fed different fluoroquinolones
88
4.57 Mean kidney arylesterase concentration (KU/L±SE) of broiler fed different fluoroquinolones at different days after oral therapy
88
4.58 Analysis of variance of broiler arylesterase concentration of broiler heart fed different fluoroquinolones
89
4.59 Mean arylesterase concentration (KU/L±SE) of broiler heart fed different fluoroquinolones at various days after a therapeutic dose
89
4.60 Analysis of variance of paraoxonase concentration of broiler serum fed different fluoroquinolones
90
4.61 Mean paraoxonase concentration (PON1; U/L±SE) of broiler serum fed different fluoroquinolones
90
iv
LIST OF TABLES … CONTINUED Table No.
Title Page No.
4.62 Analysis of variance of paraoxonase concentration of broiler liver fed different fluoroquinolones
91
4.63 Mean paraoxonase concentration (PON1; U/L±SE)of broiler liver fed different fluoroquinolones
91
4.64 Analysis of variance of paraoxonase concentration of broilers kidney fed different fluoroquinolones
92
4.65 Mean paraoxonase concentration (PON1; U/L±SE) of broilers kidney fed different fluoroquinolones
92
4.66 Analysis of variance of paraoxonase concentration of broiler heart fed different fluoroquinolones
93
4.67 Mean paraoxonase concentration (PON1; U/L±SE) of broiler heart fed different fluoroquinolones
93
4.68 Analysis of variance of broiler serum catalase exposed to different fluoroquinolones at different days
94
4.69 Mean broiler serum catalase (KU/L±SE) exposed to different fluoroquinolones at different days
94
4.70 Analysis of variance of broiler muscle catalase exposed to different fluoroquinolones at different days
95
4.71 Mean broiler muscle catalase (KU/L±SE) exposed to different fluoroquinolones at different days
95
4.72 Analysis of variance of broiler liver catalase exposed to different fluoroquinolones at different days
96
4.73 Mean broiler liver catalase (KU/L±SE) exposed to different fluoroquinolones at different days
96
4.74 Analysis of variance of broiler kidney catalase exposed to different fluoroquinolones at different days
97
4.75 Mean broiler kidney catalase (KU/L±SE) exposed to different fluoroquinolones at different days
97
4.76 Analysis of variance of broiler heart catalase exposed to different fluoroquinolones at different days
98
4.77 Mean broiler heart catalase (KU/L±SE) exposed to different fluoroquinolones at different days
98
4.78 Analysis of variance of different fluoroquinolones concentration from muscle of broiler before and after cooking
99
4.79 Mean muscle concentration (ppm±SE) of different floroquinolones in the broilers before and after cooking at various days after therapy
99
4.80 Analysis of variance of different fluoroquinolones concentration of liver from broiler before and after cooking
100
4.81 Mean liver concentration (ppm±SE) of different fluoroquinolones in the broilers before and after cooking at various corresponding days
100
4.82 Analysis of variance of fluoroquinolones concentration of broilers muscle after two cooking methods
101
4.83 Mean concentration of floroquinolones in the broiler muscle after two different methods of cooking at various days
101
4.84 Analysis of variance of floroquinolones concentration of broilers liver two methods of cooking
102
v
LIST OF TABLES … CONTINUED Table No.
Title Page No.
4.85 Mean concentration of floroquinolones in the broiler liver after two different methods of cooking at various days
102
4.86 Analysis of variance of concentration of different fluoroquinolones in different groups
103
4.87 Mean concentration (ppm±SE) of different fluoroquinolones in serum of layers at different days of experimental period
103
4.88 Analysis of variance of concentration of different fluoroquinolones in the muscles of layers at various days of experimental condition
104
4.89 Mean concentration (ppm±SE) of different fluoroquinolones in the muscles of layer at various days of experimental period
104
4.90 Analysis of variance of concentration of different fluoroquinolones present in liver at various days
105
4.91 Mean concentration (ppm±SE) of different fluoroquinolones from layer liver measured at different intervals
105
4.92 Analysis of variance of concentration of different fluoroquinolones in the kidney of days at various days of experimental period
106
4.93 Mean concentration (ppm±SE) of fluoroquinolones in the kidney of layers at various days of experimental period
106
4.94 Analysis of variance of concentration of different fluoroquinolones in the egg of layer at different days of experimental period
107
4.95 Mean concentration (ppm±SE) of different fluoroquinolones in the egg of layer at different days
107
4.96 Analysis of variance of total oxidant status in serum of layers exposed to three fluoroquinolones at different days of experimental period
108
4.97 Mean serum concentration of total oxidant status (TOS; µmol/L ± SE) at in layers different time intervals after oral ingestion of fluoroquinolones
108
4.98 Analysis of variance of total oxidant status in muscles of layers exposed to three fluoroquinolones at different days of experimental period
109
4.99 Mean muscles concentration of total oxidant status (TOS; µmol/L ± SE) at in layers different time intervals after oral ingestion of fluoroquinolones
109
4.100 Analysis of variance of total oxidant status in liver of layers exposed to three fluoroquinolones at different days of experimental period
110
4.101 Mean liver concentration of total oxidant status (TOS ;µmol/L ± SE) at in layers different time intervals after oral ingestion of fluoroquinolones
110
4.102 Analysis of variance of total oxidant status in kidney of layers exposed to three fluoroquinolones at different days of experimental period
111
4.103 Mean liver concentration of total oxidant status (TOS; µmol/L ± SE) at in layers fed different fluoroquinolones at different days after therapy
111
4.104 Analysis of variance of total oxidant status in heart of layers exposed to three fluoroquinolones at different days of experimental period after therapy
112
4.105 Mean heart concentration of total oxidant status (TOS ; µmol/L ± SE)at in layers fed different fluoroquinolones at different days after therapy
112
4.106 Analysis of variance of total antioxidant capacity in serum of layer fed with different fluoroquinolones at different time intervals
113
4.107 Mean total antioxidant capacity (TAC; mmol/L±SE) in serum of layers fed different fluoroquinalones at various days after therapy
113
vi
LIST OF TABLES … CONTINUED Table No.
Title Page No.
4.108 Analysis of variance of total antioxidant capacity from muscles of layer fed different fluoroquinolones
114
4.109 Mean muscle concentration of TAC (TAC; mmol/L±SE) of layer fed different fluoroquinolones
114
4.110 Analysis of variance of total antioxidant capacity(TAC; mmol/L) from liver of layer fed different fluoroquinolones
115
4.111 Mean liver concentration of TAC (TAC; mmol/L±SE) of layer fed different fluoroquinolones at various days after therapy
115
4.112 Analysis of variance of total antioxidant from kidney of layer fed different fluoroquinolones
116
4.113 Mean total antioxidant capacity (TAC; mmol/L±SE) from kidneys of layer fed different fluoroquinolones
116
4.114 Analysis of variance of total antioxidant capacity of heart of layers fed different fluoroquinolones
117
4.115 Mean total antioxidant capacity (TAC; mmol/L±SE) of heart from layers fed different fluoroquinolones at various days after therapy
117
4.116 Analysis of variance of serum arylesterase concentration of layers fed different fluoroquinolones
118
4.117 Mean serum concentration of arylesterase (KU/L±SE) from layers fed different fluoroquinolones at various days after therapy
118
4.118 Analysis of variance of Layer muscle Arylesterase concentration in response to different fluooquinolones on different days after therapy
119
4.119 Mean muscle Arylesterase concentration (KU/L±SE) in response to different fluoquinolones on different days after therapy
119
4.120 Analysis of variance of Arylesterase in liver of layers fed different fluoroquinolones
120
4.121 Mean concentration of Arylesterase (KU/L±SE) in liver from layers fed different fluoroquinolones for different days after therapy of experimental period
120
4.122 Analysis of variance of arylesterase in kidney of layers fed different fluoroquinolones
121
4.123 Mean arylesterase concentration (KU/L±SE) in the kidney of layers fed different fluoroquinolones at various days after therapy
121
4.124 Analysis of variance of arylesterase in layer heart fed different fluoroquinolones
122
4.125 Mean concentration of arylesterase (KU/L±SE) in heart from layers fed was different flouroquinolones at different days after therapy of experimental period
122
4.126 Analysis of variance of paraoxonase concentration of serum from layer fed different fluoroquinolones
123
4.127 Mean concentration of serum paraoxonase (PON1; U/L±SE) from layers fed different fluoroquinolones at various days after therapy
123
4.128 Analysis of variance of paraoxonase concentration of muscles from layer fed different fluoroquinolones
124
4.129 Mean concentration of muscle paraoxonase (PON1; U/L±SE) from layers fed different fluoroquinolones at various days after therapy
124
4.130 Analysis of variance of paraoxonase concentration of liver from layer fed different fluoroquinolones
125
vii
LIST OF TABLES … CONTINUED Table No.
Title Page No.
4.131 Mean paraoxonase (PON1; U/L±SE) concentration of liver from layer fed different fluoroquinolones at various days after therapy
125
4.132 Analysis of variance of paraoxonase concentration of kidney from layer fed different fluoroquinolones
126
4.133 Mean paraoxonase concentration (PON1; U/L±SE) of kidney from layer fed different fluoroquinolones at different days after therapy
126
4.134 Analysis of variance of paraoxonase concentration of heart from layer fed different fluoroquinolones
127
4.135 Mean heart paraoxonase concentration (PON1; U/L±SE) of layers fed different fluoroquinolones at different days after therapy
127
4.136 Analysis of variance of catalase concentration in serum from layers fed different fluoroquinolones
128
4.137 Mean concentration of serum catalase (KU/L±SE) from layers fed different fluoroquinolones at various days after therapy
128
4.138 Analysis of variance of catalase concentration of muscles from layers fed different fluoroquinolones
129
4.139 Mean muscle catalase concentration (KU/L±SE) of layers fed different fluoroquinolones at various days after therapy
129
4.140 Analysis of variance of catalase concentration of liver from layers fed different fluoroquinolones
130
4.141 Mean concentration of catalase (KU/L±SE) from liver of layers fed different fluoroquinolones at various days after therapy
130
4.142 Analysis of variance of catalase concentration of kidney from layers fed different fluoroquinolones
131
4.143 Mean concentration (KU/L±SE) of catalase from kidney of layers fed different fluoroquinolones at various days after therapy
131
4.144 Analysis of variance of catalase concentration of heart from layers fed different fluoroquinolones
132
4.145 Mean concentration of catalase (KU/L±SE) from heart of layers fed different fluoroquinolones at various days after therapy
132
4.146 Analysis of variance fluoroquinolones concentration of muscle of layer before and after cooking
133
4.147 Mean muscle concentration (ppm±SE) of different fluoroquinolones of layer before and after cooking
133
4.148 Analysis of variance fluoroquinolones concentration of layers liver before and after cooking
134
4.149 Mean liver concentration (ppm± SE) of different fluoroquinolones of layer before and after cooking
134
4.150 Analysis of variance fluoroquinolones concentration of layers muscles after two methods of cooking
135
4.151 Mean concentration of fluoroquinolones in the muscle layer two different methods of cooking at various days
135
4.152 Analysis of variance fluoroquinolones concentration of layers liver after two methods of cooking
136
4.153 Mean concentration of fluoroquinolones in the liver layer two different methods of cooking at various days
136
viii
LIST OF FIGURES Fig. No.
Title Page No.
3.1 Standard curve for oxidant status 45 3.2 Standard curve for antioxidant capacity 47
4.1 Overall survey of antibiotic residue from leg meat of broilers from different farm houses located in and around Faisalabad district
51
4.2 Overall survey of antibiotic residue from breast meat of broilers from different farm houses located in and around Faisalabad district
52
4.3 Overall survey of antibiotic residue from liver of broilers from different farm houses located in and around Faisalabad district
53
4.4 Overall survey of antibiotic residue from lungs of broilers from different farm houses located in and around Faisalabad district
54
4.5 Overall survey of antibiotic residue from heart of broilers from different farm houses located in and around Faisalabad district
55
4.6 Overall survey of antibiotic residue in the leg meat of layer from different towns of Faisalabad
59
4.7 Overall survey of antibiotic residue in the breast meat of layer from different towns of Faisalabad
60
4.8 Overall survey of antibiotic residue in the lungs of layer from different farms located in and around Faisalabad district
62
4.9 Overall survey of antibiotic residue in the heart of layer from different farms located in and around Faisalabad district
63
4.10 Overall survey of antibiotic residue in the egg yolk of layer at different days from different farms in and around Faisalabad district
67
4.11 Overall survey of antibiotic residue in the egg white of layer at different days from different farms in and around Faisalabad district
68
ix
ABSTRACT This study was conducted for the detection and evaluation of antibiotic residues in
poultry products. This research work completed in three different phases. In the first-phase
sample, survey was done in different farm houses in and around Faisalabad, and antibiotic
residues were detected by microbiological assay. In the second phase, withdrawal period of
fluoroquinolone antibiotics was investigated in experimental birds. Samples of liver and
muscles of these experimental birds were cooked by electric and microwave ovens. All
samples (serum, muscle, liver, kidney and eggs) were extracted for fluoroquinolones and
quantified by HPLC with fluorescent detection. Health biomarkers of all samples were
analyzed by their reference methods. Percentage of positive samples for antibiotic residues
was calculated during a survey. This indicated the wide-spread use of antibiotics in most
farm houses and high residue persistence in liver, heart and lung tissues. Seasonal variations
were also investigated, and residue prevalence was observed in rainy season. In phase 2nd
withdrawal time study, the mean concentrations (mean±SE) of fluoroquinolones were
calculated. Analysis of variance and Duncan multiple range test was applied. Significant
differences were observed in concentrations (in serum, muscle, liver and kidney) of
fluoroquinolones in different days after slaughter. The concentrations were significantly high
at day 01 and then decreased and disappeared at day 03 and day 04, respectively.
Fluoroquinolones level also depleted after cooking of meat. Health biomarkers were
significantly affected by fluoroquinolones in treated birds. Deposition of these antibiotic
residues in edible tissues causes many hazards to human as well as animals. In phase III of
this experiment, muscles and liver that were positive for their residues during different days
of the washout time were subjected to two different cooking methods i. e. by electric oven
and by microwave oven. On each experimental day, muscle and liver residues, concentration
did decrease significantly with microwave and electric oven cooked samples, and were below
the maximum residue limit (MRL) value. Left over residue in meat below MRL’ s value
cause allergic or anaphylactic reaction, toxicity, killing of beneficial bacteria in animal and
human intestine and occurrence of resistant strains of bacteria in human. In addition
aftermath of this will affect economy as well as health of the individuals.
1
Chapter 1
INTRODUCTION
Poultry industry is the major part of livestock production throughout the world. It is
rapidly expanding in developing countries to fulfill the demands of animal protein in the
form of meat and eggs. In Pakistan, this industry is a major contributor of food supply (19%
of meat consumption) and maintaining the country’s economy, because it is a cheap and easy
source of protein for human consumption. This industry is a source of income of about 1.5
million people. The country’s investment is 200.00 billion in the current year (Economic
Survey of Pakistan, 2008-2009). For this purpose, this industry split into two parts; the
broiler industry, which produces birds slaughtered 6-7 week old for meat, and egg-producing
layer birds start producing eggs at the age of 16-18 for one laying cycle. Now a day the major
threat to poultry sector concerning the quality of its products (meat and eggs), is the presence
of drug residues. In USA, about seventy percent of the total antibiotics produced is being fed
to poultry birds, therefore, Senate through an act of 2002, has passed a bill No. S-2508 to
preserve the antibiotics for human treatment, to avoid the risk pathogenic resistance against
the antibiotics. These drugs used in the poultry sector as growth promoters, therapeutics and
prophylactics (Donoghue, 2003). These are naturally occurring, semi-synthetic and synthetic
compounds with antimicrobial activity that can be administered orally, parenterally or
topically (Phillips et al., 2004). The antibiotic groups that are used to treat the poultry birds
are; aminoglycosides, tetracyclines, beta lactames, quinonlones, macrolides, polypeptides,
amphenicols and sulfonamides (Stolker and Brinkman, 2005).
Quinolones are applied to prevent the infectious diseases in lungs, urinary and
digestive system of an animal. These antibacterial inhibit DNA gyrase (type II
topoisomerase), essential enzyme involving DNA supercoiling in a replication process
(Wolfson and Hooper, 1989). The quinolones have been classified according to their
antibacterial spectrum; potency and pharmacology. There is no widely accepted classification
of these at present (Gigosos et al., 2000). These are divided into two categories; the first-
generation quinolones include, nalidixic acid (NAL acid), oxolinic acid, flumequine (FLUM)
and piromidic acid (PIRM acid), which have good antibacterial activity against gram-
negative bacteria (Martin, 1998). Norfloxacin, ciprofloxacin, ofloxacin, lemofloxacin,
2
enrofloxacin and cinoxacin are the second generation antibiotics inhibit the activity of gram-
negative bacteria and gram-positive bacteria with fewer protein binding, higher drug
tolerance, lower toxicity and longer half-life (Kowalski and Plenis, 2008). Efforts have been
done to produce high-quality meat products with fewer fats and high level of proteins and
consumption according to affordable price, farmers add antibiotic growth promoters to the
bird’s feed and drinking water for controlling the number of undesirable bacteria in their
gastrointestinal tracts in order to absorb more nutrients (Yudisthira Swarga Foundation,
2007).
Antibiotics are accumulated in the poultry meat and eggs, and their residues may exist
either as parent compound or compounds derived from the parent drugs like metabolites (or
in both forms). These residues ultimately bound to macromolecules present in the biological
system, products or fluids (Weber, 1979). The deposition of antibiotics in different
components of egg has been reported by Roudaut et al. (1987) and Yoshimura et al. (1991).
It is a potentially dangerous practice since it can encourage the development of antibiotic
resistance and antibiotic-resistant strains of bacteria (Khachatourians, 1998; Simonsen et al.,
1998; Chen and Schneider, 2003). Antibiotic residues may produce allergic or anaphylactic
reactions in susceptible individuals (Tinkelman and Bock, 1984; Settepani, 1984), drug
toxicity (Black, 1984) and adverse effects that remain unknown (Shim et al., 2003). Some of
the antibiotics are directly toxic viz; chloramphenicol leads to aplastic anaemia while
quinolones lead to destruction of cartilages in young individuals, etc.
As poultry feed industry in Pakistan now deals with a diverse system of poultry
production and poultry diseases currently requiring the most extensive use of therapeutic or
prophylactic drugs, the distribution and use of veterinary medicines in food animals could
have been the focus of major regulatory initiative by the responsible agencies. Potential for
widespread use of antibiotics provoke a development of antibiotic-resistance bacteria
(Altekruse et al., 2002; Boothe and Arnold 2003; Belloc et al., 2005). Even the antibiotic-
resistance bacteria in animal products are not pathogenic to human; however, they may pass
their resistance genes to other pathogenic bacteria (Alcaine et al., 2005; Kim et al., 2005;
Lester et al., 2006).
3
As at present there are no initiatives for monitoring drug residues in food animals in
Pakistan, consequently, there is no longer available data on drug residue in meat and eggs.
This study is therefore aimed to investigate the meat and eggs sold for human consumption
for incidence of residue of different quinolones antibiotics and finally to calculate the
withdrawal time of some common drugs used in poultry feed as growth promoters while
keeping infection in control and as well as used to treat during bacterial infection.
Aims and Objectives
1. To analysis poultry products (serum, meat, liver, heart and egg) for the presence of
antibiotic residues
2. To establish the time course of residue's persistence in poultry products for antibiotics
3. To investigate the withdrawal period of antibiotics before the foods are marketed.
4
Chapter 2
REVIEW OF LITERATURE
2.1 Scope of Antibiotic Drugs in Livestock
Antibiotics were first recognized in 1920, when Fleming discovered a natural
antimicrobial product, penicillin. With this several other natural, synthetic or semi-synthetic
antibiotics have been produced. These compounds have sudden effects such as inhibition of
protein synthesis, cell wall of bacteria and interference in a replication process of DNA and
RNA, breaking the cell membranes and interfere with metabolic pathways. These antibiotics
introduced into veterinary medicines, after their use as human medicine. From that time,
these have become an important part of veterinary medicine for prevention and treatment of
different types of infections, particularly in food animals (Chander et al., 2007). The
antibiotics use in poultry for the treatment of many respiratory infections and chronic
enteritis, which often affect broiler chickens at an early age. Treatment with antibiotics is
much necessary to improve the physical strength of animals, to produce a great yield of food
animals and to improve the animal growth efficiency (Phillips et al., 2003). These preventive
measures are much necessary in poultry industry where the infection spread more quickly
because it is a difficult practice to treat each infected bird at one time. The antibiotics which
have been used in human medicine are mostly related with antibiotics that are used in
prophylaxis and treatments of many animal infections. Bager and Emborg (2001) divided
antibiotics into many groups such as Sulfonamides (with or without trimethoprim), Beta-
lactams (including cephalosporins and penicillin), macrolides, tetracycline, streptogramins,
lincosamides and quinolones (including fluoroquinolones). From these antibiotics, the
availability of quinolones exceeded more than 25 years (Mitchell et al., 1998).
2.1.1 Use of antibiotics in poultry industry
Food and Drug Administration (FDA) banned in using two antibiotics in poultry that
are related to the member of human medicine, these are enrofloxacin and sarafloxacin. Other
types of antibiotics have been synthesized and replaced them with those which are loosing
their antimicrobial activity (Herranz et al., 2007). This is because of a rise of different
5
pathogens resistant to fluoroquinoloes called campylobacter bacteria. These pathogens are
transferred to humans when they eat the poultry products that are undercooked (Hileman,
2000). Antibiotics are given to animals by injecting intramuscularly, intravenously or orally,
or by intramammary and intrauterine infusions in feed or drinking water and apply to the skin
(Mitchell et al., 1998). Since 1993, the antibiotic has been available in pure powder form,
which can be added in poultry feed as well as in drinking water for treatment of gram-
negative microorganisms (Al-Mustafa and Al-Ghamdi, 2000).
Small quantity of antibiotics is also added to animal feed as a feed additive for growth
promotion by increasing feed efficiency in food animal production. The effect of growth
promotion was discovered in the 1940. At that time chickens were fed with tetracycline
fermentation by-products. The growth of these chickens was more efficient than those were
not fed on these by-products (Stokestad et al., 1949). Growth promoting effects are achieved
by alterations of normal intestinal microbes resulting in efficient food digestion, metabolism
and absorption of nutrient. This excessive use of antibiotics for treatment of infections,
growth promotion and meat preservation leave residues in poultry products (Risch, 2002).
Therefore, safety of food animals, drug clearance from tissues, avoiding the residue
persistence and environmental safety are the basis for regular approval of antibiotics in
livestock production (Preston, 1987).
2.2 Prevalence of Antibiotic drug Residues In Poultry Products
Improper use of licensed substances or illegal use of unlicensed product resulting in
their residues in body tissues (Papich et al., 1993). These drug residues may bind to carrier
proteins or to a macromolecule in the cell and can be biologically active if they are affected
by enzymes in the gastrointestinal tract (Lindsay, 1983).
The regular and excessive use of large quantity of antibiotics was considered as a risk
factor for direct contamination of poultry meat (Endz et al., 1991, Mitama et al., 2001 and
Kabir et al., 2004). Alhendi et al. (2000) investigated the residue concentration after feeding
different levels of antibiotics to chicken and determined their withdrawal times under Saudi
conditions.
Withdrawal time is a specific period of drug withdrawal (washout time) from the
animal body which is necessary to observe before providing any animal product for human
6
consumption. When a last dose of drug given to the animal, the time should be noted and
observe the residue concentration in the tissues; skin/fat, liver; muscle, kidney, eggs, or milk
products and honey must be lower than the maximum residue limit (MRL) or equal to it.
McCaughey et al. (1990) reported that during a fecal recycling process, when the animal
treated with antibiotic, excrete drugs in feces that contaminate the feed of healthy animals
and may result in the incidence of residue's occurrence of certain antibiotic groups.
The residues of antibiotics are dominated in poultry and eggs or within the tolerances
established by the U.S. Food and Drug Administration and U.S. department of Agriculture,
when flocks are marked or eggs are sold. Therefore, commercial chicken and Turkey's flocks
tested for certain residues. If residues exceed the tolerance level, the birds are not acceptable
for market for their distribution (Herms, 2003), so they are removed. Herms (2003) has also
given the withdrawal dates vary from 0 (may feed until slaughter) to five (5) to seven (7)
days or more withdrawal dates are based on research in which birds are fed the compounds
and tested for residue after slaughter. Alambedji et al. (2008) also reported that use of anti-
infectious agents in general, and antibiotics, in particular, can lead to residues in animal
products, especially when a user fails to respect waiting periods. The risk of these residues in
food stuff of animal organ may become carcinogens (nitrofurans), allergens (penicillin and
streptomycin), and toxic (chloramphenicol) and may cause alteration in selection of bacterial
resistant to antibiotics. Paige and Kent (1987) and Paige (1994) indicated that the most
common cause of drug residue is the result of ignoring the withdrawal time.
It was observed that physiologically albumen fractions of eggs are produced and
excreted within 24 hours of laying periods by a chicken. Drug residues in albumen are
expected to result in its highest residues soon after treatment. On the other hand, the turnover
time for yolk is in the order of 68 days. There is evidence that residue's transfer into pre-
ovulatory yolks. However, do not appear to transfer back and are confiscated until the
developing yolks are ovulated (Donoghue, 2001).
Tajick and Shohreh (2006) noticed that incorrect applying of antibiotic deposit's
residue in meat, egg, milk cheese, butter and other live stock products. Human receives this
damage as residue, which can cause changes in his intestinal microflora and elimination of
useful bacterial strain. Antibiotic residues are mostly higher in liver and kidney tissues as
7
compared to muscle in poultry (Reyes-Herrera, 2005). DeWasch et al. (1998) qualified
antibiotic residues in pork and chicken muscle tissue screened with a microbiological
inhibition test. They used a culture medium of pH 6 seeded with bacillus subtilis. Their
results indicated that this test suited to screen poultry muscle and pork for residues of
antibiotics.
Nonga et al. (2009) took a survey of antimicrobial residues in twenty small-scale
broiler chicken farms located at eight different places at Morgoro, Tanznia. They analyzed
quantitatively seventy (70) broiler chicken liver samples by agar well diffusion and Delvotest
assay. At farm houses, they observed that 95% of the farmers' slaughter their chicken before
a withdrawal period because they were afraid of their economic losses and were unaware of
the adverse effects of antibiotic residues in human. 70% of farms were positive to
antimicrobial residues in chicken. Their cross-sectional study showed the extreme misuse of
antimicrobials by poultry farmers and lack of implementation of withdrawal time, leads to
occurrence of residues in poultry products.
2.3 Fluoroquinolone Residue in Poultry Products
2.3.1 Use of fluoroquinolones
Fluoroquinolones (FQs) are a group of synthetic antibiotics used in the treatment of
food producing animals (Sarmah et al., 2006). These are fluorinated quinolone (Sarkozy,
2001), was first derived from nalidixic acid. Wetlund (1990) and Samenidou et al. (2003)
defined that norfloxacin is one of the first modern quinolone antimicrobial agents, having the
fluorine atoms at C-5 and a piperazine at C-7 that has increased its potency (compared to be
other fluoroquinolones) to mycoplasmosis, colibaccilosis and pasteurellosis in chickens
(Laczay et al., 1998). Ciprofloxacin has been known for the broadest activity against all
gram-negative bacteria and streptococci except for Enterococus faecalies and streptococcus
pneumonia (Hoogkamp-Korstanje, 1984). Ridgway et al. (1984) compared the activity of
rosoxacin, norfloxacins, nalidixic acid and oxolinic acid and ciprofloxacin against the
chlamydia trachomatis, mycoplasma hominis and ureaplasma urealyticum. Out of all
ciprofloxacin was found to be at least twice as high. Furet and Pechere (1991) also reported
that pefloxacin, oxfloxacin and ciprofloxacin were active against Plasmodium, Trypanosome
cruzi, and Leishmnia donovani. However, toxoplasma gondii was not susceptible. The
8
fluoroquinolones has been reported to be more active in alkaline environment (pH>7.4) for
the gram-negative bacteria (Blaser and Luthy, 1988), while Fernandes (1988) argued that
susceptibility of fluoroquinolones to gram-positive bacteria is not affected by pH.
Microbiological inhibition test for detection of antibiotic residues was carried out (Okerman
et al., 2007) for routine screening of quinolone residues and the limit of detection (LOD) of
10 different quinolones and fluorouinolones was estimated. For this purpose, two media were
prepared, one at pH 6 and the other at pH 8, each seeded with one of the following test
strains of Bacillus subtilis, Escherichia coli or Bacillus cereus. The results indicated that
LODs of these antibiotics were highest on plates seeded with B. cereus that was selective for
detection of tetracycline residues. Likewise, the LODs of other fluoroquinolones, i.e.
ciprofloxacin, enrolfoxacin, danofloxacin, marbofloxacin, sarafloxacin and norfloxacin were
also lower at pH 8. Nine of the 10 quinolones did show the LOD more easily with E. coli, but
the difloxacin did show the LOD with B. subtilis (Nelson et al., 2007). Meinin et al. (1995)
suggested that bactericidal effect of antibiotic is time not concentration. Several studies
indicated that when high doses of fluoroquinolones administered over a short period
optimized their therapeutic effects (Drusano et al., 1993 and Forrest et al., 1993).
FQs are very effective drugs for treatment many infectious diseases (Brown, 1996),
such as severe respiratory and intestinal infections in poultry and many domestic animals in
China (Zeng et al., 2005). The whole group of these antibiotics has bactericidal effects by
inhibiting the activity of bacterial topoisomerase (DNA gyrase) result in cell death (Asahina
et al., 1992; Bryskier and Chantot, 1995 and Appelbaum and Hunter, 2000).
Fluoroquinolones produce bacterial cell alternations, include decrease cell division,
filamentation and cell lysis (Foerster, 1987 and Sarkozy, 2001).
2.3.2 Distribution of fluoroquinolones in animal tissue and their half life
Fluoroquinolones are highly distributed in animal tissues, and their concentrations are
higher in tissues than in plasma. Due to this characteristic, these drugs suitable to livestock
for the treatment of infection, especially in poultry. These are widely distributed in tissues
and are present in high concentration in excretory organs (in the liver and in the bile). These
are the lipophilic in nature and metabolized to their active metabolites in liver who affect
pharmacologically like parent drugs and remain in the body (Prescott and Baggot, 1993).
9
That is why they have a very long half-life (Sun et al., 2003) because of their lipophilic
property. Their target tissues are mainly visceral organs and fat (Prescott et al., 2000).
Bioavailability of these antibiotics was found to be 30-90% in poultry, when administered
orally (Chen et al., 1994). Moutsfchieva et al. (2009) took a comparative study of
pharmacokinetics of pefloxacin in chickens, pheasants and pigeons, 10 mg/kg body weight of
pefloxacin were given to birds. After blood sampling for 10 h and 12 h in chicken and
pigeons, antimicrobial activity of parent drug and its metabolite using Escherichia coli B14
as test organism was checked, serum concentration of pefloxacin was higher in chickens than
in pheasants and pigeons. Maximum retention time of pefloxacin in chicken was 9.71 ± 0.13.
From this experiment, they concluded that there were significant differences in kinetics of
pefloxacin in the experimental birds. Pant et al. (2005) investigated tissue distribution of
pefloxcin and its metabolite norfloxacin in broiler birds. They gave 10 mg/kg body weight of
pefloxacin as a single dose once daily for four days. Residue patterns were characterized, and
tissue concentrations were determined 10 and 15 days after the last dose administered, the
mean peak plasma pefloxacin concentration was measured after 4 hours. The concentration
declined gradually after 8, 12 and 48 hours, and that of norfloxacin, concentration also
declined at this time. Pefloxacin absorbed and eliminated with half-lives of 1.19 ± 0.22 and
8.74 ± 1.48 hours, respectively and norfloxacin eliminated with half-life of 5.66 ± 0.81 hours.
After 24 hours, concentration of pefloxacin and norfloxacin in liver, muscle, kidney and skin,
and fats were determined. Concentration of pefloxacin in liver was 3.20 ± 0.40 µg/g, in
muscle 1.42 ± 0.18 µg/g kidney 0.69 ± 0.04 and skin plus fat was 0.06 ± 0.02 µg/g of
norfloxacin was detected in liver, kidney and skin plus fats but not present in muscles.
Elimination half-life of ofloxcin was 4.46 h and mean residence time 7.43 h. it was found
that ofloxacin more rapidly absorbed, widely distributed and more quickly eliminate than
other flouroquinolones (Kalaiselvi et al., 2006). Ofloxacin absorbes better than ciprofloxacin,
pefloxacin or enoxacin (Neu, 1988). Ciprofloxacin absorbed primarily from duodenum and
jejunum when administered orally to monogastric animals (Wolfson and Hooper, 1989).
The time to reach peak serum concentration after single oral bolus of enrofloxacin
administered was 2.5 hours in chicken (VanCustem et al., 1990). Ciprofloxacin is eliminated
slowly from chickens. The half-life of ciprofloxacin was 10.29 ± 0.45 h reported by Anadon
et al. (1995). The absorption half-life was 0.21 h and time to reach maximum concentration
10
was 45.5 min (Atta and Sharif, 1997). It was investigated by Reyes-Herrera et al. (2005) that
enrofloxacin and ciprofloxacin accumulate in high concentration in non-edible tissues such
as feathers. Feather meal use as protein sources into diets of other food animals, such as
cattle, swine, rainbow front and salmon (Bertsch and Coello, 2005). The biological half-life
(t½) of most fluoroquinolones ranges from 3 to 6 hrs. Half-life of enrofloxacin in the
chickens was 7.3 hrs (Giles et al., 1991). Enrofloxacin decreases a mortality rate in poultry
with respiratory tract infections similarly difloxacin, ofloxacin and demofloxacin (Hinz and
Rottmann, 1990). Norfloxacin and norfloxacin nicotanate not been formulated in powder
form and given to poultry in feed and drinking water (Foerster, 1987 and Sarkozy, 2001).
2.3.3 Maximum residue limits of fluoroquinoloes in poultry products
European Union established maximum residue limits (MRLs) for some quinolones,
which are legally permitted and accepted for animal species. The maximum residue limit has
been established in the European Union for seven quinolones: danofloxacin, difloxacin,
enrofloxacin, flumequinne, marbofloxacin, oxonilic acid, and sarafloxacin (Van Hoof et al.,
2005). So MRLs value for enrofloxacin and ciprofloxacin in poultry and porcine ranged from
100 to 300 µg/kg depending on on-target tissue (Kowalski and Plenis, 2008). Concentration
of ciprofloxacin in unfertilized eggs of avian scavenger was 2.45 ± 1.28 µg/ml and fertilized
egg was 4.43 ± 2.25 µg/ml, and enrofloxacin in unfertilized eggs was 6.47 ± 3.08µg/ml
(Lemus et al., 2009). Food and Drug administration banned the use of enrofloxacin in poultry
in the United States in July 2005, because of emergence of antibiotic-resistant campylobacter
(FDA, 2005). European Union maximum residue limit (MRL) for norfloxacin in edible
tissues was 50 ppb (Anadon et al., 1992). Preslaughter withdrawal time more than 10 days
which ensure that the sum of the concentration of pefloxacin and norfloxacin would be less
than 50 ppb in edible tissues (Pant et al., 2005). Maximum residue limits of some
fluoroquinolones are given in table 1.
11
Table 2.1: A maximum residue limits of fluoroquinolones in poultry products:
Quinolone residue
Poultry product
bChina (ng/g)
dTaiwan (ppm)
cJapan (ppm)
JECFA (µg/kg)
aEuropean Union (µg/g)
Flumequine Chicken, Meat Muscle Skin + fat Liver Kidney
0.05 0.5 500 1000 1000 3000
400 250 800
1000
Oxolinic acid Chicken Muscle Skin + Fat Liver, Kidney
1.0 100 50 50 150
Norflloxcin Meat, Egg
0.02
Danofloxacin Chicken Muscle Skin + Fat Liver, Kidney
200 100 400
0.2 0.2 200 100 400
200
Enrofloxacin Ciprofloxacin
serum Muscle Liver Fat Kidney
100 400 100 400
0.05 400 50 100 200 100 300
Ofloxacin Chicken Meat
0.05
Sarafloxacin Chicken Meat Fat Liver + Kidney
10 20 80
20 80
10
100
Difloxacin Chicken Meat Muscle Skin+fat Liver Kidney
300 400 600 1900
300 400 600
1900
Reference; a1990. European commission. Regulation no. 2377/1990. bZeng et al., 2005 c2006. The Japan Food Chemical Research Foundation. Maximum residue limit (MRLs) list of agricultural in
foods. d 2008. Department of Health, Executive Yuan. Tolerances for Residue of veterinary dugs. DOH Food
No.97040692. Sep. 5, Taipei. ( in China).
12
2.3.4 Persistence of fluoroquinolone residues in poultry products
The study of norfloxacin, ciprofloxacin, ofloxacin, lemofloxacin, enrofloxacin and
cinoxacin and their use in veterinary practice shows potential hazard to consumer due to
persistence of residues in edible tissues. 242 muscle and 714 liver samples of poultry were
randomly selected in Saudi Arabia.120 samples were positive for norfloxacin residue, 42
(35.0%) raw muscles and 68 56.7%) raw liver. Their concentration ranged from 0.08 to 1.00
µg/g. Mean concentration in raw muscle was 0.25 ± 0.28 and in raw liver 0.11 to 1.03 µg/g.
Norfloxacin mean concentrations in cooked fluid was 0.06 ± 0.04 µg/g from these muscles,
was a major risk to public health resulting bacterial resistance and hypersensitivity reactions
to fluoroquinolones (Al-Mustafa and Al-Ghamdi, 2000). The bacterial pathogens are
transferred to human body when they eat undercooked chicken (Rose et al., 1998a).
Consumption of contaminated raw or under cooked products (both eggs and meat) cause
salmonella infections in human (Devies et al., 1998 and Cox et al., 2000).
2.4 Resistance Developed by Antibiotic Residue
Use of antibiotics result in the selection and development of antibiotic-resistant
bacteria (Morris and Masterton, 2002). The development of resistant strain of bacteria has
decreased the effect of many antibiotics that were used in the treatment of infection in
humans as well as animals. These strains are produced by genetic mutations or by acquired
resistant genes that are involved in the production of enzyme that degrades antibiotics such as
β-lactomase (degrade β-lactams), efflux pump that drain out antibiotics by altering cell wall
permeability to protect from adverse physiological environment or production of molecules
targeted antibiotics (Chander et al., 2007). The development of multidrug resistant (MDR)
strain has caused the slow treatment of infections and these are much fetal to human and farm
animals. Bacteria from these farm animals are often resistant to multiple antibiotics which are
routinely used among farm houses (Ladely et al., 2007 and Aarestrup et al., 2008).
Methicillin-resitant Staphylococcus aureus (MRSA) is a deadly resistant strain of bacteria to
multiple antibiotics and these can be found in some farm operations and marketed meats, and
also in human population (Klevens et al., 2007). MRSA strain of bacterial which was also
collected ST398 found in livestock (pigs, poultry and cattle), as shown in Europe (Van Rijen
et al., 2008), these strain transfer from pig to pig farmers and their families and in Europe
13
and Asia MRSA has been found in retail meats with farmer associated ST398 strains (Van
Loo et al., 2007 and Chan et al., 2008).
Fluoroquinolones associated resistances occur by alteration in bacterial cell wall and
mutation of DNA gyrase which occurs rarely (Chamberland et al., 1989). The permeability
changes of bacterial cell or alteration of active transport (efflux) pump result in decreasing
intracellular concentration of fluoroquinolones (Kaatz et al., 1991). Consumption of poultry
causes infections in human by fluoroquinolone resistant campylobacter species. 95% of these
infections are caused by compylobacter jejuni and 5% by campylobacter coli (Nelson et al.,
2007). These infections are mild, self limiting diarrheal, but severe infection can also occur
(Altekruse et al., 1999). Plasma mediated resistance was observed in a single isolate Shigella
dysenteria in Bangladesh (Neu, 1988).
Sub-therapeutic levels of antibiotic in broiler feed (to promote growth) result in
resistant strains of bacteria in birds (Mumtaz et al., 2000). Antibiotic-resistant strains of
bacteria are mostly food-born pathogen, including Salmonella spp., E. coli and
Campylobacter spp. These Strains are isolated from farm animals (Aarestrup, 2005). These
resistant bacteria are excreted and discharge into sewage or soil and other parts of our
environments (Witte, 1998). Zhao et al. (2001) reported that resistant strains of salmonella
are common in food animals. In United States 20% of meat samples have salmonellae, where
as other strains are found in chicken, pork, beef and shellfish. According to Sobel et al.
(2000), Salmonella enteritidis PT4 is particularly found in eggs. It is 2.5 fold more common
than Salmonella typharium which is resistant to ampicillin, amoxicillin, tetracycline,
sulphonamide and amynoglycoside. Salmonellae are less susceptible to fluoroquinolones.
These strains have some degree of clonality and resistance resulted from de novo mutations
(Allen and Poppe, 2002). The development of resistant in salmonella cause hazardous effects
in human like, stomach pain, diarrhea, pyrexia, vomiting, enteric fever, food poisoning,
hypersensitivity, super-infections, abnormal development of teeth and bones in children,
bone marrow depression and aplastic anemia (Mumtaz et al., 2000).
2.5 Detection Methods for Antibiotic Residues
A post-screening test that should have a low cast price tag is very much needed for
the early and accurate qualitative and quantitative detection and characterization of the
14
residue with minimal chances of false detection (Aureli et al., 1996). There are commonly
used detection methods of antibiotic residues in animal products. Summary of different types
of detection methods, their advantages and disadvantages are given in the table 2.
Table 2.2: Summery of Different Techniques Applied for Detection of Drug Residues
(Ref. Toldra and Reig, 2006)
Screening Tests Confirmatory Tests Microbiological Assay
Chromatography analysis
Immunological Assays
Swab Test on Premises (STOP) (Dey et al., 1998)
Calf antibiotic and sulfonamide test (CAST) (Dey et al., 1998, Clarence et al., 1998)
Fast Antimicrobial Screening Test (FAST) (Dey et al., 1998)
Premi® Test ( Cantwell and O’Keeffe, 2006)
Four Plate Test (FPT) (Okerman et al., 1998)
High performance thin-layer liquid Chromatography (HPLTC) (Stead, 2000)
High performance Chromatography (HPLC) (Stead, 2000)
Gas Chromatography (Borner et al., 1995)
ELISA Test Kits (Stead, 2000)
Radioimmunoassay (Stead, 2000)
Biosensor (Stead, 2000)
Multiarray (Toldra and Reig, 2006)
Advantages
These are used on large scale in animal farm houses. Easy handling Slowest Method Broad Spectrum Economical Basic laboratory equipment Easily available
Highly sensitive method Atomization results in high productivity Specificity based on detector system Short time is required to obtain the results Higher recoveries Large no. of samples for a single analyte
Easy to handle Test kits are available for large no. of samples Short time is required to obtain the results Full atoumization results in high productivity Higher sensitivity and specificity.
Disadvantages Difficult to standardize Some assays can not insure the MRLs. Maximum time required for test results. Removing the false +ve results due to protein bacterial inhibitor, e.g. lysozyme in eggs Having low sensitivity.
High Cost Need expertise and specialized equipment Need all steps of extraction, filtration and addition of internal standards. Interference produced some false positive results.
It is very expensive technique Its storage is limited for few months Only one kit have to search for one type of samples. In biosensor, analysis is restricted to chip available.
2.5.1 Microbiological assays
Aerts et al. (1995) and Haasnoot et al. (1999) indicated the microbiological methods
are suitable for rapid and large-scale screening because of their convenience and broad-
15
spectrum characteristics. These are the bioassay techniques used in the screening methods
due to their low-cost and can be handled easily (Bogialli and Corcia, 2009). A screening
method is the first step for analysis of the sample to confirm the presence or absence of
antibiotic residues (Aerts et al., 1995).
2.5.1.1 Agar diffusion assay
In routine, most widely used microbiological methods for residue analysis is being
done by the agar diffusion tests (Myllyniemi, 2004). This screening method is a simple in
which agar is inoculated in a standardized manner, and then the sample is applied to the agar
surface. During the first hours of diffusion, the concentration of antibiotics within the agar
medium at the edge of the sample is relatively high and later on diminishes sharply at
increasing distances from the sample. With time, as diffusion progresses, the slope of the
concentration gradient levels off due to the broader gradient of decreasing concentrations
within the agar medium (Barry, 1976).
2.5.1.2 Multi-plate test
This test is applied on test plate containing nutrient agar with an inoculated top layers.
The test samples are placed on top layer or in agar wells. The growth inhibiting area
represents the presence of antibiotic residues in a sample. This is the oldest screening method
commonly applied in slaughter houses (Bogaert and Wolf, 1980). These methods are
applicable to many antibiotics, including quinolones (Pikkemaat et al., 2007). Examples of
such methods are;
Four-plate test, in which comparing contaminated undiluted meat samples with
aqueous solution of analytical standards of antibiotics to get the limit of detection. In this
ttest, frozen pieces of chicken, pork and beef muscle tissues were applied on paper disk
(diameter 6 mm) impregnated with antibiotic standard solutions. Inhibition zones of standard
with meat and meat sample without standard mixture were obtained (Okerman et al., 1998).
Fluoroquinolones ciprofloxacin and enrofloxacin identified by this method (Myllyniemi et
al., 1999). Further, Myllyniemi et al. (2001) changed this method into sthe six-platem
methods for detection of some antibiotic by using six test bacterium-plate growth medium
combinations. The results of growth inhibition zones were recorded as 50 ppercent, which
included muscle (242) and liver (714) samples of poultry were taken for microbiological
16
assay and transfer their pieces (50-100 µg) into wells in Mueller Hinton agar plates
previously seeded with a reference strain of E. coli (ATCC 35218), Staphylococcus aureus
(ATTCC 25923), Bacillus subtillis (B.B.L. 6633) and Pseudomonas aseruginosa (ATCC
27853). The plates were kept oovernightin iincubator, and their positive results (of which 120
samples) were observed for antibiotic residue (Al-Mustafa and Al- Ghamdi, 2000). Cornet et
al. (2005) frequently used a screening method, one-plate microbiological test seeded with
Bacillus subtilis BGA to monitor antibiotic residues in kidney tissue of slaughter animals.
A surprisingly small number ofstudies has been conducted to develop alternative
microbiological methods for residue analytics, deal though automated methods could
significantly simplify and speed upthe process (Myllyniemi, 2004).
2.5.1.3 Swab Test on Premises (STOP)
This test was developed in 1979 by Food Safety and Inspection Service. It was a
simple and inexpensive method, easy to handle and applied on kidney, liver and muscle
samples. Cotton Swabs were socked with tissue fluid and placed on a STOP agar plate
seeded with Bacillus subtilis ATCC 6633, incubated overnight (16-18 hours). Presence of an
inhibition zone indicated the occurrence of antibiotics in animal carcasses (Johnston et al.,
1981; USDA/FSIS, 1984). Many antibiotics and Sulfonamides are assayed by this method
(Dey et al., 2005). Other forms of this test is the Fast Antimicrobial Screening test (FAST),
was developed in 1989 to improve the sensitivity and detection of wider range of antibiotic
and sulfonamides using Bacillus megaterium as a test organism (Dey et al., 1998). Present
literature available for the development of FAST analysis (USDA/FSIS, 1994). The field data
analysis revealed that FAST is significantly better for detecting positive results than STOP
especially for sulfonamide residues, due to its convenience and minimum detection time (18
hours or 6 hours) (Dey et al., 2005)
2.5.1.4 Test-Tube Method
In which a test tube containing growth medium was inoculated with spores of
sensitive bacterium and analyzed by redox indicator. The color change tells the bacterial
growth at specific temperature and pH. The presence of antibiotic prevents the color change
or delay color change. This test is applied in screening of milk and other matrices (Suhren
and Heeschen, 1996; Stead et al., 2004; Kilnic et al., 2007). Practically, these are an efficient
17
method and alternative to multiplate assay. The assay result can obtain within 4 hours. Spores
are used instead of vegetative cells and easy to distribute commercially. Various test tube kits
are available. For example, Premi Test (DSM), Explorer (Zeu-immuno-tech) and kidney
inhibition swab (KIS) test (Charm Sciences). Some literature exists on Premi test (Pikkmaat,
2009).
2.5.1.5 Premi Test (DSM)
The Premi test (DSM Nutritional products, Galeen, The Netherlands) was developed as
an attractive method for the detection of antibiotics in meat juices (Reybroeck, 2000). The
principal of this method is based on test-tube screening assay. The inhibition of growth of a
thermophilic bacterium (Bacillus stearothermophilus) which is sensitive to many antibiotics
(Stead et al., 2004). This method was applied on matrices of poultry muscles and eggs
(Pikkemaat et al., 2007). Five β-lactames were analyzed by this method. These were
penicillin, ampicillin, amoxicillin, oxacillin and cloxacillin (Popelka et al., 2005). This test
assay is not applicable to fluoroquinolone because of insensitivity of test organisms to these
antibiotics (Pikkemaat et al., 2007).
2.5.2 Extraction methods and confirmatory analysis
There are some rapid methods for determining the interaction of antimicrobial agents
and organism, intermediate and end products of bacterial metabolism and the interaction of
the organisms with various energy sources. As microbiological tests are unspecific, and
indicate only the presence of an inhibiting agent, the physico-chemical methods are very
specific and quantitative, but they need more time to process particularly when the estimation
of the antimicrobial being sought is not known (Amsterdam, 1996).
Extraction methodologies of fluoroquinolone have been done on the basis of their
acidic and piperazinyl (ring) characteristics. Acidic quinolones (AQ) are oxolinic acid,
nalidixic acid, enrofloxacin, ciprofloxacin, flumequine, piromidic acid, and sarafoxacin.
Piperazinyl quinolone (PQ) are danofloxacin, difloxacin, benofloxcin, norfloxacin, ofloxacin,
marbofloxacin and pipimidic acid. These quinolones can be soluble in polar-organic solvents,
aqueous acidic and basic media, but insoluble in non-polar ones (Hernandez-Arteseros et al.,
2002). So, various extraction strategies according to these solvent types are given in table 3,
4, 5 and 6.
18
These solvent types involve the processes; first is the lixiviation (separation of
soluble substances from insoluble one) with organic solvents of a medium to polar solvents
such as acetone, ethanol or methanol, ethyl acetate or acetonitrile. Secondly, the partition
between samples homogenized with buffer solution and a immiscible or organic solvent,
such as chloroform, ethyl acetate or dichloromethane, and thirdly, extraction of sample with
acid or base in water and organic mixture or even aqueous buffer solution (Hernandez-
Arteseros et al., 2002).
These solvents give various recoveries. The extraction of quinolones from muscles
and re-extraction of organic phase (with basic solution), neutralized it and passed through
mixed-mode solid phase extraction (SPE) cartridge gives recovery near 100% (Baliac et al.,
2006). Dichloromethane gives low recovery, and it is suitable for routine analysis and for
extraction of acidic quinolone (Hernandez-Arteseros et al., 2002) and extraction procedure is
very long and dichloromethane is not recommended because it is harmful to the environment
and human and to avoid the use of this solvent, other solvent methodology has been
developed, which consists of a mixture of acid solutions and acetnitrile followed by SPE with
polymeric cartridge (Baliac et al., 2006). Acetonitrile gives lower recovery than acetone.
Methanol-water and acetonitrile-water mixture containing HCl, trichloroacetic acid (TCA),
trifuoroacetic acid, HPO3, HClO4-H3PO4 and buffer solution pH 3.6-4 are more suitable for
frequent extraction and higher recovery (Hernandez-Arteseros et al., 2002). Recovery studies
are mostly done on spiked samples and 90% of reviewed papers have used spiked samples.
Schneider and Donoghue (2000) analyzed six fluoroquinolones in whole eggs. They obtained
good sensitivity and satisfactory recovery (65-110%).
The methods of high-performance liquid chromatography (HPLC) with various
detection systems have been determined. These are fluorescent detector, ultraviolet detector
and mass spectrometer (Okerman et al., 2001; Naeem et al., 2006; Martin et al., 2007 and
Schneider et al., 2007) as given in table 3,3, 4, 5 and 6. It is a rapid method with good
recovery. The papers have been reviewed including studies on various matrices as; poultry
(muscle tissues, liver, kidney and eggs), bovine and porcine muscles. The samples of
fortified chicken (muscle tissues, liver, kidney and eggs) after sslaughters were analyzed
according to these developed methods (Samanidou et al., 2005).
19
Table 2.3: Extraction of quinolone with water immiscible organic solvent
Poultry sample Antibiotic (Quinolone) Sample treatment solvents Separation technique References Eggs Flumequine
Nalidixic acid 1) Added dry Na2SO2
2) Extraction with ethyl acetate, evaporated. 3) oxalic acid pH:3 4) washed with n-hexane
Liquid chromatography with fluorescent detection (Recovery: 75%)
Riberzani et al., 1993
Chicken: Fat, liver, skin, muscle Ciprofloxacin Enrofloxacin
1) Added Hydrogen phosphate buffer pH:7.4 2) Extraction with dichloromethane.
Liquid chromatography with UV-detection. (Recovery: 70%)
Anadon et al., 1995
Chicken: muscle Ciprofloxacin, danofloxacin, difloxacin, enrofloxacin
1) Added diethylmalonic acid, pH: 7 2) Extraction with dichloromethane
Liquid chromatography with fluorescent detection. (Recovery: 30-92%)
Hernandez-Arteseros et al., 2000.
Chicken: muscle Ciprofloxacin, sarafloxcin, oxolinic acid, danofloxacin, flumequine, enrofloxacin
1)Extraction with dichloromethane 2)1M NaOH, centrifuged, add phosphoric acid, pH:3
Liquid chromatography with UV-detection. (Recovery: 65% ciprofloxacin, 69% danofloxacin, 89% enrofloxacin, 90% sarafloxacin, 100% flumequine, 119% oxolinic acid
Baliac et al., 2004
Chicken: muscle Ciprofloxacin, enrofloxacin, balofloxacin
1)Extraction with dichloromethane, 0.1M phosphate buffer 2) added mobile phase acetonitrile, triethylamine
Liquid chromatography with fluorescent detection
Ovando et al., 2004
Egg Ciprofloxacin, sarafloxcin, oxolinic acid, danofloxacin, flumequine, enrofloxacin, norfloxacin
1) Added Conc.NH3, shake. 2) Added acetonitrile, vortexd. 3) Extraction with dichloromethane
Liquid chromatography with fluorescent detection. (Recovery: close to 100%)
Hassouan et al., 2007
20
Table 2.4: Extraction of quinolones with water miscible organic solvent
Poultry sample Antibiotic (Quinolone) Solvent type used Separation technique Reference Eggs Ciprofloxacin, Enrofloxacin 1) Extraction with acetonitrile.
2) Rota-evaporation, added mobile phase
Liquid chromatography with UV-detection. (Recovery: Ciprofloxacin: 36-50% Enrofloxacin 29-85%).
Gorla et al., 1997
Chicken: liver Flumequine Oxolonic acid Sarafloxacin
1) Extraction with acetone 2) added acetone-H2O (NaCl).washed with n-hexane. Extraction with CHCl3 , phosphate buffer pH: 9. Washed with CHCl3.
Amide liquid chromatography with fluorescent-detection. (Recovery: Flumequine: 88% Oxolinic acid: 97% Sarafloxacin: 95% )
Maxwell and Cohen, 1998
Chicken: Liver, eggs, muscle. Flumequine Oxolonic acid Nalidixic acid
1) Extraction with acetonitrile and dry Na2SO4. 2) Evaporation, added, phosphate buffer pH: 11. Solid phase extraction, evaporated, added oxalic acid.
High pressure liquid Chromatography with Fluorescent-detection. (Recovery: Flumequine: 48-70% Oxolinic acid: 44-64% Nalidixic acid: 42-63%)
Rose et al., 1998b
Eggs Sarafloxacin 1) Extraction with acetonitrile. Added aqueous NaCl. Extraction with acetonitrile. 2) washed with hexane, added ethanol
Amide liquid chromatography with fluorescent-detection. (Recovery: 87-102%)
Maxwell et al., 1999
Poultry muscle Ciprofloxacin Enrofloxacin Flumequine
1) Added buffer pH: 9, mixed, then added acetonitrile, ultrasound extracton. 2) Centrifuged, supernatant separated, evaporated, then phosphate buffer. 3) added n- hexane, upper layer discarded, water phase filtered.
High pressure liquid chromatography with Fluorescent-detection (Recovery: Ciprofloxacin; 65.52% Enrofloxacin; 73.42)
Kirbis et al., 2005
Poultry: muscle Flumequine Oxolonic acid Nalidixic acid Sarafloxacin Ciprofloxacin Enrofloxacin Danofloxacin Norfloxacin
1) Extraction with acetonitrile. Added TRIS buffer pH: 7. Evaporation. 2) Added n-hexane,
Liquid chromatography/ tandem mass spectrometry with on-line solid-phase extraction.
Tang et al., 2006
21
Poultry: muscle, liver, kidney Enrofloxacin 1) Extraction with 30mM NaH2PO4 buffer, homogenization, centrifugation, then clean up with C18 (SPE) cardriges. 2) Evaporation, then dissolved in mobile phase 30mM NaH2PO4
and acetonitile (8:2 v/v)
High pressure liquid Chromatography with UV-detection
Sadia, 2006
Poultry: muscle, liver, kidney Ciprofloxacin, Enrofloxacin 1) Extraction with 25% ammonium solution and acetonitrile. 2) Added 1M ammonium acetate, diethylether and hexane, centrifuged. 3) Separation of upper aqueous layer, then added 1N NaOH.
Liquid chromatography/ tandem mass spectrometry
Martin et al., 2007
Chicken: muscle Danofloxacin Difloxacin Enrofloxacin Sarafloxacin
1) Extraction with acetonitrile, 0.1 M citrate, MgCl2 pH: 6.5, centrifuged, supernatant evaporated. 2) Residue resuspended in 0.1M malonate.
Liquid chromatography with fluorescent-detection. (Recovery: 63-95%)
Schneider et al., 2007.
Chicken: muscle Flumequine Oxolonic acid Nalidixic acid Sarafloxacin Ciprofloxacin Enrofloxacin Danofloxacin Difloxacin Norfloxacin
1) Extraction with acetonitrile, centrifuged, supernatant separated and evaporated. 2) Residues re-dissolved in to 0.02M ammonium acetate solution pH:9
Liquid chromatography with fluorescent-detection. (Recovery: Oxolinic acid: 123% Flumequine:108% Nalidixic acid: 107% Norfloxacin: 89% Sarafloxacin: 87% Enrofloxacin: 77% Danofloxacin: 75% Ciprofloxacin: 70% Difloxacin: 48%
Stoilova and Petkova, 2010
22
Table 2.5: Extraction of flouroquinolone with Acidic solution
Poultry Sample Antibiotics (Quinolone) Extraction Solvents Separation Technique Reference
Chicken: liver Danofloxacin 1) Extraction with methanol-water (1:1) (HClO4, H3PO4) in 55C. 2) add NH 3 pH 8.5.Extraction with dichloromethane, evaporated, residues dissolved in mobile phase
Liquid chromatography with Ion spray Mass spectrometry
Schneider et al., 1993
Chicken muscles Enrofloxacin (ENR), ofloxacin (OFL), benofloxacin (BEN).
1) Extraction with acetonitrile-water (1:4) (EDTA, Mcllaviane buffer)
Liquid Chromatography with UV- detection. (Recovery: 73-91%)
Yamamoto et al., 1993
Chicken: Muscle, liver
Enrofloxacin (ENR), darnofloxacin (DAN), ofloxacin (OFL), benofloxacin (BEN).
1) Extraction with acetonitrile-H2O (3:7 ) (HPO3) 2) Solid phase extraction.
Liquid chromatography with Fluorescent detection: (Recovery: ENR: 84-85 DAN : 81-83 OFL : 82-85 BEN: 85-90
Horie et al., 1994
Chicken: liver, skin, kidney, muscle
Danofloxacin, n-desmethyl-danofloxacin
Extraction with methanol-water (1:1) (HClO4, H3PO4) in 50C.
Liquid chromatography with fluorescent detection Recovery: Danofloxacin; 89% n-desmethyl-danofloxacin; 94%
Lynch et al.,1994
Chicken muscles Ciprofloxacin Enrofloxacin
1) Extraction with ethanol. 2) Added with triethylamine, evaporated, added phosphate buffer pH: 7.4. Washed with hexane. Solid phase extraction.
Liquid chromatography with fluorescent detection (Recovery: Ciprofloxacin; 40-53% Enrofloxacin; 73-77%
Chairriere et al., 1996
Poultry eggs, muscle Ciprofloxacin Enrofloxacin
1) Extraction with ethanol Liquid chromatography with fluorescent detection (Recovery: Ciprofloxacin; 90-98% Enrofloxacin; 91-99%)
Schwaiger et al., 1997.
23
Poultry Sample Antibiotics (Quinolone) Extraction Solvents Separation Technique Reference
Chicken: Muscle, Egg
Ciprofloxacin, Enrofloxacin Norfloxacin, Marbofloxacin Sarafloxacin
1) Extraction with 2% acetic acid and acetonitrile 2) Added sodium sulfate (or acetonitrile evaporated to dryness, redissolved in 0.5 M disodium hydrogen phosphate pH11)
Liquid chromatography with fluorescent detection (Average Recovery: 45-50%)
Rose et al., 1998b
Chicken: Raw: Muscle, Liver, Cooked: Muscle Liver
Norfloxacin 1) Homogenized sample with 2.0M sodium phosphate/sulfate pH 6.1 mobile phase methanol acetonitrile: 0.4 M citric acid (3:1:10)
Liquid chromatography with UV detection
Al-Mustafa and Al-Ghamdi, 2000
Eggs, muscle, kidney Ciprofloxacin Enrofloxacin Norfloxacin Marbofloxacin, Difloxacin
1) Extraction with aqueous HCl 2) solid phase extraction with methanol-phosphate buffer
Liquid chromatography – Diode array detector. (Recovery: 64-99%)
Gigossos et al., 2000
Chicken: Muscle Ciprofloxacin Enrofloxacin
1) Extraction with acetonitrile-water. 2) Washed with phosphate buffer pH: 7.4.
Liquid chromatography with fluorescent detection (Recovery: 92-111%)
Palmada et al., 2000
Chicken: Liver, Muscle, Fat + Skin
Ofloxacin 1) Extraction with 0.15M Hcl 2) Solid phase extraction with methanol, water and Na2HP4 pH: 9 3) Water, acetonitrile and triethylamine, mobile phase
Reverse phase high performance liquid chromatography UV detection (Recovery, 80-100%)
Maraschiello et al., 2001
Eggs Ciprofloxacin, Entrofloxacin Sarafloxacin,
1) Extraction with 2M H3PO4 and acetonitrile 2) Supernatant evaporated then add potassium phosphate buffer pH 2.5 3) Mobile phase triflouro acetic acid and anetonitrile
Liquid chromatography with fluorometric detection (Recovery: >80% egg yolk; ciprofloxacin 83-91% enrofloxacin 99-108% Sarafloxacin 94-95% Egg albumin; ciprofloxacin 87-106% enrofloxacin 87-92% Sarafloxacin 96-107%)
Chu et al., 2002
24
Poultry Sample Antibiotics (Quinolone) Extraction Solvents Separation Technique Reference
Eggs Ofloxacin Norfloxacin Ciprofloxacin Enrofloxacin
1) Extraction with methanol and 1MHCl 2) Clean up with 1mM mono potassium phosphate + 50% methanol (V/V) 3) Mobile phase, 4mM phosphoric acid (pH 3.5) + methanol 50% (V/V)
Liquid chromatography with fluorescent detector (Recovery: ofloxacin and enrofloxacin 91-94% Ciprofloxacin and norfloxacin 62-66%
Shim et al., 2003
Chicken: Muscle, Liver Nalidixic acid, oxolinic acid, flumequine, piromidic acid, darnofloxacin, enrofloxacin, sarafloxacin
1) Extraction with 0.3\5 metaphosphoric acid: acetonitrile (1: 10v/v) 2) Added n-hexane 3) Acetonitrile layer added in n-propanol dryness in water bath at 40°C 4) Clean up with 100% methanol, 0.05M NaH2P4 (pH 2.5) (7:3 V/V)
HPLC-Fluorescent Su et al., 2003
Eggs Norfloxacin, enrofloxicin demofloxacin, sarafloxacin, difloxacin, pefloxacin, lomefloxacin, ciprofloxacin, ofloxacin
1) Extraction of egg while with acetic acid absolute ethanol (1:99 v/v) 2)Extraction of yolk with acetonitrile, vortexed, mixed 3) Added acetic acid absolute ethanol (1: 99 v/v) Added hexane to egg yolk 5) mobile phase: A acetonitrile: aqueous solution. (9: 91 V/V)
Liquid chromatography with fluorescent detection (Recovery Egg white: 74.7-85.6% Egg yolk: 79.1-91.2%
Zeng et al., 2005
Egg Enrofloxacin Ciprofloxacin Oxolinic acid Flumequine
1) Extraction with 2% acetic acid in acetonitrile 2) Evaporation, dryness, redissolved residue in disodium hydrogen phosphate buffer pH 4
HPLC with UV detection Amjad et al., 2006
Eggs Ofloxacin, norfloxacin, ciprofloxacin, enrofloxacin, sarafloxacin
1) Dilution with 25mM phosphate buffer solution (pH 4.1) mobile phase phosphate buffer (pH 2.1) / acetonitrile/ methanol (72:8:20, v/v)
In tube solid phase microextraction coupled to high performance liquid chromatography (HPLC)
Huang et al., 2006
25
Poultry Sample Antibiotics (Quinolone) Extraction Solvents Separation Technique Reference
Poultry: Liver, kidney, muscle, eggs
Ciprofloxacin, Enrofloxacin Levofloxacin, Norfloxacin, Ofloxacin, Flumequine, oxolinic acid, Nalidixic acid
1) Extraction with 0.3% m-phosphoric acid: acetonitrile (1: 10 v/v) clean up: 10% methanol, 0.05M NaH2PO4 (pH 2.5) (7:3v/v
HPLC-UV detector Naeem et al., 2006
Poultry: Msucle Ciprofloxacin, denofloxacin, enrofloxacin, sarafloxacin, difloxacin.
1) Extraction with 0.03 HP3: acetonitrile (75:25) 2) Clean up with 1% formic acid: acetonitrile (60:40), hexane 3) Water 2% trifluoro acetic acid in water and acetonitrile (25:75)
HPLC with ultraviolet and mass spectrometric (Recovery > 70%)
Bailac et al., 2006
Eggs Ciprofloxacin, lomefloxacin Extraction with acetonitrile, 0.1% formic acid
HPLC-fluonescent detector Recovery : 67-98%
Herranz et al., 2007
Eggs Norfloxacin, enrofloxacin ciprofloxacin, danofloxacin
1) Added trichloroacetic acid, vortexed, added anhydrous sodium sulphate 2) Extraction with acetonitrile
HPLC-fluoresent detection (Recovery > 73.7%)
Cho et al., 2008
Muscle Ciprofloxacin, enrofloxacin Sarafloxacin, danofloxacin, difloxacin
1) Extraction with acetonitrile and NaCl 2) Added acetonitrile and 10% ammonia Mobile phase: Acetonitrile and phosphoric acid with hexane -1 – sulfuric acid solution (20:80 v/v)
HPLC-Fluorescent detection Ciprofloxacin; 51.7% Enrofloxacin; 64.5% Sarafloxacin; 71.2% Danofloxacin; 78.9% Difloxacin; 80.9
Posyniak and Mitrowska, 2008
Muscle, liver, skin with fat, kidney
Six Quinolone 1) Extraction with 2.5% trichloro acetic acid and acetonitrile 2) Solid phase extraction with methanol 3) Residues dissolved in 2 mM sodium tetraborate decahydrate
Capillary electrophoresis – UV detection (Recovery 83-86.6%)
Kowalski and Plenis, 2008
Muscle tissues 18 Quinolone 1) Extraction with acetnitrile and 1% formic acid
Liquid chromatography/tandem mass spectrometry (Recovery: 51-95.8%)
Chang et al., 2010
26
Table 2.6: Extraction of quinolones with Basic or Neutral buffer solution
Poultry sample Antibiotic(Quinolone) Solvent type used Separation technique Reference Eggs .Flumequine (FLU) Oxolinic
acid (OXO) Sarafloxacin (SAR) 1) Extraction with acetnitrile (NH3). 2) Added aq. NaCl. Washed with ethanol-hexane.
Amide Liquid chromatography – Fluorescent detection (Recovery: FLU:78-106% OXO:85-115% SAR: 83-114%)
Cohen et al., 1999.
Chicken: liver Ciprofloxacin (CIP) Enrofloxacin (ENR) Enrofloxacin (SAR) Difloxacin (DIF)
1) Homogenization with aq. NaOH. Added aq. H3PO4. 2) Extraction with methanol-H2O (1:9).
Phenyl Liquid chromatography –Fluorescent detection. CIP: 88-91 ENR: 91-94 DIF: 86-89
Holtzapple et al., 1999.
Chicken: liver, eggs Ciprofloxacin, Danofloxacin, Des-ethylene ciprofloxacin. Enrofloxacin, Norfloxacin, Enrofloxacin.
1) Extraction with acetnitrile (NH3). 2) Added aq. NaCl, washed with ethanol-hexane, evaporation, added phosphate buffer pH: 9.
Liquid chromatography-Fluorescent (Recovery: 65-110%)
Schneider and Donoghue, 2000.
Chicken: muscle Ciprofloxacin, Difloxacin, Enrofloxacin, Danofloxacin, Flumequine, Oxolinic acid, Nalidixic acid, Sarafloxacin, Marbofloxacin
1) Homogenization with Tris. Buffer pH: 9.1. Washed with hexane.
PLRP-S liquid chromatography with Fluorescent detection. (Recovery: 59-77%)
Yorke and Froc, 2000
Chicken: muscle Ciprofloxacin, Difloxacin, Enrofloxacin, Danofloxacin, Flumequine, Oxolinic acid, Nalidixic acid, Sarafloxacin, Marbofloxacin, Norfloxacin.
1) Extraction with phosphate buffer pH: 7.4 2) Solid phase extraction
Liquid chromarography-APCI-mass spectrometry. (Recovery: 80-100%)
Delepine and Hurtaud-Pessel, 2000
Chicken: muscle, Eggs Ciprofloxacin Enrofloxacin, , Flumequine Oxolinic acid
1) Eggs: Extraction with aq. NaOH- phosphate buffer saline Chicken muscle: Extraction with phosphate buffer saline
Liquid chromatography-Fluorescent detection (Recovery: 75-85)
Bisschop et al., 2000
Chicken muscle Enrofloxacin 1) Added aqueous NaOH/ acetonitrile, sonication. 2) Centrifugation., Upper layer separated
Liquid chromatography –Mass Spectrometry.
Lolo et al., 2006
27
Poultry sample Antibiotic (Quinolone) Solvent type used Separation technique Reference
Chicken muscle Enrofloxacin, Ciprofloxacin. 1) Added aqueous NaOH/ acetonitrile, sonication. 2) Centrifugation., Upper layer separated
Liquid chromatography –Mass Spectrometry (Recovery: 65-101%)
Lolo et al.,2007
Chicken: muscle, serum Sarafloxacin, Ciprofloxacin, Enrofloxacin, Danofloxacin, Norfloxacin, Difloxacin.
1) Extraction with acetonitrile, conc. NH4OH, centrifuged. 2) added diethylether, hexane, 1M NaCl, lower layers evaporated, residues dissolved in phosphate buffer pH:9
Liquid chromatography-Fluorescent- Mass spectrometry (Recovery: 71-99% for serum, 68-90% for muscles)
Schneider, 2007.
28
2.6 Seasonal Effect of Antibiotic Residue Persistence in Poultry Tissues
and Eggs
Antibiotic are used in poultry farms throughout the year by the farmers for maximum
production. These are used under the conditions such as inadequate housing, prevalence of
diseases, effect of hot environment (as in summer season), stress, chicks of low quality and
poor biosecurity measures (Sirdar, 2010).
Naeem et al. (2006) observed the effect of seasonal variation on the existence of
antibiotic residue in poultry products. The estimated quinolone residues were more in
summer season than in winter, because the drug administration is more in summer than
winter. He also observed presence of residues in liver and kidneys in both seasons. Pavlov et
al. (2008) analyzed 107 liver samples and resulted in 6% positive in winter season and 3% in
summer. This was due to the ignorance of time withdrawal.
933 egg samples of 175 farms were analyzed in April (21.1%), June (45.8%) and
August (33.1%). Among these 62.2% of total farms showed the prevalence ofantibiotic
residues inegg samples collected inJune, because ofsignificant change inweather (start of
rainy season) and increase usage inhumidity and poor hygienic condition during raa rainy
season, and antibiotic administration ishigher (Sirdar, 2010).
2.7 Cooking Effect on Depletion and Removal of Antibiotic Residue
from Poultry Products
Stability of wide range of drug residues in food animals is influenced by cooking to
varying degrees (Rose et al., 1995a). Time and temperature of cooking effect amount of
residue in foods. Increasing temperature and time limit decrease the amount of drug residue
and affecting its metabolites. These can cause human toxicology (Javadi et al. 2009). Rose et
al. (1997c) found the small activity of benzyl penicillin in hamburger, pork chops and steaks.
He also gave cooking treatments (boiling, microwaving, roasting and grilling) to
contaminated chicken breast, whole leg, whole egg and liver, and observed the decrease in
enrofloxacin concentration in chicken pieces after boiling and microwaving because of loss
of water contents in boiling and increases its concentration after grilling and roasting due to
29
moisture retention. Wasting of juices from edible tissues result in reduction of enrofloxacin
residues (Javadi et al., 2011)
There was no change in concentration of streptomycin residue in boiled and fried
eggs (O’Brien et al., 1980). It was observed that high temperature completely ddegrades
teteracyclines (Honikel et al., 1978). Cooking of contaminated meat and eggs for 60 minutes
can cause the inactivation of residue of oxyteteracyclines (Yonova, 1971). Cooking effect on
theconcentration of flumiquine andoxolinic acid, and their decomposition was observed.
Cooking temperature had no effect on quinolone, butconcentration of these compounds
increased by diffusion from thekidney andliver (Steffenak et al., 1994). Norfloxacin
concentration in poultry tissues during cooking process (Al-Mustafa andAl-Ghamdi, 2000).
Effect of frying, boiling, roasting andgrilling on enrofloxacin contaminated chicken tissues
wasnoted by Lolo et al. (2006). He observed that there was increased residue concentration
during grilling androasting because moisture contents of tissues decreased, denrofloxacin
concentration remained the same. There wasalso no effect on residue of neomycin (Katz and
Levine, 1978), levamisole (Rose et al., 1995b) or ivermectin (Rose et al., 1998a) in cooking
process.These antibiotic residues cause allergic reaction, bacterial resistance strains may
produce whenconsumers eat contaminated animal products (O’Brien et al.,1981).
2.8 Occurrence of Antibiotic Residue in Poultry Products in Pakistan
Pakistan's poultry industry is largely depending on antibiotic in disease protection, in
improving feed conversion ratio to promote growth of birds and weight gain, but farmers are
unaware of the use and time withdrawal of these antibiotics in birds, especially broilers
possessing residues in their meat (Mumtaz et al., 2000). Jabbar (2004) evaluated antibiotic
residues in poultry meat and eggs by STAF (swab test on animal food) procedure. His result
showed 66% residue in eggs and 60% of residues in meat. He used Bacillus subtilis as a test
organism. The large inhibition zone (≥2mm) on STAF plate showed the presence of residue
in the given samples.
Sadia (2006) investigated enrofloxacine residue in 100 tissue samples (muscles 33,
liver 33and kidneys 34) by performing swab test, and estimated 39% residues in liver, 21%
in kidneys and 9% in muscles. Amjad et al. (2006) and Naeem et al. (2006) quantified
30
residues of quinolones in poultry products (from local market) by HPLC method with UV-
detector. They observed the highest levels of ciprofloxacin in liver, kidneys and eggs.
Maqbool (1988) suggested withdrawal periods ofthe layer birds treated with
sulphachloropyrazine for 12 days and broilers for 17 days, and detected the drug
concentration ineggs, tissues and plasma sample by using colorimetric method and declared
that 0.1 ppm concentration in eggs was affordable for human consumption. Saleem (1990),
Ahmad (1996) and Akhtar (1996) also investigated the withdrawal time in poultry birds
treated with sulphonamide antibiotics and estimated the residues inpoultry products
spectophotometerically, and suggested that 3 days were enough time after post treatment for
the marketed birds. Residues of sulfonamides were also estimated in marketed poultry
products (Farooq, 1998; Afzal, 1998; Tabasum, 1998). Kalsoom (2000) determined
trimethoprim ranged over-236.6 ppm in eggs and 820.1-2073.5 ppm in meat.Occurrence of
residues of tetracycline (Munir, 2000; Iqbal, 2000) oxytetracycline (Shahid, 2006; Ashraf,
2000) and doxycline (Sabeh, 2000 and Azhar, 2000) were also observed in chicken meat and
eggs.
31
Chapter 3
MATERIAL AND METHOD
PHASE I (Surveillance)
3.1 Selection of Birds
The broilers chicken, layers and eggs were purchased from the different farm houses,
randomly located in and around the city of Faisalabad. Total number of samples (Different
parts of layer meat, eggs) and Broiler (different parts of meat) obtained at different areas
throughout the year has also been mentioned in table 3.1 and 3.2 respectively.
3.2 Sampling
The birds were slaughtered and the samples of muscle tissue, liver and heart were
collected. The muscle tissues and organs were trimmed of excessive fat and connective tissue
and egg white and egg yolks were separated and stored in separate polyethylene bags. All
samples were stored at -20C for microbiological screening test.
Table 3.1: Number of samples of Layer birds collected from different farm houses in and around Faisalabad.
Areas
Types of Samples
LM BM Li H Lu Eggs
Dalovaal 25 25 25 25 25 25 Pancera 35 35 35 35 35 35 Jaranwala 35 35 35 35 35 35 Gojre 25 25 25 25 25 25 Toba 35 35 35 35 35 35 Dijkot 37 37 37 37 37 37 Avaagat 25 25 25 25 25 25 Satyana 35 35 35 35 35 35 Sumanderi 25 25 25 25 25 25 Jhumrah 25 25 25 25 25 25 Amin-pur-Bangla 25 25 25 25 25 25
Jabiraa 25 25 25 25 25 25 Khidderwala 25 25 25 25 25 25
Shahkot 25 25 25 25 25 25
Mamu Kanjun 25 25 25 25 25 25
Total 427 427 427 427 427 427
LM = Leg Muscles, BM= Breast Muscles, Li= Liver, H= Heart, Lu= Lung
32
Table 3.2: Number of samples of Broiler birds collected from different farm houses in
and around Faisalabad.
Areas
Types of Samples
LM BM Li H Lu
Bernalah 25 25 25 25 25
Pancera 25 25 25 25 25
Jaranwala 37 37 37 37 37
Gojre 35 35 35 35 35
Toba 31 31 31 31 31
Dijkot 30 30 30 30 30
Satyanah 25 25 25 25 25
Sumanderi 25 25 25 25 25
Jhumrah 25 25 25 25 25
Amin-pur-Bangla 35 35 35 35 35
Jabiraa 35 35 35 35 35
Khidderwala 25 25 25 25 25
Shahkot 25 25 25 25 25
Mamu Kanjun 25 25 25 25 25
Chak Sadhar 25 25 25 25 25
Makuwana 37 37 37 37 37
Khurrianwala 35 35 35 35 35
Total 500 500 500 500 500
LM = Leg Muscles, BM= Breast Muscles, Li= Liver, H= Heart, Lu= Lung
3.3 Swab Test on Animal Food (STAF)
The STAF is a microbiological in-plant screening test for the detection of antibiotic
reissues in animal tissues (Rehman and Jabbar, 2006)
33
a) Test Principle
STAF is based on the principal that if an animal tissue contains a residue of
previously administered antibiotic, then fluid from the tissue will inhibit the growth of a
sensitive organism on a bacterial culture plate. In this test, cotton swabs saturated with tissue
fluid and are placed on a culture plate where surface has been seeded with spores of harmless
organism bacillus subtilis. This organism is known to be sensitive to commonly used
antibiotics. The swabs on cultured plate incubated over night to allow the growth around the
swab. The presence of clear zone of inhibition is evidence that sample tissue contain
antibiotic residue (Dey et al., 1998).
b) Methodology
Preparation of STAF plate:
i) 32.0 g of nutrient agar was mixed in 1L distilled water and autoclaved at 1210C for 15
min
ii) Cooled media in a water bath at 48 0C.
iii) Aseptically added 1 ml of 2 x 107 spores/ml B. subtilis spore suspension in 100 ml of
melted agar. Mixed it well under sterilized condition.
iv) 20 ml of the agar was poured into each 6 x 6 inch plate and tilt plates to ensure even
distribution and plates were allowed to harden on a flat, level surface. They were sealed
in double plastic bags and refrigerated to prevent moisture and evaporation, these plates
can be used for a period of 10 working day.
Lid of each STAF plate was labeled with the following information:
a. STAF Plate Number b. Date c. Sample type
Test Procedure
i. Frozen sample were allowed to thaw completely at room temperature for a sufficient
period such that the ice crystal were no longer present within the sample.
ii. Opened a sterile cotton swab pack, removed one swab and inserted the swab shaft about
½ “to ¾” in animal tissue (liver, muscle, heart and lungs). Sample swabs were saturated
with tissue fluid obtained by macerating the tissue with sharp end of the swab shaft.
This allowed contacting the macerated tissue until a maximum tissue fluid absorbed
into it (At least 30 minutes but not more than 2 hr).
iii. For eggs samples swabs were dipped in albumin and yolk.
34
iv. Allowed the refrigerated STAF plates to warm to room temperature for about 10 min.
Each plate was checked for the absence of contamination, cracking of agar or dryness.
v. Placed a neomycin 5µg disc on the agar surface of STAF plate.
vi. Removed the swab from sample tissue, broke the shaft approximately two inches from
the swab end. Gently placed the sterile sample swab on the surface of the STAF plate
with broken end of the shaft (1 inch) from neomycin disc.
vii. Made sure not to break the agar surface and made sure the sample swab had a uniform
contact with agar.
viii. The plates were incubated at 30ºC for 16-18 hours. Then observed and measure
inhibition zones with roller.
Composition of nutrient agar:
Nutrient agar (0.4 % dextrose) was used as test medium. The composition of nutrient
agar is given in the following table 3.3
Beef extract 3.0 g
Peptone 5.0 g
NaCl 5.0 g
Agar 15.0g
Dextrose 4.0g
Distilled Water 1000 ml
c) Spore Suspension of Bacillus Subtilis JS 2004
Bacillus subtilis was obtained from the department of Microbiology, Faculty of
Veterinary Sciences, University of Agriculture, Faisalabad. Growth of Bacillus subtilis was
obtained by using a single colony of pure isolate and streaking out on the surface of nutrient
agar plate then incubated at 37ºC for 16 hours. Cultural morphology and biochemical
characteristics of Bacillus Subtilis JS 2004 have been presented in Table 1.
Roux flasks were freshly prepared with nutrient agar and 5 ml of pure growth isolate,
as confirmed by Gram’s staining for its purity was streaked out homogenously over the
surface of nutrient agar. These flasks were kept at room temperature for six (6) days. From
these flasks, growth was harvested by adding 20 sterile glass beads and 20 ml of normal
saline; dislodge the bacterial growth after gentle agitation.
35
Bacterial suspension was transferred to a sterile centrifuge tubes aseptically and
heated in boiling water at 100ºC for 10 minutes. Heated suspension was washed three times
with sterile distal water and centrifuge at 5ºC for 20 min at 20,000 x g, while decanting
supernatant. This stock suspension was stored at 4ºC for the preparation of working spore
suspension.
Using Bread Smear Method (total count) bacterial population in semi-pure and pure-
culture was measured in a 0.01 ml fluid on bread slides which has marked area of 1 cm2
slides were treated with KOH to remove fat layer and after drying these slides were stained
with Gram’s stain. Bacterial counts were made in randomly selected areas of microscope
under oil immersion lens.
The diameter of are microscope filed at 100 x lens was measured in micron (µ): a =
(D/2)
Small division of stage micrometer = 10 µ
Number of division of stage micrometer = 32
32 x 10 = 320
Therefore r = 320/2 = 160
a = (160)2 x 3.14 = 80384
Area of scaled microscope slide A = 1 cm2 x 108µ
Microscopic Factor (MF) = A/a 108/80384 = 1244
For the calculation of total spores:
Spores/ml = Average number of spore x MF x dilution Factor
Number of spore = 82, 160, 240
Dilution Factor = 100
Spores/ml = 160 x 1244 x 100 = x 2 107 Spores/ml
In order to increase the reliability of the results, Negative Control of the STAF test was also used throughout the experiment period.
36
PHASE II (WITHDRAWAL TIME)
On the detection of residue of commonly used antibiotics by microbiological assay
(Swab test), control studies for individual antibiotics were conducted to know withdrawal
time in broiler and layer birds (with their eggs) separately for different quinolones.
3.4 Experimental Protocol
a) Selection of Poultry Birds
3.4.1 Layer
Forty eight (48) 35-week-old healthy white leghorn layer birds at their peak egg
production period were selected for experiment. The birds were obtained from selected farm
house of University of Agriculture, Faisalabad. The hens were placed in experimental shed
with free access to feed and water ad libitum. They were fed with commercially produced
ration ad libitum. They were all acclimatized for one week before the start of experiment and
their eggs were collected. After one week these birds were divided in to four groups, three
(03) experimental and one (01) control group, having twelve (12) birds in each group. Each
group kept in to its separate experimental cage. The control group fed on antibiotic free
ration and provided purified drinking water. The experimental groups were classified
according to medication (ciprofloxacin, norfloxacin and ofloxacin) they receive.
b) Medication
Each antibiotic was given to experimental birds in drinking water (1ml/4L) as shown
in table 3.4.
Table 3.3: Fluoroquinolone antibiotics (1ml/4L) added in layers and broiler drinking water (ad libitum).
Group(N) Name of antibiotic Manufacturer name
C (12) Control (antibiotic free water)
Cip. (12) Ciprobak (ciprofloxacin) Attabak Pharma industries
Nor. (12) Anflox (Norfloxacin) Anglian Nutrition product company.
Ofl. (12) Oflobak(Ofloxacin) Attabak Pharma industries
Each treated group received each drug (in drinking water) for five (05) consecutive
days.
37
c) Sampling
On 6th, 8th and 9th day, four (04) birds from each antibiotic group and control (total
no. of 16 birds at each experimental day) were decapitated for serum, leg muscle, liver and
kidney for estimation of antibiotic residue and for withdrawal time. Serum samples were
separated from blood by centrifugation at 500g for 10 min, in small aliquot. All samples were
preserved at -20C° in polyethylene bags for their extraction of drug and analysis.
Egg samples of control and experimental birds were also collected at each day of
slaughter, and stored in polyethylene bags at -20 0C for analysis.
3.4.2 Broilers
Forty eight (48), Five (05) week-old healthy broiler birds (Hubbards) were obtained
from selected farm house of University of Agriculture, Faisalabad. The birds were housed in
a separate shed. They were fed on commercially produced antibiotic free ration and provided
purified dinking water ad libitum. They were all acclimatized for one week before the start of
experiment. After one week, the birds were grouped in to four; one (01) control and three
(03) experimental (according to medication). Each group housed separately. The control
group (12 birds) fed on antibiotic free ration and drinking water for five (05) consective days.
The experimental groups were placed separately and each group medicated with each
antibiotic (ciprofloxacin, norfloaxacin and ofloxacin) for five days.
a) Medication
Each antibiotic was given to experimental birds in drinking water (1ml/4L) as shown
in table 6. Birds administered each antibiotic (at therapeutic concentration) for five (05)
consecutive days.
b) Sampling
On day six (06), eight (08) and 9th (9), four (04) birds from each antibiotic group and
control (no. of 16 birds at each day) were slaughtered and their serum, leg muscle, liver and
kidney were collected for estimation of antibiotic residue and for withdrawal time. Serum
samples were separated from blood by centrifugation at 500g for 10 min, in small aliquot. All
samples were preserved at -20 0C in polyethylene bags for their extraction and analysis.
38
3.4 Cooking Operation
3.4.1 Cooking in Electric Oven
i. 20 different tissue sample were selected (liver and muscles of control and
experimental birds of 1st day sampling) for roasting.
ii. 10 g of each sample was placed on a metal baking tray and cooked in electric oven
(Memmert, Germany) at 200 0C for specific time (25 min for liver samples, 40 min fro
muscle samples) as described by Javadi et al., 201.
iii. Cooked samples without juice were collected for extraction.
3.4.2 Cooking in Microwave Oven
i. 20 different tissue sample were selected (liver and muscles of control and
experimental birds of 1st day sampling) for microwaving.
ii. 10 g of each sample was placed in microwave oven and cooked under full power (900
w) for a specific time (3 min for all samples) as reported by Javadi et al., (2011).
iii. Cooked samples without juice were collected for extraction.
Extraction Methodology
Sample Preparation steps of tissues, serum and egg samples were done before
extraction by high pressure liquid chromatography (HPLC) analysis, by adopting different
strategies.
Sample preparation and extraction steps of raw and cooked tissues: (Su et al., 2003)
a) Homogenization and Filtration steps:
i. Frozen tissue samples of all birds (experimental and control) were thawed
completely.
ii. 5 g of thigh muscle, liver and kidney tissue were weighed.
iii. Each sample was homogenized for 3 minutes in homogenizer by adding 30mL of
0.3% metaphosphoric acid : acetonitrile (1:10, v/v)
iv. The homogenized mixture was filtered under suction process by Buchner funnel.
v. Added 50 mL of n-hexane which is saturated by 8mL of acetonitrile. Vortexed for
5minutes. Removed acetonitrile (lower layer) by separatory funnel in to
concentration bottle kept in a water bath at 40 0C.
39
vi. Added 5 mL of n-propanol to inhibit sudden boiling in concentration bottle
vii. The filtrate concentrated to dryness in water bath.
b) Sample Clean up steps:
i. Added 10 ml of demonized water in sample concentrate. The solution loaded to
Bond Elute C18 cartridge. (Strata® phenomenex).The cartridge was already activated
by 5mL of methanol and rinsed with 10mL of deionized water.
ii. The bottle containing concentrate was washed twice with 5mL 10% methanol and
loaded to cartridge. Flow-through was discarded. The eluent was collected and
dried at water bath at 40 0C.
iii. Residue was dissolved in 1mL mobile phase and filtered by 0.45µm membrane
(Sartorius AG, Weender Landstasse 94-108, and 7075 Goettingen Germany). The
filtrate was ready for HPLC analysis.
c) HPLC analysis:
i. Separation of fluroquinolones by HPLC was performed by C18 column (4.6mm x
250mm; 5µm)
ii. Fluorescent detector system was operated, the excitation and emission wavelength
were 260 and 286 nm respectively.
iii. Acetonitrile and 0.05 M sodium dihydrogen phosphate (pH 2.5; 35:65 v/v) were
used as a mobile phase having 3.5mM sodium dodesyl sulphate (SDS).
iv. Flow rate was adjusted to 1.0 mL/min.
v. 20 µL of sample solution was injected.
Sample preparation and extraction steps of egg: (Hassouan et al., 2007)
i. 1g of homogenized whole egg sample (experimental and control) was taken in 10mL
centrifuge tube and added 250µL of concentrated ammonia, vortexed for 5 second.
ii. Added 2mL of acetonitrile and votexed for 10 seconds.
iii. Centrifuged at 500 x g for 5 minutes.
iv. Supernatant was separated into another glass centrifuge tube and added to 4mL of
dichloromethane in to it. Vortexed for 10 seconds at high speed.
v. Centrifuged at 4000rpm for 5 minutes. The upper aqueous layer was separated and
filtered through 0.45µm membrane. Filtrate was ready for HPLC analysis.
40
HPLC analysis:
i. The excitation and emission wavelength was adjusted at 280nm and 460nm
respectively.
ii. Mobile phase consisted of aqueous formic acid (2.0%), methanol and acetonitrile
(75:13:12)
iii. Temperature of column (C8) was maintained at 35 0C.
Sample preparation and extraction steps of serum: (Olutosin et al., 2004)
i. 1mL of serum sample (control and experimental) was taken in test tube, added
100µL of orthophosphoric acid. Vortexed for 5 seconds.
ii. Added 2mL of acetonitrile, vortexed for 10 seconds and centrifuged at 500 x for 5
minutes.
iii. Supernatant was removed and added to 3mL of methylene chloride to supernatant.
Votexed for 10 seconds at high speed then centrifuged at 4000rpm, 4 0C.
iv. The aqueous was separated and transferred into vial. This sample was ready for
HPLC analysis (Olutosin et al., 2004)
HPLC analysis:
iv. The excitation and emission wavelength was adjusted at 280nm and 460nm
respectively.
v. Mobile phase consisted of aqueous formic acid (2.0%), methanol and acetonitrile
(75:13:12)
vi. Temperature of column (C18) was maintained at 35 0C.
a) Preparation of Standard Solution
Preparation of stock solution:
Stock solutions (100µg/ml) of ciprofloxacin, norfloxacin and ofloxacin (reagent
grade) were prepared by the following method.
1000ppm = 1000mg/L (1000µg/1mL) or 10mg/10mL or 0.01g/10mL
41
Preparation of standards of antibiotics for serum samples from 1000ppm stock
solution:
Standards of ciprofloxacin, ofloxacin and norfloxacin of 0.1, 0.5, 1.0, 2.0 and 5.0 ppm
concentration of were prepared by using the following formula
C1V1 = C2V2
C1 (known concentration)
C2 (required concentration)
V1 (unknown volume)
V2 (known volume)
Preparation of standards of antibiotics for tissues and eggs from 1000 ppm stock
solution:
Standards of 1000, 500, 100, and 50 ppm concentration of ciprofloxacin, 1000, 500,
250, 100 and 50 ppm of ofloxacin, and 500, 250, 100, 50 and 10 ppm of norfloxacin were
prepared by using the formula as given above.
Preparation of Standard Curve:
Standard solutions were mixed with their respective mobile phases and analyzed by
HPLC with fluorescent detector. Standard curve was prepared by different peak areas of
fluoroquinolones with respect to their concentrations.
Calculated response factor (R.F) from standards by following formula:
R.F = Concentration of Std. / Peak Area of Std.
Concentrations of samples calculated by following formula;
Concentration = R.F / Peak Area of sample
42
Validation and Calibration of the Assay
Using FDS guideline, validation of the bioanalytical was performed. Spiked standard
samples at different concentration level were extracted in duplicate as described in the
method of this assay to be used for the calibration of this easy.
To check the accuracy as well as precision of this assay, four spiked samples at four
different concentrations were first extracted and than analyzed in duplicate and this
procedure was repeated on alternate days to estimate the interday variability. Likewise,
recovery estimate were determined.
For quality control (QC) samples to determine inter and intra-assay accuracy and
precision were prepared using different concentration. The following table prepared using
different concentration. The following table (Table 3.5) did show the inter and intra day
accuracy and precision of the different fluoroquinolones. For serum, intra day accuracy of
ciprofloxacin did range from 96-101 percent and its precision coefficient of variance (CV)
was between 4.6 to 9.5 percent. For serum norfloxacin, intra day accuracy did range from 94-
99 percent while precision CV was 5.0 to 6.8 percent. Likewise, for serum ofloxacin, the
accuracy percent did range from 90-96 percent with precision CV percent of 6.0-10.5 (Table
3.5). The muscle and serum interday accuracy and precision has been presented in table 3.5.
During analysis of serum and tissue samples concentration, it was noticed that after
every sixty to eighty (60-80) analysis of samples particularly after extraction did show a
change in column performance that was indicted by 15-20 percent decrease in the relative
time of ciprofloxacin, norfloxacin and ofloxacin. Therefore, after analysis of 50 samples
column performance was restored by flush with pure acetonitrile for about two to three (2-3
h) hours. This treatment was also ensured when column was even not in use for couple of
days. The calibration curve obtained for ciprofloxacin (range between 50-1000 ppm),
ofloxacin (range from 50-1000 ppm) and for norfloxacin (range from 10-500 ppm) were
evaluated by the regression coefficient, slop and by the intercept. This analysis was used for
the calculation of unknown samples (serum, tissue and eggs) for using the peak ratios of each
day in the internal standard.
43
Table 3.4: Accuracy (%) and precision CV (%) of different quinolones during intra and inter assay
Drugs Accuracy (%) Precision CV (%)
Serum Muscle Serum Muscle
Ciprofloxacin
Intraday 96-101 91-97 4.6-9.5 3.8-7.6
Interday 95-98 93-99 4.3-8.9 3.6-9.2
Norfloxacin
Intraday 94-99 92-96 5.0-6.8 4.0-8.0
Interday 95-101 95-102 4.5-7.0 3.9-9.0
Ofloxacin
Intraday 90-96 96-103 6.0-10.5 5.7-10.0
Interday 95-104 99-106 5.8-7.8 7.5-11.0
3.5 Health Biomarkers
Total antioxidants, oxidants, paraoxonase, arylesterase and catalase were analyzed by
their respective reference methods and concentrations were calculated by the given formulas.
a) Homogenization of tissues:
Before health biomarker analysis, tissue samples of muscle, liver, heart and kidney
were homogenized according to the following method (Ruiz-Gutierrez et al., 2001).
b) Preparation of buffer solution:
Buffer for homogenization was prepared by taking 0.25 M of sucrose (85.5 g) of
sucrose in 1000 mL of distilled H2O), 1mM EDTA (0.372 g of EDTA in 1000 mL of distilled
water), 15 mM Tris-HCL (0.235 g of Tris-HCL in 1000 mL of distilled H2O, pH 7.4), and
1mM of DL-dithiothreitol (0.154 g of DL-dithiothreitol in 1000 mL of distilled H2O)..
44
c) Procedure:
Tissue homogenate (10% w/v) was prepared by adding 1000 µL of buffer in 0.1 g of
tissue sample in a homogenizer. Each homogenate was centrifuged for 20 min at 800 × g; the
supernatant was separated (Ruiz-Gutierrez et al., 2001) and used for analysis the total
oxidant and anti-oxidant status, paraoxonase, aryesterase and catalase of organs of all
experimental birds (layers and broilers).
3.5.1 Total Oxidant Status (TOS; mMol/L)
The total oxidant status of all samples (serum/homogenized tissues) was determined
using a method developed by Erel (2005).
a) Preparation of reagent:
Reagent 1 (R1)
Dissolved 114 mg of xylenol orange and 8.18 g of NaCl in 900 ml of 25mM H2SO4
solution. 100mL of glycerol was added to this solution. The final reagent was composed of
150 µM xylenol orange, 140 mM NaCl and 1.35 M glycerol. The pH of reagent was 1.75 and
was stable for at least 6 months at 4°C.
Reagent 2 (R2)
Dissolved 1.96 g of ferrous ammonium sulfate and 3.17 g of O-dianisidine
dihydrochloride in 1000 mL of 25 mM H2SO4 solution. The final reagent was composed of 5
mM ferrous ammonium sulfate and 10 mM O-dianisidine dihydrochloride. This reagent was
stable for at least 6 months at 4°C.
b) Principle:
Oxidants present in the sample oxidize the ferrous ion-O-dianisidine complex to
ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are present in the
reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic
medium. The color intensity, which can be measured spectrophotometrically, is related to the
total amount of oxidant molecules present in the sample. The assay is calibrated with
hydrogen peroxide and results are expressed in terms of µM hydrogen peroxide equivalent
per liter (µmol H2O2 equiv. /L).
c) Procedure:
35 µL of each sample (serum /homogenized tissue) was taken into eppendorfs which
were arranged in two rows. In both the rows 225 µL of reagent 1 (R1) was added and noted
45
the reading of the first row. In second row 11 µL of reagent 2 (R2) was added and time was
noted. After 4 minutes of incubation, absorbance of second row was taken at (Bichromatic)
wavelength of 500 nm and 800 nm (Biosystem, BTS-330, Biosystems, S.A. Costa Brava,
Barcelona, Spain). The total oxidant concentrations of samples were determined from the
standard curve.
y = 0.0803x + 0.2039 R² = 0.9673
0
0.5
1
1.5
2
2.5
0 5 10 15 20 25 30
Ab
sorb
ance
Concentration (µ Mol H2O2 equi./L)
Fig 3.1:-Standard curve for Total oxidant status
d) Calculations:
The assay was calibrated with H2O2 and the results were expressed in micromolar
hydrogen peroxide equivalent per liter (µ mol H2O2 Equiv./L). The concentration of total
oxidants in the sample was determined from the standard curve and expressed as, micromole
H2O2 Equiv. /L.
e) Sensitivity:
The sensitivity for this assay was < 3%.
46
3.5.2 Total Antioxidant Capacity (TAC; mmol/L)
Total antioxidant capacity of serum and organs samples was measured, by adopting
Erel (2004) method.
a) Principle:
The reduced ABTS molecule is oxidized to ABTS+ (deep green) in the presence of
hydrogen peroxide in an acidic medium (acetate buffer; pH=3.6) where it remains stable for
long time. More concentrated acetate buffer with high pH (pH=5.8) caused the bleaching of
color. Antioxidants present in the sample speed up the rate of bleaching to a degree
proportional to their concentration and the bleaching rate is inversely related to the TAC of
the sample.
b) Preparation of Reagents:
Reagent 1
Reagent 1 (0.4 mol/L acetate buffer solution; pH 5.8) was prepared by dissolving 32.8
g of sodium acetate (CH3COONa) in 1000 ml of deionized water in a flask. In another flask,
22.8 ml of reagent graded glacial acetic acid was diluted with deionized water upto 1000 ml
(final concentration reached to 0.4 mol/L). 940 mL of the sodium acetate solution were
mixed in 60 mL of acetic acid and pH was maintained at 5.8 with the help of pH meter. The
buffer solution was stable for 6 months at 4 ºC.
Reagent 2
The regent 2 (30 mmol/L acetate buffer solution; pH= 3.6) was prepared as follow:
2.46 g of sodium acetate was dissolved in 1000 mL of deionized water (final concentration=
30 mmol/L). Reagent grade glacial acetic acid (1.705) was diluted to 1000 mL with
deionized water. Acetic acid sodium acetate buffer was prepared by mixing 75 ml of sodium
acetate solution with acetic acid solution (925 mL) and pH was maintained at 3.6 with the
help of pH meter. Then 278 µL of commercial hydrogen-peroxide solution (35%, Merk) was
diluted to 1000 ml with buffer solution (final concentration: 2 mmol/L). After that ABTS
(0.549g) was dissolved in 100 mL of prepared solution (final concentration; 10 mmol/L).
After an hour incubation at room temperature the characteristic color of ABTS.+ appeared.
The color was stable for 6 months at 4 ºC.
47
c) Procedure:
Wavelength of spectrophotometer (Biosystem, BTS-330, Biosystems, S.A, Costa
Brava, Barcelona, Spain), was adjusted at 660 nm.
Five (5 µL) of sample (serum /homogenized tissue) was taken in eppendorfs and 200
µL of reagent 1 was added to them. First sample was run as blank. Now 20 µL of R2 was
added in all the eppendorfs and timer was started for 5 minutes. After 5 minutes incubation
absorbance was taken spectrophotometrically.
y = -0.2663x + 0.4937 R² = 0.9335
0
0.1
0.2
0.3
0.4
0.5
0.6
0 0.5 1 1.5 2
Ab
sorb
ance
Concentration mmol Trolex equivalent /L
Fig 3.2:- Standard curve for Total antioxidant capacity
d) Calculations:
The reaction rate was calibrated with Trolex (Vit, E) and concentrations were
expressed in mmol Trolex equivalent /L. The concentration of total antioxidant was obtained
from standard curve.
e) Linearity/Sensitivity:
The assay was linear up to 6 mmol Trolex equivalent/l and sensitivity for this
assay was < 3%.
48
3.5.3 Paraoxonase Activity (PON1; Unit/L)
The enzymatic activity of Paraoxonase was determined with the method of Juretic et
al. (2006).
a) Principle:
Paraoxonase activity was determined based on the principle of rate enzymatic
hydrolysis paraoxon (O, O-diethyl-O-p-nitrophenylphosphate) to p-nitrophenol. The amount
of p-nittrophenol generated was monitored with continuous recording of spectrophotometer.
b) Preparation of Reagents:
0.1 M Tris-HCl (pH 8.0) buffer solution was prepared by the addition of 1.21 g of
tris-base in 80 mL of distilled water. The pH of the solution was adjusted to 8.0 with drop by
drop addition of 0.1 M HCl solution under pH meter and made the volume to 100mL. Further
2 mmol CaCl2-Tris- HCl solution was prepared by the addition of 0.0022 g of CaCl2
followed by 3 µL of paraoxon substrate (2.0 mM) in the prepared solution of Tris-HCL.
Finally total volume was made to 100 mL by the addition of distilled water.
c) Procedure:
The 10 µL each sample (serum /homogenized tissue) was taken in eppendorf then 350
µL of paraoxon substrate and reagent (2 mmol/L CaCl2 and 0.1 mol/L Tris-HCl buffer with
pH 8.0 and 2.0 mmol/L paraoxon as substrate) was added into it. The absorbance of sample
was taken after 5 minutes of incubation on spectrophotometer (Biosystems, BTS-330, S.A.
Costa Brava, Barcelona, Spain) with a wavelength 405 nm against paraoxonase substrate
reagent which was taken as a sample blank. The reaction was stable up to 5 minute.
d) Calculations:
The final enzymatic activity of paraoxonase was expressed in Unit/L and the activity
of paraoxonase was measured by using following formula:
Paraoxonase activity (U/mL) = 017.0
/minAbsorbance× 50
3.5.4 Arylesterase Activity (K Unit/L)
The enzymatic activity of arylesterase was determined by adopting the methodology
as described by Juretic et al. (2006).
49
a) Principle:
The phenylacetate was used as a substrate for the measurement of enzymatic activity
of arylesterase and increase in concentration of phenol was measured on subsequent
hydrolysis.
b) Preparation of Reagents:
Buffer solution of 0.1 M Tris-HCl of pH 8.0 was prepared by adding 1.211 g of tris
base in 80 mL of distilled water. The pH was adjusted to desired level with the help of 0.1 M
HCl solution under pH meter. For the preparation of 2 mmol CaCl2-Tris-HCl solution,
0.0022 g of CaCl2 was added in prepared Tris-HCl solution then added 3 µL of phenyl
acetate (2 mM). Finally added distilled water to make volume up to 100 mL.
c) Procedure of Assay:
Added 10 µL of each sample (serum /homogenized tissue) in 350 µL reagent of
arylesterase substrate (2.0 mMol/L phenylacetate and 2.0 mMol/L CaCl2 in 0.1 mol/L Tris-
HCl buffer with pH 8.0) in eppendorf. The reaction mixture was allowed to stand for
incubation for five minutes and then absorbance was noted on spectrophotometer (Biosystem
BTS-330, S.A. Costa Brava, Barcelona, Spain) with a wavelength of 270 nm. The enzyme
activity became stable within 5 minutes.
d) Calculations:
The enzymatic activity of arylesterase was calculated by using following formula
Arylesterase activity (KU/L) = 017.0
/minAbsorbance × 50
3.5.5 Catalase Activity
Catalase activity was determined by using the method of Goth (1991). It is a
hydrogen peroxide based spectrophotometer assay.
a) Principle:
One unite of catalase decomposes 1 micro mol of hydrogen peroxide/ 1 min.
Chemicals and reagents used:
Hydrogen per oxide = 65 micro mol/ml Sodium potassium buffer = 60 mmol
50
b) Procedure:
0.2ml of each sample (serum /homogenized tissue) was incubated in 1.0 ml substrate
containing 65µmol per ml hydrogen peroxide in 60 mmol sodium-potassium phosphate
buffer, ph 7.4, at room temperatue for 60 seconds. Serum catalase activity is linear up to 100
KU/L. If the catalase activity exceeded 100 KU/L the sample was diluted with the phosphate
buffer (2- to 10 fold) and the assay was repeated. One unite of catalase decomposes 1
micromol of hydrogen per oxide/1 min under these conditions.
The enzymatic reaction was stopped by addition of 1.0 ml of 8.5 mol/ L 3- amino-1, 2, 4-
triazole.
The enzymatic reaction was stopped with 1.0 ml of 32.4 mmol/l ammonium
molybdate ((NH4)6 Mo7O24.4 H2O) and the yellow complex of molybdate and hydrogen
peroxide was measured at 405 nm against blank 3.
Blank 1 contained 1.0 ml substrate, 1.0 ml molybdate and 0.2 ml of sample.
Blank 2 contained 1.0 ml substrate, 1.0 ml molybdate and 0.2 ml buffer.
Blank 3 contained 1.0 ml buffer, 1.0 ml molybdate and 0.2 ml buffer.
c) Calculations:
Serum catalase activity (KU/l) = 3)(blank A - 2)(blank A
1)(blank A - (sample)A × 271
3.6 Statistical Analysis
Mean frequency percentage for surveillance data was calculated. Two way analysis of
variance was applied (Steel et el., 1997). In case of significant differences, Duncan Multiple
Range (DMR) test was applied. For cooking methods, three way analysis of variance was
applied and then DMR (Duncan, 1955).
NOTE: Overall care and wellbeing of the birds needed for research were maintained and look after by a certified veterinarian from University of Agriculture, Faisalabad, Pakistan.
51
Chapter 4
RESULTS
PHASE I: (Surveillance)
4.1 Survey of Broilers 4.1.1 Survey of thigh muscle samples of broilers:
In eight areas located in and around Faisalabad district, antibiotics residues were not
detected and in contrast only in one area, 100% animal samples were positive for antibiotic
residue (Table 4.1). In nine areas of Faisalabad district, antibiotics residues was detected that
ranged from 24 percent (at Samundari) to 100 percent at Kidderwala as shown in table 4.1.
Overall, drug residue in broiler meat was found to be 30% of the sample treated in the present
study (Fig. 4.1).
Table 4.1: Survey of antibiotic residue from leg meat of broilers from different farm houses located in and around Faisalabad.
Areas Positive Negative Barnala (n=25) 0 25 (100%) Pancera (n=25) 15 (60%) 10 (40%) Jarnawala (n=37) 21 (56.8%) 16 (43.2%) Gojra (n=35) 0 35 (100%) Toba (n=31) 25 (80.6%) 6 (19.4%) Dijkot (n=30) 0 30 (100%) Styanah (n=25) 12 (48%) 13 (52%) Sumanderi (n=25) 6 (24%) 19 (76%) Jhumra (n=25) 9 (36%) 16 (64%) Aminpur Bangla (n=35) 0 35 (100%) Jabiraa (n=35) 0 35 (100%) Khidderwala (n=25) 25 (100%) 0 Shahkot (n=25) 23 (92%) 2 (8%) Mamu Kanjun (n=25) 0 25 (100%) Chak Sadhar (n=25) 0 25 (100%) Makkuwana (n=37) 16 (43.2%) 21 (56.8%) Khurrianwala (n=35) 0 35 (100%) Total (n=500) 152 (30.4%) 348 (69.6)
30%
70%
Fig. 4.1: Overall survey of antibiotic residue from leg meat of broilers from
different farm houses located in and around Faisalabad.
52
4.1.2 Survey of breast muscle samples of broilers:
In the present survey, antibiotics residue was not detected from eight different areas
except one where it was found to be 100% positive in all animal samples (Table 4.2). In other
area of Faisalabad district, the minimum and maximum percentage for residue ranged 20 to
88% positive. Overall, residue was positive in 32.0% of the breast meat of broiler in the
present study (Fig. 4.2).
Table 4.2: Survey of antibiotic residue from breast meat of broilers from different farm houses located in and around Faisalabad.
Areas Positive Negative Barnala (n=25) 0 25 (100%) Pancera (n=25) 17 (68%) 8 (32%) Jarnawala (n=37) 21 (56.8%) 16 (43.2%) Gojra (n=35) 0 35 (100%) Toba (n=31) 25 (80.6%) 6 19.4%) Dijkot (n=30) 0 30 (100%) Styanah (n=25) 10 (40%) 15 (60%) Sumanderi (n=25) 19 (76%) 6 (24%) Jhumra (n=25) 5 (20%) 20 (25%) Aminpur Bangla (n=35) 0 35 (100%) Jabiraa (n=35) 0 35 (100%) Khidderwala (n=25) 25 (100%) 0 Shahkot (n=25) 22 (88%) 3 (8%) Mamu Kanjun (n=25) 0 25 (100%) Chak Sadhar (n=25) 0 25 (100%) Makkuwana (n=37) 15 (40.5%) 22 (59.5%) Khurrianwala (n=35) 0 35 (100%) Total (n=500) 159 (31.8%) 341 (68.2)
32%
68%
Positive
Negative
Fig. 4.2: Overall survey of antibiotic residue from breast meat of broilers from different farm houses located in and around Faisalabad.
53
4.1.3 Survey of liver samples of broilers:
Drug residue from liver of broiler did reveal a 99% presence in almost all areas of
district Faisalabad (Table 4.3). Therefore, overall presence of drug residue was maximum in
liver of broiler under study (Fig. 4.3).
Table 4.3: Survey of antibiotic residue from liver of broilers from different farm houses located in and around Faisalabad.
Areas Positive Negative Barnala (n=25) 25 (100%) 0 Pancera (n=25) 25 (100%) 0 Jarnawala (n=37) 35 (94.6%) 2 (5.4%) Gojra (n=35) 35 (100%) 0 Toba (n=31) 31 (100%) 0 Dijkot (n=30) 30 (100%) 0 Styanah (n=25) 25 (100%) 0 Sumanderi (n=25) 25 (100%) 0 Jhumra (n=25) 25 (100%) 0 Aminpur Bangla (n=35) 33 (94.3%) 2 (5.7%) Jabiraa (n=35) 35 (100%) 0 Khidderwala (n=25) 25 (100%) 0 Shahkot (n=25) 25 (100%) 0 Mamu Kanjun (n=25) 25 (100%) 0 Chak Sadhar (n=25) 25 (100%) 0 Makkuwana (n=37) 37 (100%) 0 Khurrianwala (n=35) 35(100%) 0 Total (n=500) 496 (99.2%) 4 (0.8%)
99%
1%
Positive
Negative
Fig. 4.3: Overall survey of antibiotic residue from liver of broilers from different
farm houses located in and around Faisalabad.
54
4.1.4 Survey of lungs samples of broilers:
Survey of antibiotic residue from lungs of broilers was found to be maximum in
100% of samples in nine different areas of Faisalabad district (Table 4.4). Very few samples
of lungs were negative (6%) ranging from minimum of 4 percent to maximum of 20% in this
study (Fig. 4.4).
Table 4.4: Survey of antibiotic residue from lungs of broilers from different farm houses located in and around Faisalabad.
Areas Positive Negative Barnala (n=25) 20 (80%) 5 (20%)
Pancera (n=25) 25 (100%) 0 Jarnawala (n=37) 32 (86.5%) 5 (13.5%) Gojra (n=35) 28 (80%) 7 (20%)
Toba (n=31) 28 (90.3%) 3 (9.7%) Dijkot (n=30) 30 (100%) 0 Styanah (n=25) 24 (96%) 1 (4%)
Sumanderi (n=25) 24 (96%) 1 (4%) Jhumra (n=25) 25 (100%) 0 Aminpur Bangla (n=35) 35 (100%) 0
Jabiraa (n=35) 31 (88.6%) 4 (11.4%) Khidderwala (n=25) 25 (100%) 0 Shahkot (n=25) 25 (100%) 0
Mamu Kanjun (n=25) 25 (100%) 0 Chak Sadhar (n=25) 25 (100%) 0 Makkuwana (n=37) 32 (86.5%) 5(13.5%) Khurrianwala (n=35) 35 (100%) 0
Total (n=500) 469 (93.8%) 31 (6.3%)
94%
6%
Positive
Negative
Fig. 4.4: Overall survey of antibiotic residue from lungs of broilers from different
farm houses located in and around Faisalabad.
55
4.1.5 Survey of heart samples of broilers:
In broiler heart, antibiotic residue was found to be maximum (100%) in tested
samples in four areas of Faisalabad district (Table 4.5). Overall, 85% of hear samples did
have residue. Only 15% did not show any residue (Fig. 4.5).
Table 4.5: Survey of antibiotic residue from heart of broilers from in and around of Faisalabad.
Areas Positive Negative Barnala (n=25) 16 (64.0%) 9 (36%) Pancera (n=25) 24 (96%) 1 (4%) Jarnawala (n=37) 30 (81.1%) 7 (18.9%) Gojra (n=35) 24 (86.6%) 11 (31.4%) Toba (n=31) 27 (87.1%) 4 (12.9%) Dijkot (n=30) 28 (93.3%) 2 (6.7%) Styanah (n=25) 23 (92%) 2 (8%) Sumanderi (n=25) 22 (88%) 3 (12%) Jhumra (n=25) 25 (100%) 0 Aminpur Bangla (n=35) 31 (88.6%) 4 (11.6%) Jabiraa (n=35) 25 (71.4%) 10 (28.6%) Khidderwala (n=25) 25 (100%) 0 Shahkot (n=25) 25 (100%) 0 Mamu Kanjun (n=25) 25 (100%) 0 Chak Sadhar (n=25) 23 (92%) 2 (8%) Makkuwana (n=37) 29 (78.4%) 8 (21.6%) Khurrianwala (n=35) 25 (71.4%) 10 (28.6%) Total (n=500) 427 (85.4%) 73 (14.6%)
85%
15%
Positive Negative
Fig. 4.5: Overall survey of antibiotic residue from heart of broilers from different
farm houses located in and around of Faisalabad.
56
4.1.6. Inhibition zones of drug residues:
Overall inhibition zones was highest (12.154 mm) for liver, 7.653 mm for heart,
3.484 mm for leg muscle and 2.993 mm for breast muscle (Table 4.6).
Table 4.6: Mean diameter of inhibition zones (mm) of different tissues of broilers from different areas located in and around of Faisalabad.
Area Leg muscles
Breast muscles
Liver Hearts
Barnala (n=25) 0 0 12.24 5.56
Pancera (n=25) 4.68 5.64 13.2 9.28
Jarnawala (n=37) 3.84 4.57 11.14 7.03
Khurrianwala (n=35) 0.00 0.00 13.23 6.91
Gojra (n=35) 3.94 3.77 13.49 6.74
Toba (n=31) 5.97 7.13 11.77 8.16
Chak Sadhar (n=25) 0.00 0.00 13.44 7.20
Dijkot (n=30) 6.08 3.31 14.77 8.73
Makkuwana (n=37) 2.70 2.54 13.70 6.46
Styanah (n=25) 4.45 2.51 12.24 7.98
Aminpur Bangla (n=35) 2.86 2.11 11.76 7.92
Jhumra (n=25) 2.56 2.39 10.95 8.12
Jabiraa (n=35) 0.00 0.00 11.53 5.71
Khidderwala (n=25) 9.52 7.72 10.24 7.20
Shahkot (n=25) 9.15 7.19 10.81 10.04
Mamu Kanjun (n=25) 0.00 0.00 9.96 9.40
Overall Means 3.484 2.993 12.154 7.653
57
4.1.7 Month wise survey of broilers:
In the present survey, it was observed that highest percentage of leg muscle samples
(77.2%) did show residue in August, 78.0% in breast muscle samples, 100% in liver samples
during the month of May, June, August and September, 98.4 percent of heart samples during
August and 100% lungs samples during the month of September in the present study (Table
4.7).
Table 4.7: Surveys of antibiotic residue in different tissue and organ of broiler during different months of experimental period
Type of
Test results +ve/-ve
Months
March (n=32)
April (n=43)
May (n=139)
June (n=70)
August (n=123)
September(n=81)
Leg Muscles
+ve 2
(6.3%) 5
(11.6%) 47
(33.8%) 7
(10%) 95
(77.2%) 48
(59.3%)
-ve 30
(93.7%) 38
(88.4%) 92
(66.2%) 63
(90%) 28
(22.8%) 33
(40.7%)
Breast Muscles
+ve 0
(0%) 0
(0%) 90
(64.7%) 7
(10%) 96
(78%) 39
(48.1%)
-ve 32
(100%) 43
(100%) 49
(35.3%) 63
(90%) 27
(22%) 42
(51.9%)
Liver +ve
30 (93.7%)
41 (95.3%)
139 (100%)
70 (100%)
123 (100%)
81 (100%)
-ve 2
(6.3%) 2
(4.7%) 0
(0%) 0
(0%) 0
(0%) 0
(0%)
Heart +ve
10 (31.2%)
23 (53.5%)
132 (95%)
64 (91.4%)
121 (98.4%)
78 (96.3%)
-ve 22
(68.8%) 20
(46.5%) 7
(5%) 6
(8.6%) 2
(1.6%) 3
(3.7%)
Lungs +ve
21 (65.6%)
29 (67.4%)
133 (95.7%)
68 (97.1%)
120 (97.6%)
81 (100%)
-ve 11
(34.4%) 14
(32.6%) 6
(4.3%) 2
(2.9%) 3
(2.4%) 0
(0%) Values in parenthesis are percentage
58
4.1.8 Seasonal survey of broilers:
Overall high percentage of residue in the leg and breast muscles, liver, heart and
lungs were observed during the rainy season of the year. Overall minimum and maximum
residue did range from 9.33% of leg muscle samples during spring to 100% in liver samples
during summer (Table 4.8).
Table 4.8: Surveys of antibiotic residue in different tissue and organs of broilers during different seasons
Type of tissue
Spring
(n=75)
Summer
(n=209)
Rainy Season
(n=204)
Positive Negative Positive Negative Positive Negative
Leg Muscles 7
(9.33%)
68
(90.67%)
54
(25.84%)
155
(74.16%)
143
(70.10%)
61
29.90%)
Breast Muscles 0 75
(100%)
97
(46.41%)
112
(53.59%)
135
(66.18%)
69
(33.82%)
Liver 71
(94.67%)
4
(5.33%)
209
(100%)
0 204
(100%)
0
Heart 33
(44%)
42
(56%)
196
(93.78%)
13
(6.22%)
199
(97.55%)
5
(2.45%)
Lungs 50
(66.67%)
25
(33.33%)
201
(96.17%)
8
(3.83%)
201
(98.33%)
3
(1.47%)
Values in parenthesis are percentage
59
4.2 Surveys of Layers
4.2.1 Survey of leg muscle samples of layers:
At present in Pakistan published data on antibiotics residue in chicken meat and eggs
is scanty. This part of the research highlights some results of the surveys that were carried
out in the laboratory by analyzing the meat from layers and eggs collected from different
farmers or enterprises situated in and around Faisalabad during various months of 2008. The
antibiotics residue was analyzed by using Swab Test on animal food (STAF). In leg meat of
layers from four small cities did show 100% positive antibiotic residue while in three cities
almost no residue (0%) was detected in the present study (Table 4.9). Overall positive
samples for antibiotic from leg meat of layer birds were 38% (Fig. 4.6).
Table 4.9: Survey of antibiotic residue in the leg meat of layer from different towns of located in and around of Faisalabad
Areas Positive Negative Pancera (n=35) 6 (17.1%) 29 (82.9%) Dalowal (n=25) 0 25 (100%) Jarnawala (n=35) 6 (17.1%) 29 (82.9%) Gojra (n=25) 25 (100%) 0 Toba (n=35) 5 (14.3%) 30 (85.7%) Dijkot (n=37) 18 (48.6%) 19 (51.4%) Awagat (n=25) 1 (4%) 24 (96%) Styanah (n=35) 15 (42.9%) 20 (57.1%) Sumanderi (n=25) 11 (44%) 14 (56%) Jhumra (n=25) 1 (4%) 24 (96%) Aminpur Bangla (n=25) 0 25 (100%) Jabiraa (n=25) 0 25 (100%) Khidderwala (n=25) 25 (100%) 0 Shahkot (n=25) 25 (100%) 0 Mamu Kanjun (n=25) 25 (100%) 0 Total (n=427) 163 (38.17%) 264 (61.8%)
38%
62%
Positive Negative
Fig. 4.6: Overall survey of antibiotic residue in the leg meat of layer from different
towns located in and around of Faisalabad.
60
4.2.2 Survey of breast muscle samples of layers:
Antibiotic residue from all breast meat of layer was positive (100 percent) from the
birds collected from four different cities during the experimental period. Presence of drug
residue in breast meat of layer (Table 4.10) did differ in percentage between towns when
compared with the antibiotic residue present in leg meat of layer. Overall presence of residue
was 40% of the breast meat samples of layers (Fig. 4.7).
Table 4.10: Survey of antibiotic residue in the breast meat of layer from different towns located in and around of Faisalabad
Areas Positive Negative Pancera (n=35) 8 (22.9%) 27 (77.1%) Dalowal (n=25) 0 25 (100%) Jarnawala (n=35) 6 (17.1%) 29 (82.9%) Gojra (n=25) 25 (100%) 0 Toba (n=35) 8 (22.9%) 27 (77.1%) Dijkot (n=37) 12 (32.4%) 25 (67.6%) Awagat (n=25) 2 (8%) 23 (92%) Styanah (n=35) 20 (57.1%) 15 (42.9%) Sumanderi (n=25) 13 (52%) 12 (48%) Jhumra (n=25) 2 (8%) 23 92(%) Aminpur Bangla (n=25) 0 25 (100%) Jabiraa (n=25) 0 25 (100%) Khidderwala (n=25) 25 (100%) 0 Shahkot (n=25) 25 (100%) 0 Mamu Kanjun (n=25) 25 (100%) 0 Total (n=427) 171 (40%) 256 (60%)
40%
60%
Positive Negative
Fig. 4.7: Overall survey of antibiotic residue in the breast meat of layer from
different towns of Faisalabad
61
4.2.3 Survey of liver samples of layers:
As shown in table 4.11, a hundred percent liver samples from layer did show the
presence of residues in them.
Table 4.11: Survey of antibiotic residue in the liver of layer from different farms located in and around Faisalabad.
Areas Positive Negative Pancera (n=35)
35 (100%)
-
Dalowal (n=25)
25 (100%)
0
Jarnawala (n=35)
35 (100%)
-
Gojra (n=25)
25 (100%)
0
Toba (n=35)
35 (100%)
-
Dijkot (n=37)
37 (100%)
0
Awagat (n=25)
25 (100%)
0
Styanah (n=35)
35 (100%)
-
Sumanderi (n=25)
25 (100%)
0
Jhumra (n=25)
25 (100%)
0
Aminpur Bangla (n=25)
25 (100%)
0
Jabiraa (n=25)
25 (100%)
0
Khidderwala (n=25)
25 (100%)
0
Shahkot (n=25)
25 (100%)
0
Mamu Kanjun (n=25)
25 (100%)
0
62
4.2.4 Survey of lung samples of layers:
Maximum of 100% antibiotic residue was positive for lung samples from layers in
nine different locations (Table 4.12) indicating almost 84% positive samples for the presence
of such drugs (Fig. 4.8).
Table 4.12: Survey of antibiotic residue in the lungs of layer from different farms located in and around Faisalabad.
Areas Positive Negative Pancera (n=35) 32 (91.4%) 3 (8.6%) Dalowal (n=25) 17 (68%) 8 (32%) Jarnawala (n=35) 17 (48.6%) 18 (51.4%) Gojra (n=25) 25 (100%) 0 Toba (n=35) 32 (91.4%) 3 (8.6%) Dijkot (n=37) 37 (100%) 0 Awagat (n=25) 25 (100%) 0 Styanah (n=35) 1 (2.9%) 34 (97.1%) Sumanderi (n=25) 25 (100%) 0 Jhumra (n=25) 23 92(%) 2 (8%) Aminpur Bangla (n=25) 25 (100%) 0 Jabiraa (n=25) 25 (100%) 0 Khidderwala (n=25) 25 (100%) 0 Shahkot (n=25) 25 (100%) 0 Mamu Kanjun (n=25) 25 (100%) 0 Total (n=427) 359 (84.1) 68 (15.9)
Fig. 4.8: Overall survey of antibiotic residue in the lungs of layer from different
farms located in and around Faisalabad district
63
4.2.5 Survey of heart samples of layers:
Heart samples from layers did show the presence of antibiotic residue to all most all
samples (100%) at least from seven (7) locations throughout the experimental period (Table
4.13). Overall antibiotic residue was present in 62% of the total sample (n=427) studied
during the different locations (Fig. 4.9).
Table 4.13: Survey of antibiotic residue in the heart of layers from different farms located in and around Faisalabad.
Area Positive Negative Pancera (n=35) 30 (85.7%) 5 (14.3%) Dalowal (n=25) 16 (64%) 9 (36%) Jarnawala (n=35) 15 (42.9%) 20 (57.1%) Gojra (n=25) 25 (100%) 0 Toba (n=35) 8 (22.9%) 27 (77.1%) Dijkot (n=37) 33 (89.2%) 4 (10.4%) Awagat (n=25) 25 (100%) 0 Styanah (n=35) 4 (11.4%) 31 (88.6%) Sumanderi (n=25) 25 (100%) 0 Jhumra (n=25) 2 (8%) 23 (92%) Aminpur Bangla (n=25) 25 (100%) 0 Jabiraa (n=25) 23 (92%) 2 (8%) Khidderwala (n=25) 25 (100%) 0 Shahkot (n=25) 25 (100%) 0 Mamu Kanjun (n=25) 25 (100%) 0 Total (n=427) 306 (72%) 121 (28%)
72%
28% Positive Negative
Fig. 4.9: Overall survey of antibiotic residue in the heart of layer from different
farms located in and around Faisalabad district
64
4.2.6. Inhibition zone of tissues and various organs: The highest inhibition zones was observed in the liver (12.10 mm) samples followed
by heart (7.31 mm), breast muscle (3.49 mm) and in leg muscles (3.06 mm) to be the
minimum in this study (Table 4.14).
Table 4.14: Mean diameter of inhibition zones (mm) of different tissues of layers
from different areas of Faisalabad
Area Leg muscles
Breast muscles
Liver Hearts
Dalovaal 0.00 0.00 11.30 4.38
Pancera (n=25) 0.91 1.43 12.29 5.60
Jarnawala (n=37) 0.20 2.23 10.74 6.06
Gojra (n=35) 6.04 8.23 15.00 7.19
Toba (n=31) 1.09 1.49 11.94 5.26
Dijkot (n=30) 3.35 3.70 14.65 8.32
Awagat (n=25) 2.31 0.54 16.57 7.37
Styanah (n=25) 4.48 5.28 12.88 9.40
Sumanderi (n=25) 2.80 3.60 15.12 9.28
Aminpur Bangla (n=35) 0.00 0.00 10.60 6.84
Jhumra (n=25) 2.21 2.37 10.34 5.97
Jabiraa (n=35) 0.00 0.00 10.04 5.36
Khidderwala (n=25) 9.36 10.40 9.08 9.68
Shahkot (n=25) 8.24 6.40 9.48 9.80
Mamu Kanjn (n=25) 7.92 8.68 11.48 9.12
Overall Mean 3.06 3.49 12.10 7.31
65
4.2.7 Month wise survey of layers:
Data collected from March to September was tabulated for various organs of layer for
the presence of residues. Survey indicated that for all organs studied did show a minimum
residue for leg muscle, breast muscle and liver samples during April and heart during March.
The maximum drug residue positive samples of leg muscles and breast muscle during
September while maximum liver samples for drug residue occurence was during July,
August and September months. Heart and lungs sample percentage was highest during July
and August during this study (Table 4.15).
Table 4.15: Survey of antibiotic residue in various organs of laying hens during various months of experimental conditions
Type of tissue
Test results +ve / -ve
March
(n=42)
April
(n=25)
May
(n=90)
June
(n=40)
July
(n=38)
August
(n=74)
September
(n=118)
Leg Muscles
+ve 7
(16.7%)
0 (0)
36 (40%)
2 (5%)
15 (39.5%)
24 (32.4%)
87 (73.7%)
-ve 35
(83.3%) 25
(100%) 54
(60%) 38
(95%) 23
(60.5%) 50
(67.6%) 31
(26.3%)
Breast Muscles
+ve 2
(4.8%)
0 (0)
41 (45.6%)
4 (10%)
14 (36.8%)
30 (40.5%)
90 (76.3%)
-ve 40
(95.2%) 25
(100%) 49
(54.4%) 36
(90%) 24
(63.2%) 44
(59.5%) 28
(23.7%)
Liver +ve
42 (100%)
0 (0%)
90 (100%)
40 (100%)
38 (100%)
74 (100%)
118 (100%)
-ve 0
(0%) 25
(100%) 0
(0%) 0
(%0) 0
(0%) 0
(0%) 0
(0%)
Heart +ve
17 (40.5%)
15 (60%)
79 (87.8%)
34 (85%)
38 (100%)
74 (100%)
115 (97.5%)
-ve 25
(59.5%) 10
(40%) 11
(12.2%) 6
(15%) 0
(0%) 0
(0%) 3
(2.5%)
Lungs +ve
36 (85.7%)
19 (76%)
81 (90%)
37 (92.5%)
38 (100%)
74 (100%)
114 (96.4%)
-ve 6
(14.3%) 6
(24%) 9
(10%) 3
(7.5%) 0
(0%) 0
(0%) 4
(3.6%) Values in parenthesis are percentage
66
4.2.8 Seasonal survey of layers:
During three different seasons, rainy season did show the maximum residue positive
samples of all tissues and organs of layers which ranged from 57-81% to 100% of samples.
Only maximum liver samples did show residue during summer (Table 4.16) season.
Table 4.16: Surveys of antibiotic residue in different tissue and organs of layer during three different seasons
Type of tissue
Spring
(n=67)
Summer
(n=168)
Rainy Season
(n=192)
Positive Negative Positive Negative Positive Negative
Leg Muscles 7
(10.45%)
60
(89.55%)
53
(31.55%)
115
(68.45%)
111
(57.81%)
81
(52.19%)
Breast Muscles 2
(2.99%)
65
(97.01%)
59
(35.12%)
109
(64.88%)
120
(62.50%)
72
(37.50%)
Liver 42
(62.69%)
25
(37.31%)
168
(100%)
0 192
(100%)
0
Heart 32
(46.76%)
35
(52.24%)
151
(89.88%)
17
(10.12%)
189
(98.44%)
3
(1.56%)
Lungs 55
(82.09%)
12
(17.91%)
156
(92.86%)
12
(7.14%)
188
(97.92%)
4
(2.08%)
Values in parenthesis are percentage
67
4.2.9 Survey of egg yolk samples:
Forty (40) percent of egg yolk samples did show residue positive tests only in one
location throughout the experiment and was almost nil in samples of nine different locations
(Table 4.17). Overall, there was only 9% positive egg yolk samples for drug residue (Fig.
4.10) in the present study.
Table 4.17: Survey of antibiotic residue in the egg yolk of layer at different days from different farms in and around Faisalabad district
Areas Positive Negative Pancera (n=35) 14 (40%) 21 (60%) Dalowal (n=25) 0 (0%) 25 (100%) Jarnawala (n=35) 0 35(100%) Gojra (n=25) 0 (0%) 25 (100%) Toba (n=35) 5 (14.3%) 30 (85.7%) Dijkot (n=37) 0 (0%) 37 (100%) Awagat (n=25) 0 (0%) 25 (100%) Styanah (n=35) 7 (20%) 28 (80%) Sumanderi (n=25) 0 (0%) 25 (100%) Jhumra (n=25) 5 (16%) 20 (84%) Aminpur Bangla (n=25) 5 (16%) 20 (84%) Jabiraa (n=25) 0 (0%) 25 (100%) Khidderwala (n=25) 0 (0%) 25 (100%) Shahkot (n=25) 2 (8%) 23 (92%) Mamu Kanjun (n=25) 0 (0%) 25 (100%) Total (427) 38 (8.7%) 389 (91.3%)
Fig. 4.10: Overall survey of antibiotic residue in the egg yolk of layer at different
days from different farms in and around Faisalabad district
68
4.2.10 Survey of egg white samples:
Egg white of layers did show a wide variation for the presence of antibiotics residue.
From eight locations, antibiotic residue was absent (0%) while on other location its presence
ranged from 4-92% of egg white samples (Table 4.18). Overall 25% of the samples did show
a positive results and rest of 75% were negative for residue (Fig. 4.11).
Table 4.17: Survey of antibiotic residue in the egg white of layer at different days
from different farms in and around Faisalabad district Areas Positive Negative Pancera (n=35) 15 (42.9%) 20 (57.1%) Dalowal (n=25) 0 (0%) 25 (100%) Jarnawala (n=35) 4 (11.4%) 31 (88.6%) Gojra (n=25) 0 (0%) 25 (100%) Toba (n=35) 16 (45.7%) 19 (54.3%) Dijkot (n=37) 0 (0%) 37 (100%) Awagat (n=25) 0 (0%) 25 (100%) Styanah (n=35) 13 (37.1%) 22 (62.9%) Sumanderi (n=25) 0 (0%) 25 (100%) Jhumra (n=25) 22 (88%) 3 (12%) Aminpur Bangla (n=25) 10 (40%) 15 (60%) Jabiraa (n=25) 0 (0%) 25 (100%) Khidderwala (n=25) 0 (0%) 25 (100%) Shahkot (n=25) 23 (92%) 2 (8%) Mamu Kanjun (n=25) 0 (0%) 25 (100%) Total (n=427) 103 (25.1%) 324 (74.9%)
25.1%
74.9%
Positive
Negative
Fig. 4.11: Overall survey of antibiotic residue in the egg white of layer at different
days from different farms in and around Faisalabad district
69
4.2.11 Inhibition zones of egg white and egg yolk:
The inhibition zone of egg white were negative from eight different areas while for
Egg yolk it did not show any zone from nine different areas under study. For egg white the
inhibition ranged from 2.32 to 7.93 mm and for egg yolk it did range from 0.20 to 3.14 mm
at different areas under study.
Table 4.19 Mean diameter inhibition zones (mm) of egg white and yolk of layers
from different areas of Faisalabad
Area Egg White Egg yolk
Dalovaal (n=25) 0.00 0.00
Pancera (n=25) 5.43 3.14
Jarnawala (n=37) 2.34 0.00
Gojra (n=35) 0.00 0.00
Toba (n=31) 3.83 0.97
Dijkot (n=30) 0.00 0.00
Awagat (n=25) 0.00 0.00
Styanah (n=25) 9.00 1.72
Sumanderi (n=25) 0.00 0.00
Aminpur Bangla (n=35) 2.32 0.20
Jhumra (n=25) 7.93 2.07
Jabiraa (n=35) 0.00 0.00
Khidderwala (n=25) 0.00 0.00
Shahkot (n=25) 6.40 0.56
Mamu Kanjn (n=25) 0.00 0.00
Overall Mean 2.48 0.58
70
4.2.12 Month wise survey of eggs:
A survey of eggs reveals that egg whites as well as egg yolk samples were positive
for drug residue ranging from 57.6% and 24.24% respectively during the month of July as
compared to other months under study (Table 4.20).
Table 4.20: Survey of antibiotic residue of egg white and egg yolk of layers during
different months of a year Type
of tissue
Test results +ve/-ve
March
(n=42)
April
(n=25)
May
(n=114)
June
(n=49)
July
(n=33)
August
(n=81)
September
(n=118)
Egg White
+ve 0
(0%)
0
(0%)
9
(7.9%)
13
(26.5%)
19
(57.6%)
43
(53.1%)
23
(19.5%)
-ve 42
(100%)
25
(100%)
105
(92.1%)
36
(73.5%)
14
(42.4%)
38
(46.9%)
95
(80.5%)
Egg Yolk
+ve 0
(0%)
0
(0%)
5
(4.4%)
1
(2%)
8
(24.24%)
18
(22.2%)
2
(1.7%)
-ve 42
(100%)
25
(100%)
109
(95.6%)
48
(98%)
25
(75.76%)
63
(77.8%)
116
(98.3%)
Values in parenthesis are percentage
4.2.13 Seasonal survey of eggs:
Data organized for various seasons of the year indicated that egg white did have
33.17% positive and egg yolk 10.05% positive during rainy seasons as compared to spring
and summer (Table 4.21).
Table 4.21: Survey of antibiotic residue of egg white and egg yolk of layers during
three different season
Type of tissue
Spring
(n=67)
Summer
(n=196)
Rainy Season
(n=199)
Positive Negative Positive Negative Positive Negative
Egg White 0
(0%) 67
(100%) 41
(20.92%) 155
(79.08%) 66
(33.17%) 133
(66.83%)
Egg Yolk 0
(0%) 67
(100%) 14
(7.14%) 182
(92.86%) 20
(10.05%) 179
(89.95%) Values in parenthesis are percentage
71
PHASE II:
4.3 Withdrawal Time in Broilers
4.3.1 Fluoroquinolones in serum of broilers:
Serum concentration of different fluoroquinolones was subjected to analysis of
variance to observe the difference between drugs, days and their interaction (Table 4.22).
Days and drugs x days were significantly different. Ofloxacin and norfloxacin did increase
significantly on day 1 after therapy; however, these values did decrease significantly on day
3 after therapy (Table 4.23).
Table 4.22: Analysis of variance of concentration of different fluoroquinolones at different groups on different days.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.001 0.001 0.771NS
Days 3 0.055 0.018 21.770**
Drugs x Days 6 0.020 0.003 3.897**
Error 24 0.020 0.001
Total 35 0.095
NS = Non-significant ** = Significant P≤0.01 Table 4.23: Mean concentration (ppm±SE) of different fluoroquinolones in serum of
broilers at different days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Means
Ciprofloxacin 0.00
±0.00c
0.06
±0.034bc
0.06
±0.030bc
0.01
±0.00c
0.030
±0.013
Ofloxacin 0.00
±0.00c
0.15
±0.017a
0.00
±0.00c
0.04
±0.007bc
0.05
±0.019
Norfloxacin 0.00
±0.00c
0.10
±0.023ab
0.01
±0.004c
0.03
±0.020bc
0.04
±0.013 abc, similar alphabets on means do not different significantly at P≤0.01
72
4.3.2 Fluoroquinolones in muscle of broilers:
Analysis of variance showing a significant difference between drugs, days and in
their interaction has been presented in table 4.24. Mean fluoroquinolones concentration of
muscle from broilers did show a significant high residual quantity of norfloxacin on day 1,
which than decreased (P≤0.01) on day 3 and finally was detected in traces (Table 4.25).
Ciprofloxacin and ofloxacin did show a residual effect on day 3 which turned into traces at
the end of study.
Table 4.24: Analysis of variance of concentration of fluoroquinolones in muscle of
broiler at different days
Source of variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 322.597 161.299 104.922**
Days 3 384.858 128.286 83.448**
Drugs x Days 6 749.489 124.915 81.255**
Error 36 55.343 1.537
Total 47 1512.287
** = Significant P≤0.01 Table 4.25: Mean fluoroquinolones concentration (ppm ± SE) of muscle from broiler
at different days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.00
±0.00c
0.123
±0.006c
0.035
±0.006c
0.003
±0.003c
0.04
±0.01B
Ofloxacin 0.00
±0.00c
0.063
±0.008c
0.008
±0.005c
0.00
±0.00c
0.02
±0.007B
Norfloxacin 0.00
±0.00c
20.04
±2.11a
2.05
±0.40b
0.02
±0.004c
5.53
±2.23A AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
73
4.3.3 Fluoroquinolones in liver of broilers:
Analysis of variance of concentration of fluoroquinolons in the liver of layer has been
presented in table 4.26. Drugs, days as well as their interaction did differ significantly.
Highest concentration of norfloxacin was observed on day 1 which did decrease significantly
in day 4 at the end of experimental period (Table 4.27). Ofloxacin did not deposit in the liver,
however, ciprofloxacin did deposit in liver on day 1, decreased (P≤0.01) on day 3 and was
free of drug on day 4.
Table 4.26: Analysis of variance of concentration of fluoroquinolones in the liver of broilers at different days after therapy.
Source of variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 29656.895 14828.448 32.327**
Days 3 35222.147 11740.716 25.595**
Drugs x Days 6 65795.020 10965.837 23.906**
Error 36 16513.356 458.704
Total 47 147187.418
** = Significant at P≤0.01 Table 4.27: Mean fluoroquinolone (ppm ± SE) concentration of liver from broilers at
different days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Means
Ciprofloxacin 0.00
±0.00b
4.59
±0.32b
1.63
±0.19b
0.02
±0.01b
1.56
±0.49B
Ofloxacin 0.00
±0.00b
0.03
±0.003b
0.00
±0.00db
0.00
±0.00b
0.008
±0.004B
Norfloxacin 0.00
±0.00b
190.12
±37.02a
23.26
±2.32b
0.60
±0.07b
53.50
±22.12A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 ab, similar alphabets on means do not different significantly at P≤0.01
74
4.3.4 Fluoroquinolones in kidney of broilers:
Different fluoroquinolones were analyzed from the kidney of broiler and statistically
observed the differences between drugs, days and if any in their interaction (Table 4.28).
Mean norfloxacin concentration was high in the kidney of broilers on day 1 which quickly
disappear on day 3. Ofloxacin did have a residue of significant amount on days 1 which also
disappeared on day 4 of experimental period (Table 4.29).
Table 4.28: Analysis of variance of concentration of fluoroquinolones in the kidney of
broiler at different days of experimental period
Source of variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 8.750 4.375 1.750NS
Days 3 0.032 0.011 425.889**
Drugs x Days 6 0.001 0.000 7.972**
Error 36 0.001 2.500E-5
Total 47 0.034
NS = Non-significant ** = Significant P≤0.01 Table 4.29: Mean fluoroquinolones concentration (ppm±SE) in the kidney of broilers
in different groups at various time intervals
Drugs Day 0 Day 1 Day 3 Day 4 Overall Means
Ciprofloxacin 0.00
±0.00e
0.048
±0.005c
0.010
±0.00d
0.00
±0.00e
0.01
±0.005
Ofloxacin 0.00
±0.00e
0.068
±0.005b
0.003
±0.003e
0.00
±0.00e
0.02
±0.008
Norfloxacin 0.00
±0.00e
0.10
±0.005a
0.00
±0.00e
0.00
±0.00e
0.02
±0.008 a-e, similar alphabets on means do not different significantly at P≤0.01
75
4.4 Health Biomarkers in Broilers
4.4.1 Total Oxidant Status (TOS; µmol/L±SE) in Broilers
4.4.1.1 TOS in serum:
Analysis of variance of serum total oxidant status of broilers did show a
significant difference between drug, days and drugs × days interaction (Table 4.30) Mean
serum concentration of total oxidant status of broilers fed ciprofloxacin, ofloxacin and
norfloxacin did increase (P≤0.01) on days after drug is withdrawn and stayed high
throughout the experimental period (Table 4.31)
Table 4.30: Analysis of variance of total oxidant status in serum of broiler birds fed with different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.050 0.025 74.475**
Days 3 0.190 0.063 190.250**
Drugs x Days 6 0.018 0.003 8.750**
Error 24 0.008 0.000
Total 35 0.265
** = Significant at P≤0.01 Table 4.31: Mean total oxidant status (TOS; µmol/L ± SE) in serum of broiler
birds fed with different fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.250
±0.007f
0.360
±0.017de
0.350
±0.012e
0.310
±0.012e
0.318
±0.014B
Ofloxacin 0.246
±0.006f
0.470
±0.006ab
0.445
±0.009abc
0.415
±0.144bc
0.395
±0.026A
Norfloxacin 0.255
±0.006f
0.485
±0.003a
0.450
±0.012abc
0.405
±0.014cd
0.398
±0.027A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-e, similar alphabets on means do not different significantly at P≤0.01
76
4.4.1.2 TOS in broiler muscles:
Total oxidant status of broiler muscles was analyzed after fed with three different
fluoroquinolones and analyzing them at different days. Drug and days was significantly
different (Table 4.32). Oxidant status did increase on day 1 after each drug therapy and
stayed in muscles and there was high level of TOS on day 4 of the experimental period
(Table 4.33)
Table 4.32: Analysis of variance of total oxidant status in broiler muscles fed with
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.014 0.007 9.561**
Days 3 0.027 0.009 12.241**
Drugs x Days 6 0.009 0.001 1.955NS
Error 24 0.017 0.001
Total 35 0.067
NS = Non-significant ** = significant at P≤0.01 Table 4.33: Mean total oxidant status (TOS; µmol/L ± SE) of liver from broilers fed
different fluoroquinolones.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.237
±0.020
0.355
±0.0.009
0.310
±0.012
0.320
±0.023
0.308
±0.014A
Ofloxacin 0.277
±0.022
0.290
±0.012
0.230
±0.012
0.278
±0.009
0.260
±0.009B
Norfloxacin 0.257
±0.024
0.323
±0.019
0.310
±0.012
0.285
±0.009
0.291
±0.011A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01
77
4.4.1.3 TOS in broiler liver:
Broiler birds were fed different fluoroquinolones orally for five days and then were
sampled for liver to measure total oxidant status. Data collected was analyzed by the
analysis of variance and it did reveal that drugs, days and their interaction was significantly
different (Table 4.34). No change in TOS was observed in the liver of broiler when these
birds were fed ciprofloxacin from day 1 to day 5. Ofloxacin did decrease TOS on day 3 and
TOS was stable on day 4 (Table 4.35). Norfloxacin did increase TOS on day 1 after the
ingestion of drug and TOS remained high at day 4 of experimental period.
Table 4.34: Analysis of variance of total oxidant status in the liver of broilers fed different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.092 0.046 155.913**
Days 3 0.031 0.010 34.425**
Drugs x Days 6 0.046 0.008 25.961**
Error 24 0.007 0.000
Total 35 0.176
** = Significant at P≤0.01 Table 4.35: Mean total oxidant status (TOS; µmol/L ± SE) of liver from broilers fed
different fluoroquinolones.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.245
±0.008b
0.289
±0.011b
0.255
±0.003b
0.247
±0.009b
0.260
±0.006B
Ofloxacin 0.258
±0.006b
0.289
±0.014b
0.155
±0.020c
0.145
±0.009c
0.209
±0.019C
Norfloxacin 0.240
±0.004b
0.375
±0.009a
0.355
±0.003a
0.350
±0.006a
0.248
±0.015A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
78
4.4.1.4 TOS in broiler kidney:
Total oxidant status (TOS) of kidney from broilers fed different fluoroquinolones was
analyzed by analysis of variance and has been presented in table 4.36. Drugs, days and drugs
x days interaction were significantly different. Mean total oxidant status of kidney treated did
increase on day 1 of ofloxacin and norfloxacin treated broiler in the present study. On day 3,
TOS of kidney from broilers did decrease (P≤0.01) as compared to day 1. Ciprofloxacin
treated broiler did not show any alteration in the TOS of kidney of broilers (Table 4.37).
Table 4.36: Analysis of variance of total oxidant status in broiler kidney fed
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.032 0.016 34.063**
Days 3 0.037 0.012 26.130**
Drugs x Days 6 0.013 0.002 4.679**
Error 24 0.011 0.000
Significant at P≤0.01 Table 4.37: Mean total oxidant status (TOS; µmol/L ± SE) in kidney of broilers fed
different fluoroquinolones.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.257
±0.016ef
0.277
±0.025def
0.255
±0.009ef
0.225
±0.003e
0.251
±0.008C
Ofloxacin 0.237
±0.014ef
0.360
±0.006ab
0.290
±0.012cdef
0.295
±0.003bcde
0.300
±0.013B
Norfloxacin 0.277
±0.019ef
0.370
±0.012a
0.350
±0.006abc
0.325
±0.020abcd
0.323
±0.015A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-f, similar alphabets on means do not different significantly at P≤0.01
79
4.4.1.5 TOS in broiler heart:
Analysis of total oxidant status of heart from broiler fed different fluoroquniolones
was analyzed for drugs, days and for their interaction and has been presented in table 4.38.
Mean TOS of heart from broiler fed three fluoroquinolones has been given in table 4.39.
Ciprofloxacin and ofloxacin did increase (P≤0.01) the TOS of heart from broilers and then
decrease (P≤0.01) on day 3of experimental period. Norfloxacin did not affect TOS.
Table 4.38: Analysis of variance of total oxidant status in heart of broiler fed
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.002 0.001 4.889*
Days 3 0.023 0.008 31.908**
Drugs x Days 6 0.010 0.002 6.837**
Error 24 0.006 0.000
Total 35 0.040
* = Significant at P≤0.05 ** = Significant at P≤0.01 Table 4.39: Mean of total oxidant status (TOS; µmol/L ± SE) in heart of broiler
fed different fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.347
±0.006c
0.398
±0.005ab
0.340
±0.006c
0.340
±0.017c
0.354
±0.009AB
Ofloxacin 0.337
±0.009c
0.438
±0.010a
0.350
±0.006c
0.330
±0.006c
0.364
±0.014A
Norfloxacin 0.333
±0.008c
0.355
±0.009bc
0.330
±0.006c
0.355
±0.009bc
0.344
±0.005B
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
80
4.4.2. Total Antioxidant Capacity (TAC; mmol/L±SE) in Broilers
4.4.2.1 TAC in serum of broiler:
Analysis of variance of serum total antioxidant capacity (TAC) did reveal that days
and drugs x days interaction were significantly different (Table 4.40). Ciprofloxacin and
norfloxacin did decrease (P≤0.01) serum total antioxidant capacity in broiler on day 1 and
was then almost similar in both groups tell day 4. Ofloxacin did decrease (P≤0.05). Total
antioxidant capacity in serum of broiler on day 1 and another significant decrease on day 4
was also observed in the present study (Table 4.41).
Table 4.40: Analysis of variance of serum concentration of total antioxidant capacity in broiler at different days of experimental period
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.005 0.002 3.201NS
Days 3 0.034 0.011 15.919**
Drugs x Days 6 0.014 0.002 3.247*
Error 24 0.017 0.001
Total 35 0.070
* = Significant at P≤0.05 ** = Significant at P≤0.01 NS = Non-Significant Table 4.41: Mean serum concentration of total antioxidant capacity (TAC;
mmol/L±SE) in broiler showing its effects on different days of experimental period
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.475
±0.023a
0.410
±0.006abc
0.420
±0.006abc
0.435
±0.014abc
0.434
±0.009
Ofloxacin 0.470
±0.033a
0.385
±0.014bc
0.450
±0.017b
0.365
±0.014c
0.418
±0.015
Norfloxacin 0.464
±0.031a
0.370
±0.012c
0.375
±0.003bc
0.410
±0.012abc
0.406
±0.013
abc, similar alphabets on means do not different significantly at P≤0.05
81
4.4.2.2 TAC in broiler muscle:
Total antioxidant capacity (TAC) of muscles from broiler fed different
fluoroquinolones at different days of experimental period was analyzed by analysis of
variance (Table 4.42). Drugs, days and their interactions were significantly different. At day
0, the total antioxidant capacity did not differ significantly between organs. Ciprofloxacin did
increase (P≤0.01) on day 4 while ofloxacin did increase significantly on day 3. Norfloxacin
did not alter the TAC throughout the experimental period (Table 4.43).
Table 4.42: Analysis of variance of total antioxidant capacity in muscles of broiler fed fluoroquinolones in different groups at different days of experimental period
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.035 0.017 37.397**
Days 3 0.043 0.014 30.374**
Drugs x Days 6 0.050 0.008 17.904**
Error 24 0.011 0.000
Total 35 0.139
** = Significant at P≤0.01 Table 4.43: Mean total antioxidant capacity (TAC; mmol/L±SE) of broiler muscles
fed different fluoroquinolones during different days
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.456
±0.014cd
0.440
±0.012d
0.460
±0.017cd
0.620
±0.006a
0.494
±0.023A
Ofloxacin 0.455
±0.011cd
0.435
±0.003d
0.545
±0.020b
0.505
±0.003bc
0.485
±0.014A
Norfloxacin 0.463
±0.016cd
0.400
±0.012d
0.405
±0.003d
0.450
±0.014d
0.424
±0.008B
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-d, similar alphabets on means do not different significantly at P≤0.01
82
4.4.2.3 TAC in broiler liver:
Total antioxidant capacity measured from broiler liver that was fed three different
fluoroquinolones were subjected to analysis of variance for different days (Table 4.44).
Drugs, days and their interaction were significantly different. TAC did not differ significantly
on day 0 between different fluoroquinolones. Ciprofloxacin did alter the TAC and day 1 that
increased again on day 4. Ofloxacin and norfloxacin could not alter the TAC in liver of
broiler at different days of experimental period (Table 4.45).
Table 4.44: Analysis of variance of total antioxidant capacity (TAC) of broiler liver obtained from different groups fed fluoroquinolons at different days
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.067 0.034 47.404**
Days 3 0.049 0.016 23.138**
Drugs x Days 6 0.057 0.009 13.378**
Error 24 0.017 0.001
Total 35 0.191
** = Significant at P≤0.01 Table 4.45: Mean total antioxidants capacity (TAC; mmol/L±SE) of liver from
broiler fed fluoroquinolons during different days.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.467
±0.004a
0.275
±0.038b
0.2700
±0.023b
0.445
±0.003a
0.364
±0.029B
Ofloxacin 0.484
±0.005a
0.440
±0.006a
0.450
±0.012a
0.475
±0.009a
0.458
±0.005A
Norfloxacin 0.467
±0.002a
0.435
±0.009a
0.463
±0.010a
0.450
±0.021a
0.454
±0.007A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 ab, similar alphabets on means do not different significantly at P≤0.01
83
4.4.2.4 TAC in broiler kidney:
Total antioxidant capacity (TAC) of kidney from broiler fed different
fluoroquinolones was analyzed statistically by analysis of variance (Table 4.46). Drugs, days
and their interaction were significantly different. Mean TAC of kidney from broilers fed
fluoroquinolones at various days after therapy did decrease on day 1 in ciprofloxacin and
ofloxacin treated broilers. Ciprofloxacin treated broilers did show an increase (P≤0.01) in
TAC on day 3 and than on day 4 of experimental period. Norfloxacin did increase (P≤0.01)
TAC in kidney of broilers on day 4 in the present study (Table 4.47).
Table 4.46: Analysis of variance OF TAC (mmol/L±SE) in broiler kidney
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.092 0.046 99.552**
Days 3 0.114 0.038 81.872**
Drugs x Days 6 0.042 0.007 14.977**
Error 24 0.011 0.000
Total 35 0.259
** = Significant at P≤0.01 Table 4.47: Mean total antioxidants capacity (TAC; mmol/L±SE) of kidney from
broiler fed fluoroquinolons during different days.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.453
±0.016b
0.305
±0.009e
0.370
±0.012cd
0.475
±0.009b
0.401
±0.021B
Ofloxacin 0.453
±0.016b
0.235
±0.014f
0.355
±0.009de
0.354
±0.009de
0.349
±0.234C
Norfloxacin 0.453
±0.016b
0.420
±0.006bc
0.465
±0.009b
0.553
±0.019a
0.473
±0.016A
ABC, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-f, similar alphabets on means do not different significantly at P≤0.01
84
4.4.2.5 TAC in broiler heart:
Broiler heart total antioxidants capacity (TAC) did differ significantly for drugs, days
and in their interaction when fed for three different fluoroquinolones and analyzed by
analysis of variance (Table 4.48). Mean TAC of heart from broiler did show a significant
decrease on day 1 and than an increase on day 3 of experimental period when treated with
ciprofloxacin (Table 4.49). Norfloxacin did increase the TAC in the heart of broiler on day 4
of the experimental period.
Table 4.48: Analysis of variance of total antioxidant capacity in broiler heart fed
different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.025 0.012 18.436**
Days 3 0.067 0.022 33.267**
Drugs x Days 6 0.032 0.005 8.103**
Error 24 0.016 0.001
Total 35 0.140
** = Significant at P≤0.01 Table 4.49: Mean total antioxidants capacity (TAC; mmol/L±SE) of heart from
broiler fed fluoroquinolons during different days.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.345
±0.012bc
0.180
±0.006d
0.305
±0.009c
0.367
±0.009abc
0.299
±0.221B
Ofloxacin 0.345
±0.012bc
0.305
±.009bc
0.395
±.009ab
0.370
±0.012abc
0.354
±0.011A
Norfloxacin 0.345
±0.012bc
0.322
±0.034c
0.325
±0.009bc
0.400
±0.023a
0.355
±0.016A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
85
4.4.3 Arylesterase Concentration (KU/L±SE) in Broilers
4.4.3.1 Arylesterase in Serum of broilers:
Analysis of variance of serum arylesterase concentration was significantly
different between days, but did not differ significantly between different drugs (Table
4.50). Mean concentration of serum arylesterase did not differ significantly between three
fluoroquinolones, thus showing no change throughout the study period (Table 4.51).
Table 4.50: Analysis of variance of serum arylesterase concentration of broiler fed
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 13.680 6.840 0.777NS
Days 3 157.221 52.407 5.957**
Drugs x Days 6 43.643 7.274 0.827NS
Error 24 211.142 8.798
Total 35 425.686
NS = Non-significant ** = Significant at P≤0.01 Table 4.51: Mean serum arylesterase (KU/L±SE) concentration of broiler fed
different fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 59.14
±2.52
56.59
±1.17
56.67
±1.74
49.76
±0.29
55.55
±1.27
Ofloxacin 59.36
±2.22
56.72
±0.86
56.14
±0.82
55.35
±2.01
56.84
±0.85
Norfloxacin 60.16
±2.51
57.06
±1.02
56.49
±1.02
54.76
±2.02
56.87
±0.89
86
4.4.3.2 Arylesterase in broiler Muscles:
Drug, days and drug × days interaction was significantly different, when data on
muscle arylesterase was analyzed by the analysis of variance (Table 4.52). Mean muscle
arylesterase did decrease (P≤0.01) on day 1 after drug therapy for ofloxacin and
norfloxacin was stable on day 4 of experimental period (Table 4.53) Ciprofloxacin did
decrease (P≤0.01) muscle arylesterase on day 3 and was stable on day 4 of experimental
condition.
Table 4.52: Analysis of variance of muscle arylesterase concentration of broiler fed
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 1765.444 882.722 169.263**
Days 3 1172.375 390.792 74.935**
Drugs x Days 6 629.744 104.957 20.126**
Error 24 125.162 5.215
Total 35 3692.724
** = Significant at P≤0.01 Table 4.53: Mean muscle arylesterase concentration (KU/L±SE) of broiler fed
fluoroquinolones at different days of therapeutic dose.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 86.70
±1.51a
83.91
±0.96abc
77.21
±1.53cd
75.58
±1.74d
80.86
±1.52B
Ofloxacin 87.74
±1.45a
64.08
±1.34e
61.68
±1.75e
58.93
±0.16e
67.85
±3.38C
Norfloxacin 88.74
±1.59a
84.57
±0.58ab
85.11
±1.42ab
79.77
±0.70bcd
84.05
±0.92A
ABC, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-e, similar alphabets on means do not different significantly at P≤0.01
87
4.4.3.3 Arylesterase in broiler liver:
Liver arylesterase concentration of broiler was analyzed by analysis of variance to
monitor the difference between drug, days and their interaction (Table 4.54). Drug, days and
their interaction were significantly different. Only ciprofloxacin did the liver arylesterase
concentration of broilers on day 3 and was stable at day 4. Ofloxacin and norfloxacin did not
affect the liver arylesterase concentration of broiler throughout the study period (Table 4.55).
Table 4.54: Analysis of variance of liver arylesterase concentration of broiler fed
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 67.414 33.707 6.857**
Days 3 153.003 51.001 10.375**
Drugs x Days 6 152.022 25.337 5.154**
Error 24 117.981 4.916
Total 35 490.419
** = Significant at P≤0.01 Table 4.55: Mean liver arylesterase concentration (KU/L±SE) of broiler fed
different fluoroquinolones at different days of therapeutic dose.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 74.73
±0.81a
73.02
±1.99a
63.17
±2.01b
69.09
±0.87ab
69.99
±1.49B
Ofloxacin 76.70
±0.61a
75.59
±0.54a
70.59
±2.38a
71.42
±1.06a
72.33
±0.75A
Norfloxacin 76.53
±0.69a
72.99
±0.82a
74.89
±0.94a
70.41
±1.07a
73.25
±0.66A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 ab, similar alphabets on means do not different significantly at P≤0.01
88
4.4.3.4 Arylesterase in broiler kidney:
Analysis of variance of kidney arylesterase concentration did change significantly
for drugs, days and for their interaction (Table 4.56). Mean kidney arylesterase did
decrease on day 1 of ciprofloxacin treated broiler and then decrease on day 4 after
treatment. Ofloxacin and norfloxacin did not effect the kidney arylesterase concentration
in the present study (Table 4.57).
Table 4.56: Analysis of variance of kidney arylesterase concentration of broiler fed
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 145.461 72.731 17.998**
Days 3 397.752 132.584 32.809**
Drugs x Days 6 350.239 58.373 14.445**
Error 24 96.987 4.041
Total 35 990.439
** = Significant at P≤0.01 Table 4.57: Mean kidney arylesterase concentration (KU/L±SE) of broiler fed
different fluoroquinolones at different days after oral therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 76.68
±0.15a
66.65
±1.73cde
72.54
±0.57abc
63.45
±2.19de
69.83
±1.66B
Ofloxacin 79.18
±0.12a
74.12
±0.90a
61.92
±0.96e
68.06
±0.87bcd
70.19
±1.76B
Norfloxacin 77.63
±0.18a
73.89
±1.52ab
74.94
±0.40a
71.56
±1.73abc
74.26
±0.75A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-e, similar alphabets on means do not different significantly at P≤0.01
89
4.4.3.5 Arylesterase in broiler heart:
Broiler heart arylesterase concentration was significantly different between days,
however, drugs and days did not differ significantly (Table 4.58). Ciprofloxacin did decrease
the heart arylesterase concentration, however, ofloxacin and norfloxacin did change the heart
arylesterase concentration after a therapeutic dose (Table 4.59).
Table 4.58: Analysis of variance of broiler arylesterase concentration of broiler
heart fed different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 20.791 10.395 1.530NS
Days 3 145.171 48.390 7.121**
Drugs x Days 6 51.997 8.666 1.275NS
Error 24 163.092 6.795
Total 35 381.051
NS = Non-significant ** = Significant at P≤0.01 Table 4.59: Mean arylesterase concentration (KU/L±SE) of broiler heart fed different
fluoroquinolones at various days after a therapeutic dose.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 57.50
±0.62
53.61
±3.49
54.75
±2.02
48.1
±0.84
53.51
±1.37
Ofloxacin 58.56
±0.72
53.90
±0.14
54.85
±0.96
54.76
±2.02
55.27
±0.65
Norfloxacin 59.00
±0.70
53.94
±0.39
54.95
±1.51
53.19
±1.11
54.91
±0.66
90
4.4.4 Paraoxonase Concentration (PON1; U/L±SE) in Broilers
4.4.4.1 Paraoxonase in serum of broilers:
Serum paraoxonase contents of broiler fed different fluoroquinolones did show a
significant difference between drugs, days and their interaction (Table 4.60). Mean serum
paraoxonase concentration of broilers at various days after therapy has been given in
table 4.60 in ciprofloxacin treated birds, serum paraoxonase did decrease on day 1 and
then on day 3 after drug therapy. While ofloxacin treated broiler did show a significant
decrease only on day1 after treatment (Table 4.61).
Table 4.60: Analysis of variance of paraoxonase concentration of broiler serum fed
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 1465.672 732.836 49.541**
Days 3 1255.558 418.519 28.293**
Drugs x Days 6 720.601 120.100 8.119**
Error 24 355.017 14.792
Total 35 3796.849
** = Significant at P≤0.01 Table 4.61: Mean paraoxonase concentration (PON1; U/L±SE) of broiler serum fed
different fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 80.91
±0.76a
66.18
±4.25b
52.17
±2.03c
51.68
±4.02c
62.73
±3.86B
Ofloxacin 80.91
±0.76a
72.34
±1.03ab
73.55
±2.32ab
70.59
±3.39ab
74.35
±1.50A
Norfloxacin 80.91
±0.76a
78.43
±0.76a
75.12
±0.88ab
75.94
±0.38ab
77.60
±0.74A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
91
4.4.4.2 Paraoxonase in broiler liver:
Liver paraoxonase concentration obtained from broiler was analyzed by the
analysis of variance and has been given in table 4.62. Drugs, days and their interaction
were significantly different. Mean liver paraoxonase concentration did decrease
significantly on day 4 after therapy. Ofloxacin and norfloxacin treatment did not show
any alteration in their liver paraoxonase contents in the present study (Table 4.63)
Table 4.62: Analysis of variance of paraoxonase concentration of broiler liver fed
different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 374.242 187.121 22.083**
Days 3 277.211 92.404 10.905**
Drugs x Days 6 1309.133 218.189 25.750**
Error 24 203.362 8.473
Total 35 2163.948
** = Significant at P≤0.01 Table 4.63: Mean paraoxonase concentration (PON1; U/L±SE) of broiler liver fed
different fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 63.09
±1.77ab
62.52
±1.67ab
59.06
±1.49ab
36.63
±1.14c
55.32
3.35B
Ofloxacin 65.49
±1.77ab
62.00
±2.51ab
60.00
±2.38b
66.21
±0.60a
62.83
±1.08A
Norfloxacin 62.67
±1.82ab
60.02
±0.77ab
57.13
±1.47b
64.60
±1.76ab
61.21
±1.08A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
92
4.4.4.3 Paraoxonase in broiler kidney:
Analysis of variance of kidney paraoxonase concentration from broilers fed different
fluoroquinolones has been presented in table 4.64. Drugs and drugs × days interaction were
significantly different. Mean kidney paraoxonase concentration did decrease significantly on
day1 in ciprofloxacin while a significant increase was observed on day 1 in norfloxacin
treated broiler after therapy (Table 4.65).
Table 4.64: Analysis of variance of paraoxonase concentration of broilers kidney fed
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 7030.437 3515.218 339.823**
Days 3 62.700 20.900 2.020NS
Drugs x Days 6 2376.395 396.066 38.288**
Error 24 248.262 10.344
Total 35 9717.794
NS = Non-significant ** = Significant at P≤0.01 Table 4.65: Mean paraoxonase concentration (PON1; U/L±SE) of broilers kidney fed
different fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 64.07
±1.49b
39.41
±2.72c
34.08
±1.45c
37.00
±0.24c
43.14
±3.42C
Ofloxacin 62.07
±1.36b
59.70
±2.89b
60.12
±3.16b
61.97
±1.91b
60.96
±1.09B
Norfloxacin 61.87
±1.26b
81.16
±0.25a
81.22
±0.79a
81.99
±1.91a
77.36
±2.73A
ABC, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
93
4.4.4.4 Paraoxonase in broiler heart:
Analysis of variance of heart paraoxonase concentration for drugs, days and their
interaction are given in table 4.66. Drug, days and their interaction was significantly
different. Mean heart paraoxonase concentration was significantly low on day 1 of
ciprofloxacin and norfloxacin treated broiler after therapy (Table 4.67).
Table 4.66: Analysis of variance of paraoxonase concentration of broiler heart fed
different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 57463.604 28731.802 2621.628**
Days 3 10439.297 3479.766 317.511**
Drugs x Days 6 13265.795 2210.966 201.739**
Error 24 263.029 10.960
Total 35 81431.725
** = Significant at P≤0.01 Table 4.67: Mean paraoxonase concentration (PON1; U/L±SE) of broiler heart fed
different fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 86.44
±2.60b
70.75
±1.01d
66.45
±1.68d
68.45
±1.62d
73.02
±2.50B
Ofloxacin 82.40
±2.34a
84.83
±0.27a
82.08
±2.54a
84.45
±1.85bc
159.45
±13.09C
Norfloxacin 81.22
±2.69b
73.56
±1.68d
75.17
±2.03cd
70.75
±0.52d
76.48
±1.97A
ABC, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-d, similar alphabets on means do not different significantly at P≤0.01
94
4.4.5 Catalase Concentration (KU/L±SE) in Broilers
4.4.5.1 Catalase in serum of broilers:
Analysis of variance of serum catalase in broilers did reveal that drugs, days and their
interaction was significantly different (Table 4.68). Mean serum catalase concentration of
broiler did decrease in drug treated birds on day 1 and then increased on day 3 after therapy
(Table 4.69). A further increase in serum catalase was observed in ciprofloxacin treated birds
on day 4 while ofloxacin and norfloxacin did not change serum catalase concentration.
Table 4.68: Analysis of variance of broiler serum catalase exposed to different
fluoroquinolones at different days.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 3390.293 1695.146 4.222*
Days 3 22861.110 7620.370 18.980**
Drugs x Days 6 8508.419 1418.070 3.532*
Error 24 9635.831 401.493
Total 35 44395.652
** = Significant at P≤0.01 Table 4.69: Mean broiler serum catalase (KU/L±SE) exposed to different
fluoroquinolones at different days.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 360.00
±8.00a
252.47
±10.70c
334.15
±12.58b
364.11
±11.93a
327.82
±14.37B
Ofloxacin 366.
±8.65a
320.37
±9.50b
357.67
±19.30a
339.05
±13.41b
344.41
±7.50AB
Norfloxacin 360.55
±7.05a
320.31
±12.78b
366.89
±4.64a
355.68
±11.80a
350.86
±6.92A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
95
4.4.5.2 Catalase in broiler muscle:
Muscle catalase concentration of broiler fed different fluoroquinolones at various
days after a therapeutic dose was analyzed by analysis of variance (Table 4.70). Mean
muscle catalase concentration of broiler did decrease on day 1 after therapy while it did
increase on day 3 and was almost similar to day 0 various at the end of experiment (Table
4.71).
Table 4.70: Analysis of variance of broiler muscle catalase exposed to different
fluoroquinolones at different days.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 21.699 10.849 0.029NS
Days 3 7726.657 2575.552 6.845**
Drugs x Days 6 945.152 157.525 0.419NS
Error 24 9030.081 376.253
Total 35 17723.589
NS = Non-significant ** = Significant at P≤0.01 Table 4.71: Mean broiler muscle catalase (KU/L±SE) exposed to different
fluoroquinolones at different days.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 248.82
±6.88
217.01
±10.03
258.80
±10.20
244.12
±8.49
242.19
±6.10
Ofloxacin 235.89
±7.09
211.74
±17.86
244.67
±6.84
259.84
±7.30
241.27
±7.15
Norfloxacin 242.08
±8.84
219.12
±2.55
251.47
±22.92
241.73
±9.53
240.28
±6.77
96
4.4.5.3 Catalase in broiler liver:
Analysis of variance of liver catalase concentration was not significantly different
for drugs, days and in their interaction (Table 4.72). Mean liver catalase concentration of
broiler did decrease on day 1 in ofloxacin and norfloxacin groups and then increased on
day 3 after drug administration in the present study (Table 4.73).
Table 4.72: Analysis of variance of broiler liver catalase exposed to different
fluoroquinolones at different days.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 1064.573 532.287 1.498NS
Days 3 2099.389 699.796 1.970NS
Drugs x Days 6 1317.272 219.545 0.618NS
Error 24 8527.024 355.293
Total 35 13008.259
NS = Non-significant Table 4.73: Mean broiler liver catalase (KU/L±SE) exposed to different
fluoroquinolones at different days.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 261.74
±10.16
252.32
±7.73
258.08
±3.64
254.41
±9.34
256.64
±3.76
Ofloxacin 266.40
±11.18
243.35
±4.30
279.63
±5.97
272.49
±5.79
264.30
±5.16
Norfloxacin 261.87
±11.10
236.76
±18.85
250.09
±20.33
255.56
±5.59
251.04
±7.05
97
4.4.5.4 Catalase in broiler kidney:
Catalase concentration of kidney after fluoroquinolone therapeutic dose was
analyzed by analysis of variance and been presented in table 4.74. Drugs, days and their
interaction were significantly different. Mean kidney catalase concentration did increase
on day 3 after therapy and then decrease on day 4 of experimental period (Table 4.75).
Table 4.74: Analysis of variance of broiler kidney catalase exposed to different fluoroquinolones at different days.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 2009917.536 1004958.768 7.346**
Days 3 5265837.158 1755279.053 12.831**
Drugs x Days 6 5967339.675 994556.613 7.270**
Error 24 3283268.576 136802.857
Total 35 1.653E7
** = Significant at P≤0.01 Table 4.75: Mean broiler kidney catalase (KU/L±SE) exposed to different
fluoroquinolones at different days.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 328.45
±6.06d
307.88
±8.94e
429.38
±17.37b
355.03
±7.05c
354.75
±19.821A
Ofloxacin 318.00
±6.04e
337.96
±10.70d
480.59
±37.98a
363.32
±12.12c
374.5
±24.37A
Norfloxacin 322.64
±6.41e
302.62
±7.28e
347.06
±13.58d
332.47
±1.63d
327.65
±6.01B
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-e, similar alphabets on means do not different significantly at P≤0.01
98
4.4.5.5 Catalase in broiler heart:
Analysis of variance of heart catalase concentration were significantly different
between drugs and days, however, it did not differ significantly between drugs × days
interaction (Table 4.76). Mean heart catalase concentration from broilers fed different
fluoroquinolones did show a decreasing trend on day 3 and day 4, catalase concentration
of heart in broiler was normal as observed at day 0 (Table 4.77).
Table 4.76: Analysis of variance of broiler heart catalase exposed to different
fluoroquinolones at different days.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 1872.261 936.130 4.479*
Days 3 4082.862 1360.954 6.512**
Drugs x Days 6 1880.276 313.379 1.499NS
Error 24 5015.881 208.995
Total 35 12851.279
NS = Non-significant ** = Significant at P≤0.01 Table 4.77: Mean broiler heart catalase (KU/L±SE) exposed to different
fluoroquinolones at different days.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 225.60
±7.99
206.07
±8.31
247.77
±4.68
250.27
±8.65
232.43
±6.31A
Ofloxacin 217.50
±7.99
209.21
±8.53
216.94
±9.41
216.85
±13.04
217.15
±4.59B
Norfloxacin 248.60
±7.99
216.59
±8.42
244.12
±5.09
243.57
±7.11
232.47
±4.72A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01
99
4.6 Concentration of Fluoroquinolones before and after Cooking
4.6.1 Muscles of broilers:
Before and after cooking the concentration of different fluoroquinolones in broilers
muscle was significantly different for cooking method, drugs, and days and for their possible
interaction (Table 4.78). Ciprofloxacin and ofloxacin did reduce in their residual
concentration significantly while norfloxacin reduced from 20.04 to 0.04 after cooking, on
day 1 of experimental conditions. Likewise, concentration of fluoroquinolones did show a
significant low or almost to zero concentration on day 4 of the experimental condition (Table
4.79).
Table 4.78: Analysis of variance of different fluoroquinolones concentration from muscle of broiler before and after cooking
Source of variations Degree of Freedom
Sum of Squares
Means Squares
F-Value
Groups 1 82.715 82.715 107.608**
Drugs 2 161.923 80.962 105.328**
Days 3 193.336 64.445 83.841**
Groups x Drugs 2 160.676 80.338 104.516**
Groups x Days 3 191.524 63.841 83.055**
Drug x Days 6 375.667 62.611 81.454**
Cooking x Drug x Days 6 373.824 62.304 81.055**
Error 72 55.344 0.769
Total 95 1595.007
** = Significant at P≤0.01 Table 4.79: Mean muscle concentration (ppm±SE) of different fluoroquinolones in
the broilers before and after cooking at various days after therapy
Drugs Before Cooking After Cooking
Day 0 Day 1 Day 3 Day 4 Day 0 Day 1 Day 3 Day 4
Ciprofloxacin 0.00
±0.00e
0.12
±0.006c
0.04
±0.006d
0.00
±0.00e
0.00
±0.00e
0.01
±0.003e
0.00
±0.00e
0.00
±0.00e
Ofloxacin 0.00
±0.00e
0.06
±0.008d
0.01
±0.005e
0.00
±0.00e
0.00
±0.00e
0.00
±0.00e
0.00
±0.00e
0.00
±0.00e
Norfloxacin 0.00
±0.00e
20.04
±2.11a
2.05
±0.40b
0.02
±0.004d
0.00
±0.00e
0.04
±0.003d
0.02
±0.005d
0.00
±0.00e
a-e, similar alphabets on means do not different significantly at P≤0.01
100
4.6.2 Liver of broilers:
Concentration of fluoroquinolones in the liver of broiler was measured before and
after cooking at various days of experimental period after therapy. Analysis of variance has
been presented in table 4.80. Between cooking methods, drugs, days as well as their
interaction did predicts significant differences. Mean liver concentration of fluoroquinolones
did show a significant decrease after cooking as compared to before cooking (Table 4.81).
Table 4.80: Analysis of variance of different fluoroquinolones concentration of liver from broiler before and after cooking
Source of variations Degree of Freedom
Sum of Squares
Means Squares
F-Value
Groups 1 8081.533 8081.533 35.236**
Drugs 2 14832.696 7416.348 32.336**
Days 3 17617.756 5872.585 25.605**
Groups x Drugs 2 14824.200 7412.100 32.318**
Groups x Days 3 17604.392 5868.131 25.586**
Drug x Days 6 32905.071 5484.179 23.912**
Cooking x Drug x Days 6 32889.950 5481.658 23.901**
Error 72 16513.357 229.352
Total 95 155268.955
** = Significant at P≤0.01 Table 4.81: Mean liver concentration (ppm±SE) of different fluoroquinolones in
the broilers before and after cooking at various corresponding days
Drugs Before Cooking After Cooking
Day 0 Day 1 Day 3 Day 4 Day 0 Day 1 Day 3 Day 4
Ciprofloxacin 0.00
±0.00g
4.59
±0.32c
1.63
±0.19d
0.02
±0.01f
0.00
±0.00g
0.01
±0.006f
0.00
±0.00g
0.00
±0.00g
Ofloxacin 0.00
±0.00g
0.03
±0.003f
0.00
±0.00g
0.00
±0.00g
0.00
±0.00g
0.00
±0.00g
0.00
±0.00g
0.00
±0.00g
Norfloxacin 0.00
±0.00g
190.12
±37.02a
23.26
±2.32b
0.60
±0.07e
0.00
±0.00g
0.03
±0.005f
0.01
±0.005f
0.00
±0.00g
a-g, similar alphabets on means do not different significantly at P≤0.01
101
4.7 Concentration of Fluoroquinolones after Cooking in Electric and
Microwave Ovens 4.7.1 Muscle of Broilers:
Analysis of variance of different fluoroquinolones concentration in the muscles of
broilers by two different methods has been given in table 4.82. Cooking methods, drugs, days
and their possible interaction were significantly different. Mean concentration of different
fluoroquinolones in the muscles of broilers did show a significant decrease by the two
different methods of cooking in the present study (Table 4.83).
Table 4.82: Analysis of variance of fluoroquinolones concentration of broilers muscle after two cooking methods:
Source of variations Degree of Freedom
Sum of Squares
Means Squares
F-Value
Methods 1 0.000 0.000 4.167*
Drugs 2 0.002 0.001 108.667**
Days 3 0.003 0.001 125.056**
Methods x Drugs 2 0.000 0.000 4.667*
Methods x Days 3 0.000 0.000 1.500NS
Drug x Days 6 0.002 0.000 48.222**
Methods x Drug x Days 6 0.000 0.000 2.000NS
Error 48 0.000 0.000
Total 71 0.008
** = Significant at P≤0.01 Table 4.83: Mean concentration of fluoroquinolones in the broiler muscle after two
different methods of cooking at various days
Drugs Oven Microwave
Day 0 Day 1 Day 3 Day 4 Day 0 Day 1 Day 3 Day 4
Ciprofloxacin 0.00
±0.00
0.01
±0.003
0.00
±0.00
0.00
±0.00
0.00
±0.00
0.01
±0.00
0.00
±0.00
0.00
±0.00
Ofloxacin 0.00
±0.00
0.00
±0.00
0.00
±0.00
0.00
±0.00
0.00
±0.00
0.00
±0.00
0.00
±0.00
0.00
±0.00
Norfloxacin 0.00
±0.00
0.04a
±0.00
0.02
±0.007
0.00
±0.00
0.00
±0.00
0.03
±0.00
0.01
±0.00
0.00
±0.00
102
4.7.2 Liver of Broilers:
Liver concentration of different fluoroquinolones of broilers was determined by using
two different methods of cooking (Table 4.84). Analysis of this study did show a significant
difference in ciprofloxacin fed group between two different cooking methods at day 1 but not
on day 3 and 4 of experimental period. However, these concentrations of different
fluoroquinolones were significant low in electeric oven treated as well as microwave cooked
samples (Table 4.85).
Table 4.84: Analysis of variance of fluoroquinolones concentration of broilers liver two methods of cooking
Source of variations Degree of Freedom
Sum of Squares
Means Squares
F-Value
Methods 1 0.000 0.000 15.125**
Drugs 2 0.001 0.000 37.625**
Days 3 0.002 0.001 46.125**
Methods x Drugs 2 0.000 0.000 4.625*
Methods x Days 3 0.000 0.000 7.125**
Drug x Days 6 0.002 0.000 23.625**
Methods x Drug x Days 6 0.000 0.000 2.625*
Error 48 0.001 0.000
Total 71 0.005
** = Significant at P≤0.01 Table 4.85: Mean concentration of fluoroquinolones in the broiler liver after two
different methods of cooking at various days
Drugs Oven Microwave
Day 0 Day 1 Day 3 Day 4 Day 0 Day 1 Day 3 Day 4
Ciprofloxacin 0.00
±0.00b
0.01
±0.007a
0.00
±0.0b
0.00
±0.0b
0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
Ofloxacin 0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
Norfloxacin 0.00
±0.00b
0.03a
±0.003
0.01
±0.006a
0.00
±0.00b
0.00
±0.00b
0.02
±0.00a
0.00
±0.00b
0.00
±0.00b
ab, similar alphabets on means do not different significantly at P≤0.01
103
PHASE II: (withdrawal time of layer birds)
4.8 Layers
4.8.1 Fluoroquinolones in Serum of Layers:
During withdrawal time study, three fluoroquinolones namely ciprofloxacin, ofloxacin
and norfloxacin were given orally (in drinking water) for a period of five days in order to
determine the residual effect of each drugs. Analysis of variance of different fluoroquinolones
did reveal that drugs, days and their interaction was significantly different (Table 4.86). All
three drugs did show a decreasing trend by each day of experimental period, however, this
decrease was significant at day 3 for ciprofloxacin and norfloxacin (Table 4.87).
Table 4.86: Analysis of variance of concentration of different fluoroquinolones in different groups
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.023 0.011 7.205**
Days 3 0.140 0.047 29.927**
Drugs x Days 6 0.137 0.023 14.586**
Error 24 0.037 0.002
Total 35 0.337
** = Significant P≤0.01 Table 4.87: Mean concentration (ppm±SE) of different fluoroquinolones in serum of
layers at different days of experimental period
Drugs Day 0 Day 1 Day 3 Day 4 Overall Means
Ciprofloxacin 0.00
±0.00c
0.18
±0.052b
0.03
±0.026c
0.02
±0.002c
0.06
±0.024B
Ofloxacin 0.00
±0.00c
0.13
±0.028bc
0.08
±0.041bc
0.05
±0.18bc
0.06
±0.018B
Norfloxacin 0.00
±0.00c
0.10
±0.00bc
0.32
±0.009a
0.03
±0.003c
0.11
±0.038A AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
104
4.8.2 Fluoroquinolones in Muscle of Layers:
Concentration of different fluoroquinolones in the muscles of layers did reveals that
drugs, days and their interaction were significantly different when analyzed by analysis of
variance (Table 4.88). Mean concentration of fluoroquinolones in the muscle of layer was
significantly high (P≤0.01) in norfloxacin on day 1 and decrease (P≤0.01) on day 3 and day
4. Ofloxacin muscle concentration did decrease significantly on day 3 while decrease in
ciprofloxacin was gradual from day 1 to day 2 and then day 3 (Table 4.89).
Table 4.88: Analysis of variance of concentration of different fluoroquinolones in the muscles of layers at various days of experimental condition
Source of variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.212 0.106 22.881**
Days 3 2.402 0.801 173.039**
Drugs x Days 6 0.277 0.046 9.985**
Error 36 0.167 0.005
Total 47 3.057
** = Significant P≤0.01 Table 4.89: Mean concentration (ppm±SE) of different fluoroquinolones in the
muscles of layer at various days of experimental period
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.00
±0.00f
0.49
±0.06b
0.13
±0.03de
0.00
±0.00f
0.15
±0.05C
Ofloxacin 0.00
±0.00f
0.35
±0.03c
0.05
±0.03ef
0.00
±0.00f
0.10
±0.04B
Norfloxacin 0.00
±0.00f
0.80
±0.07a
0.22
±0.05d
0.02
±0.004f
0.26
±0.09A AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-f, similar alphabets on means do not different significantly at P≤0.01
105
4.8.3 Fluoroquinolones in Liver of Layers:
Drugs, days and their interaction were significantly different for fluoroquinolones
concentration measured at different days of experimental period (Table 4.90). On day 1,
norfloxacin concentration was significantly high in liver of layer followed by lower
concentration in liver of ciprofloxacin fed layer and lowest concentration was observed in
liver of layer fed ofloxacin (Table 4.91). Ofloxacin disappear from liver very quickly on day
3 and was lowest in ciprofloxacin and ofloxacin fed groups. Liver did show some traces only
in norfloxacin group.
Table 4.90: Analysis of variance of concentration of different fluoroquinolones present in liver at various days
Source of variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 13.225 6.613 107.966**
Days 3 14.691 4.897 79.955**
Drugs x Days 6 16.531 2.755 44.985**
Error 36 2.205 0.061
Total 47 46.652
** = Significant P≤0.01 Table 4.91: Mean concentration (ppm±SE) of different fluoroquinolones from layer
liver measured at different intervals
Drugs Day 0 Day 1 Day 3 Day 4 Overall Means
Ciprofloxacin 0.00
±0.00d
0.72
±0.17c
0.04
±0.008d
0.00
±0.00d
0.19
±0.09B
Ofloxacin 0.000
±0.00d
0.028
±0.005d
0.00
±0.00d
0.00
±0.00d
0.007
±0.003C
Norfloxacin 0.00
±0.00d
3.32
±0.032a
1.48
±0.22b
0.008
±0.005d
1.20
±0.36A ABC, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-d, similar alphabets on means do not different significantly at P≤0.01
106
4.8.4 Fluoroquinolones in Kidney of Layers:
Concentration of fluoroquinolones in the kidney of layers for different drugs, days
and for their interaction did differ significantly and has been given in table 4.92.
Fluoroquinolones concentration in the kidney of different groups did show a significant
deposit of norfloxacin on day 1 that also level off on day 3 and day 4 of experimental period
(Table 4.93).
Table 4.92: Analysis of variance of concentration of different fluoroquinolones in the kidney of days at various days of experimental period
Source of variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.032 0.016 75.545**
Days 3 0.055 0.018 86.065**
Drugs x Days 6 0.097 0.016 75.545**
Error 36 0.008 0.000
Total 47 0.192
** = Significant P≤0.01 Table 4.93: Mean concentration (ppm±SE) of fluoroquinolones in the kidney of layers
at various days of experimental period
Drugs Day 0 Day 1 Day 3 Day 4 Overall Means
Ciprofloxacin 0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
0.00
±0.00b
0.00
±0.00B
Ofloxacin 0.00
±0.00b
0.010
±0.004b
0.00
±0.00b
0.00
±0.00b
0.003
±0.001B
Norfloxacin 0.00
±0.00b
0.225
±0.025a
0.00
±0.00b
0.00
±0.00b
0.06
±0.03A AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 ab, similar alphabets on means do not different significantly at P≤0.01
107
4.8.5 Concentration of Fluoroquinolones in Eggs:
Analysis of variance of concentration of different fluoroquinolones in egg of layer did
show a significant difference between drugs, days and in their interaction (Table 4.94). Mean
concentration of ciprofloxacin and norfloxacin was high (P≤0.01) on day 1 and then suddenly
disappear from the egg on day 3 and day 4 of experimental period (Table 4.95).
Table 4.94: Analysis of variance of concentration of different fluoroquinolones in the egg of layer at different days of experimental period
Source of variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 7.917E-5 3.958E-5 8.143**
Days 3 0.000 0.000 21.000**
Drugs x Days 6 0.000 3.958E-5 8.143**
Error 36 0.000 4.861E-6
Total 47 0.001
** = Significant P≤0.01 Table 4.95: Mean concentration (ppm±SE) of different fluoroquinolones in the egg of
layer at different days
Drugs Day 0 Day 1 Day 3 Day 4 Overall Means
Ciprofloxacin 0.00
±0.00c
0.013
±0.003a
0.00
±0.00c
0.00
±0.00c
0.031
±0.001A
Ofloxacin 0.00
±0.00c
0.00
±0.00c
0.00
±0.00c
0.00
±0.00c
0.00
±0.00B
Norfloxacin 0.00
±0.00c
0.005
±0.003b
0.00
±0.00c
0.00
±0.00c
0.01
±0.0009B AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
108
4.9 Health Biomarkers in Layers
4.9.1 Total Oxidant Status (TOS; µmol/L ± SE) in Layers:
4.9.1.1 TOS in serum of layers: Analysis of variance of serum concentration of oxidant status showing the effects of
different antibiotics at different days and interaction between them has been presented in
table 4.96. Drugs, days and their interaction were significantly different. Serum total oxidant
status did increase significantly on day 1 as compared to day 0 in layers irrespective of the
fluoroquinolones ingestion by the birds (Table 4.97). On day 3, total oxidant status did
decrease significantly in ofloxacin and norfloxacin treated layer. On day 4, serum total
oxidant status further decrease (P≤0.01) in ciprofloxacin and in norfloxacin treated layer,
however, these value were still higher (P≤0.01) in ofloxacin and norfloxacin as compared to
day 0 values.
Table 4.96: Analysis of variance of total oxidant status in serum of layers exposed to three fluoroquinolones at different days of experimental period
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 1.340 0.670 137.142**
Days 3 3.581 1.194 244.365**
Drugs x Days 6 1.196 0.199 40.797**
Error 24 0.117 0.005
Total 35 6.235
** = Significant at P≤0.01 Table 4.97: Mean serum concentration of total oxidant status (TOS; µmol/L ± SE) at
in layers different time intervals after oral ingestion of fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.540
±0.030e 1.015
±0.032d 0.925
±0.032d 0.705
±0.014e 0.798
±0.057C
Ofloxacin 0.555
±0.031e 1.630
±0.029ab 0.950
±0.023d 0.980
±0.017d 1.026
±0.118B
Norfloxacin 0.500
±0.033e 1.795
±0.318a 1.495
±0.014b 1.245
±0.101c 1.270
±0.141A ABC, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-e, similar alphabets on means do not different significantly at P≤0.01
109
4.9.1.2 TOS in layer muscle:
Total oxidant status was analyzed by analysis of variance for different drugs, days
and for their interaction (Table 4.98). Days and drugs x days were significantly different.
Ciprofloxacin did increase the total oxidant status of muscle on day 1 and was stable till the
day 4. Ofloxacin did increase on day 1 and day 3 but was not significant and did decrease
(P≤0.05) on day 4. Norfloxacin did increase on day 1 and started decreasing on day 3 and
day 4 (Table 4.99).
Table 4.98: Analysis of variance of total oxidant status in muscles of layers exposed to three fluoroquinolones at different days of experimental period
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.016 0.008 2.225NS
Days 3 0.185 0.062 17.421**
Drugs x Days 6 0.061 0.010 2.850*
Error 24 0.085 0.004
Total 35 0.347
NS = Non-significant * = Significant at P≤0.05 ** = Significant at P≤0.01 Table 4.99: Mean muscles concentration of total oxidant status (TOS; µmol/L ±
SE) in layers at different time intervals after oral ingestion of fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.583
±0.040bcd
0.785
±0.043a
0.645
±0.020abcd
0.660
±0.012abcd
0.671
±0.026
Ofloxacin 0.570
±0.050bcd
0.745
±0.061ab
0.745
±0.009ab
0.490
±0.029d
0.643
±0.037
Norfloxacin 0.583
±0.034bcd
0.710
±0.017abc
0.635
±0.026abcd
0.540
±0.017cd
0.619
±0.022 abcd, similar alphabets on means do not different significantly at P≤0.01
110
4.9.1.3 TOS in layer liver:
Liver total oxidant status of layers was analyzed by analysis of variance showing its
effects for drugs, days and drugs x days interaction (Table 4.100). Groups were significantly
different. Total oxidant status of liver from layers did show an increasing trend on day 1,
though not significantly different and then it did show a non-significant decrease in total
oxidant status (Table 4.101).
Table 4.100: Analysis of variance of total oxidant status in liver of layers exposed to three fluoroquinolones at different days of experimental period
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.027 0.014 2.314NS
Days 3 0.224 0.075 12.762**
Drugs x Days 6 0.057 0.009 1.621NS
Error 24 0.140 0.006
Total 35 0.448
NS = Non-significant ** = Significant at P≤0.01 Table 4.101: Mean liver concentration of total oxidant status (TOS; µmol/L ± SE) in
layers at different time intervals after oral ingestion of fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.720
±0.042
0.910
±0.006
0.805
±0.038
0.850
±0.012
0.812
±0.023
Ofloxacin 0.710
±0.038
0.895
±0.020
0.785
±0.009
0.655
±0.118
0.761
±0.038
Norfloxacin 0.701
±0.044
0.910
±0.012
0.780
±0.012
0.640
±0.046
0.760
±0.033
111
4.9.1.4 TOS in layer kidney:
Analysis of variance of total oxidant status of kidney from layers did show a
significant difference between drugs, days and in their interaction (Table 4.102). Mean
concentration of TOS in the kidney of layers fed ciprofloxacin did show significant level on
day 0 and then on day 4 after therapy. Ciprofloxacin and norfoxacin fed layer did show a
significant decrease on days 4 of experimental period after therapy (Table 4.103). However,
ofloxacin did increase significantly till day 4 of experimental period.
Table 4.102: Analysis of variance of total oxidant status in kidney of layers exposed
to three fluoroquinolones at different days of experimental period
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.282 0.141 32.700**
Days 3 0.238 0.079 18.383**
Drugs x Days 6 0.350 0.058 13.496**
Error 24 0.104 0.004
Total 35 0.974
** = Significant at P≤0.01 Table 4.103: Mean kidney concentration of total oxidant status (TOS; µmol/L ±
SE) in layers fed different fluoroquinolones at different days after therapy
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.357
±0.014c
0.420
±0.092bc
0.510
±0.05bc
0.120
±0.017d
0.352
±0.049C
Ofloxacin 0.357
±0.014c
0.605
±0.020ab
0.580
±0.017ab
0.725
±0.20a
0.567
±0.041A
Norfloxacin 0.357
±0.014c
0.500
±0.052bc
0.565
±0.049ab
0.315
±0.020c
0.434
±0.035B
ABC, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-d, similar alphabets on means do not different significantly at P≤0.01
112
4.9.1.5 TOS in layer heart:
Total oxidant status of heart was significantly different for groups and drugs and
drugs x days interaction did not differ significantly (Table 4.104). Mean concentration of
total oxidant status of heart of layers fed different fluoroquinolones has been presented in
table 4.105. Ciprofloxiacin, ofloxacin and norfloxacin fed layers did increase the total
oxidant status of heart of day 1 after therapy and remained high throughout the experimental
period (Table 4.105).
Table 4.104: Analysis of variance of total oxidant status in heart of layers exposed to three fluoroquinolones at different days of experimental period after therapy
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.025 0.013 2.188NS
Days 3 0.900 0.300 51.933**
Drugs x Days 6 0.018 0.003 0.512NS
Error 24 0.139 0.006
Total 35 1.082
NS = Non-significant ** = Significant at P≤0.01 Table 4.105: Mean heart concentration of total oxidant status (TOS; µmol/L ± SE)
at in layers fed different fluoroquinolones at different days after therapy
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.496
±0.083
0.900
±0.017
0.860
±0.017
0.830
±0.17
0.771
±0.052
Ofloxacin 0.496
±0.083
0.925
±0.020
0.940
±0.006
0.810
±0.017
0.793
±0.057
Norfloxacin 0.496
±0.083
0.820
±0.023
0.830
±0.012
0.770
±0.012
0.729
±0.045
113
4.9.2 Total Antioxidant Capacity (TAC; mmol/L±SE) in Layers:
4.9.2.1 TAC in serum of layers:
Serum from layers given different antibiotic was analyzed statistically by analysis
of variance. Days were found to be significantly different (Table 4.106). At day 0, TAC
of serum in layer did show a high value; however, after administration of drugs, the
serum TAC values did decrease throughout the experimental period, but were not
significant statistically (Table 4.107).
Table 4.106: Analysis of variance of total antioxidant capacity in serum of layer fed with different fluoroquinolones at different time intervals.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.016 0.008 0.466NS
Days 3 0.556 0.185 10.929**
Drugs x Days 6 0.210 0.035 2.068NS
Error 24 0.407 0.017
Total 35 1.189
** = Significant at P≤0.01 NS = Non-Significant Table 4.107: Mean total antioxidant capacity (TAC; mmol/L±SE) in serum of layers
fed different fluoroquinalones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.650
±0.100
0.425
±0.107
0.300
±0.035
0.475
±0.026
0.464
±0.050
Ofloxacin 0.635
±0.104
0.135
±0.003
0.515
±0.089
0.440
±0.115
0.436
±0.069
Norfloxacin 0.653
±0.108
0.390
±0.012
0.400
±0.023
0.505
±0.043
0.488
±0.402
114
4.9.2.2 TAC in layer muscles:
Analysis of variance of muscles did show total antioxidant capacity from layers
fed different fluoroquinolones differ significantly between drugs and between days
(Table 4.108). Mean total antioxidant capacity of muscles from layers was high when fed
ofloxacin as compared to ciprofloxacin and norfloxacin in the present study (Table 4.109)
Table 4.108: Analysis of variance of total antioxidant capacity from muscles of layer fed different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.035 0.017 11.408**
Days 3 0.060 .020 13.168**
Drugs x Days 6 0.021 0.003 2.245NS
Error 24 0.037 0.002
Total 35 0.152
NS = Non-significant ** = Significant at P≤0.01 Table 4.109: Mean muscle concentration of TAC (TAC; mmol/L±SE) of layer fed
different fluoroquinolones.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.911
±0.025
0.765
±0.014
0.810
±0.017
0.800
±0.017
0.821
±0.018B
Ofloxacin 0.900
±0.021
0.860
±0.017
0.95
±0.020
0.875
±0.009
0.885
±0.010A
Norfloxacin 0.920
±0.032
0.815
±0.032
0.795
±0.038
0.749
±0.022
0.817
±0.022B
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01
115
4.9.2.3 TAC in layer liver:
The liver from layers fed different fluoroquinolones, did show a significant
difference between days when data was analyzed by the analysis of variance (Table
4.110). Mean liver total antioxidant from layer has been presented in table 4.111
Liver TAC did not change at different days of experimental period.
Table 4.110: Analysis of variance of total antioxidant capacity (TAC; mmol/L) from
liver of layer fed different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.004 0.002 2.512NS
Days 3 0.032 0.011 15.278**
Drugs x Days 6 0.008 0.001 1.778NS
Error 24 0.017 0.001
Total 35 0.060
NS = Non-significant ** = Significant at P≤0.01 Table 4.111: Mean liver concentration of TAC (TAC; mmol/L±SE) of layer fed
different fluoroquinolones at various days after therapy
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.690
±0.009
0.605
±0.014
0.630
±0.006
0.705
±0.014
0.659
±0.0137
Ofloxacin 0.687
±0.007
0.650
±0.012
0.665
±0.0260
0.720
±0.023
0.683
±0.011
Norfloxacin 0.678
±0.009
0.620
±0.012
0.680
±0.023
0.670
±0.012
0.666
±0.106
116
4.9.2.4 TAC in layer kidney:
Total antioxidant capacity of kidney from layers fed different fluoroquinolones was
significantly different for drugs, days and into their interaction (Table 4.112). Mean total
antioxidant capacity was significantly high in ofloxacin treated birds while low (P≤0.01)
in the kidney of norfloxacin treated birds on day 1 (Table 4.113). Total antioxidant capacity
did not change in layer fed ciprofloxacin therapy.
Table 4.112: Analysis of variance of total antioxidant from kidney of layer fed different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.366 0.183 5.336*
Days 3 3.162 1.054 30.734**
Drugs x Days 6 1.175 0.196 5.711**
Error 24 0.823 0.034
Total 35 5.525
* = Significant at P≤0.05 ** = Significant at P≤0.01 Table 4.113: Mean total antioxidant capacity (TAC; mmol/L±SE) from kidneys of
layer fed different fluoroquinolones
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.815
±0.026ab
0.706
±0.025abc
0.795
±0.009ab
0.555
±0.043bcd
0.718
±0.033A
Ofloxacin 1.208
±0.255a
0.450
±0.002bcd
0.073
±0.010d
0.229
±0.047cd
0.490
±0.142B
Norfloxacin 1.218
±0.201a
0.372
±0.043bcd
0.308
±0.042bcd
0.198
±0.043cd
0.521
±0.133B
ABC, similar alphabets on overall means in a column do not differ significantly at P≤0.05 a-d, similar alphabets on means do not different significantly at P≤0.01
117
4.9.2.5 TAC in layer heart:
In the heart of layers fed different fluoroquinolones, total antioxidant capacity did
differ significantly for drugs, days and drugs x days interaction (Table 4.114). Mean total
antioxidant capacity of heart from layers fed different floroquinolones has been presented in
table 4.115. Ciprofloxacin and ofloxacin treated layers did show a significant decrease in
total antioxidant capacity of heart at day 1 after therapy.
Table 4.114: Analysis of variance of total antioxidant capacity of heart of layers fed different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 0.043 0.022 27.732**
Days 3 0.047 0.016 20.029**
Drugs x Days 6 0.047 0.008 9.957**
Error 24 0.019 0.001
Total 35 0.155
** = Significant at P≤0.01 Table 4.115: Mean total antioxidant capacity (TAC; mmol/L±SE) of heart from layers
fed different fluoroquinolones at various days after therapy
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 0.677
±0.024ab
0.548
±0.010de
0.565
±0.014cde
0.505
±0.014e
0.572
±0.020C
Ofloxacin 0.687
±0.023ab
0.515
±0.026e
0.600
±0.006bcd
0.635
±0.003abc
0.605
±0.019B
Norfloxacin 0.657
±0.026ab
0.660
±0.012ab
0.610
±0.012abcd
0.685
±0.003a
0.656
±0.010A
ABC, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-e, similar alphabets on means do not different significantly at P≤0.01
118
4.9.3 Arylesterase Concentration (KU/L±SE) in Layers:
4.9.3.1 Arylesterase in serum of layers:
Serum arylesterase concentration of layers was analyzed by the analysis of
variance (Table 4.116) showing days and drug × days were significantly different. Mean
serum arylesterase in layer in response to ciprofloxacin, ofloxacin did decrease (P≤0.05)
and increased (P≤0.05) on day 3 and 4 after ingestion of a therapeutic dose for five days
(Table 4.117).
Table 4.116: Analysis of variance of serum arylesterase concentration of layers fed different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 27.001 13.500 0.108NS
Days 3 11126.008 3708.669 29.738**
Drugs x Days 6 2601.498 433.583 3.477*
Error 24 2993.038 124.710
Total 35 16747.545 NS = Non-significant * = Significant at P≤0.05 ** = Significant at P≤0.01 Table 4.117: Mean serum concentration of arylesterase (KU/L±SE) from layers fed
different fluoroquinolones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 186.70
±8.90a
139.85
±0.77bc
151.42
±5.91ab
163.24
±2.55ab
158.81
±5.13
Ofloxacin 184.77
±8.95a
140.68
±11.60bc
170.89
±3.57ab
150.97
±5.65ab
160.82
±5.88
Norfloxacin 180.07
±8.95a
116.77
±1.19c
177.95
±5.94a
166.18
±0.85ab
160.41
±8.11
abc, similar alphabets on means do not different significantly at P≤0.01
119
4.9.3.2 Arylesterase in layer muscles:
Muscles arylesterase concentration after being analyzed by analysis of variance did
show a significant difference between drugs and days (Table 4.118). Overall mean muscle
arylesterase was high in ciprofloxacin treated groups as compared to ofloxacin and
norfloxacin the present study (Table 4.119).
Table 4.118: Analysis of variance of Layer muscle Arylesterase concentration in
response to different fluooquinolones on different days after therapy.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 956.947 478.474 5.128*
Days 3 1352.221 450.740 4.831**
Drugs x Days 6 532.142 88.690 0.951NS
Error 24 2239.354 93.306
Total 35 5080.664 NS = Non-significant * = Significant at P≤0.05 ** = Significant at P≤0.01 Table 4.119: Mean layer muscle Arylesterase concentration (KU/L±SE) in response to
different fluoquinolones on different days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 142.38
±2.49
140.77
±3.63
145.59
±7.64
133.82
±0.85
140.64
±2.29A
Ofloxacin 142.83
±2.99
114.71
±3.40
132.36
±8.49
123.30
±3.30
128.18
±3.77B
Norfloxacin 142.39
±2.39
129.00
±12.67
141.18
±5.10
132.35
±1.70
136.23
±3.44AB
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01
120
4.9.3.3 Arylesterase in layer liver:
Liver arylesterase concentration measured in different days fed with different
flouroquinolones did differ for drugs, days and their interaction (Table 4.120). Mean liver
arylesterase concentration did decrease on day 1 as compared to day 0 in ciprofloxacin and
ofloxacin but no change was observed on various days in a group treated with norfloxacin
(Table 4.121)
Table 4.120: Analysis of variance of Arylesterase in liver of layers fed different
fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 52.622 26.311 0.074NS
Days 3 1311.517 437.172 1.228NS
Drugs x Days 6 1554.177 259.030 0.728NS
Error 24 8543.847 355.994
Total 35 11462.164
NS = Non-significant Table 4.121: Mean concentration of Arylesterase (KU/L±SE) in liver from layers fed
different fluoroquinolones for different days after therapy of experimental period.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 155.18
±14.44
132.36
±3.40
152.94
±16.98
158.83
±11.89
150.32
±6.31
Ofloxacin 150.27
±14.34
142.65
±14.43
152.06
±3.57
139.70
±5.94
147.90
±5.08
Norfloxacin 157.81
±14.45
153.24
±3.23
156.33
±7.39
135.59
±5.27
150.58
±4.56
121
4.9.3.4 Arylesterase in layer kidney:
Analysis of variance of arylesterase concentration in kidneys of layer fed with
different fluoroquinolones did differ significantly for drugs, days and their interaction (Table
4.122). Mean kidney arylesterase concentration was low during day 1 as compared to day 0
as well as during day 4 compared to day 3 in the present study for layers fed different
fluoroquinolones (Table 4.123)
Table 4.122: Analysis of variance of arylesterase in kidney of layers fed different
fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 148.848 74.424 0.250NS
Days 3 616.209 205.403 0.689NS
Drugs x Days 6 433.065 72.177 0.242NS
Error 24 7155.287 298.137
Total 35 8353.410
NS = Non-significant Table 4.123: Mean arylesterase concentration (KU/L±SE) in the kidney of layers fed
different fluoroquinolones at various days after therapy
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 146.99
±4.84
127.35
±3.23
139.71
±21.23
135.30
±11.88
137.32
±5.74
Ofloxacin 144.91
±4.42
139.42
±5.10
141.18
±6.79
140.30
±3.91
141.95
±2.41
Norfloxacin 141.91
±3.82
141.18
±20.38
132.65
±2.55
144.12
±3.39
141.21
±4.83
122
4.9.3.5 Arylesterase in layer heart:
Drugs and drugs × days interaction was significantly different for arylesterase
concentration from heart of layers in the present study (Table 4.124). Mean arylesterase
concentration did decrease on day 1 of ciprofloxacin and ofloxacin treated birds (Table
4.125). In ofloxacin, it did decrease (P≤0.01) on day 3 as compared to day 1 of experimental
period.
Table 4.124: Analysis of variance of arylesterase in layer heart fed different
fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 530.947 265.474 1.655NS
Days 3 19137.252 6379.084 39.773**
Drugs x Days 6 7500.867 1250.144 7.794**
Error 24 3849.339 160.389
Total 35 31018.405
NS = Non-significant ** = Significant at P≤0.01 Table 4.125: Mean concentration of arylesterase (KU/L±SE) in heart from layers fed
was different flouroquinolones at different days after therapy of experimental period.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 193.29
±3.87a
122.06
±0.85cde
147.72
±4.71bcde
139.71
±17.83cde
150.69
±8.89
Ofloxacin 195.29
±3.82a
152.94
±1.70bcd
130.89
±5.94de
163.24
±7.64abc
160.09
±7.13
Norfloxacin 193.92
±2.87a
180.00
±5.43ab
114.71
±5.10e
135.30
±10.19cde
155.82
±10.05
a-e, similar alphabets on means do not different significantly at P≤0.01
123
4.9.4 Paraoxonase Concentration (PON1; U/L±SE) in Layers:
4.9.4.1 Paraoxonase in serum of layers:
Serum paraoxonase concentration of layer exposed to different fluoroquinolones was
subjected to analysis of variance. Drugs and days were significantly different (Table 4.126).
Mean serum paraoxonase concentration was much higher at day 0 and it decreased on day 1
after therapeutic drug level was stopped (Table 4.127)
Table 4.126: Analysis of variance of paraoxonase concentration of serum from layer
fed different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 12151.850 6075.925 9.453**
Days 3 29331.567 9777.189 15.211**
Drugs x Days 6 5915.423 985.904 1.534NS
Error 24 15426.770 642.782
Total 35 62825.609
NS = Non-significant ** = Significant at P≤0.01 Table 4.127: Mean concentration of serum paraoxonase (PON1; U/L±SE) from layers
fed different fluoroquinolones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 236.15
±6.27
148.53
±17.83
145.59
±24.62
180.46
±6.87
177.68
±12.90B
Ofloxacin 226.45
±6.27
197.06
±9.85
192.18
±7.37
263.24
±14.43
222.16
±9.78A
Norfloxacin 236.85
±5.72
175.00
±7.64
164.71
±25.47
200.00
±20.37
193.96
±11.03B
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01
124
4.9.4.2 Paraoxonase in layer muscles:
Paraoxonase from muscles of layer fed three different fluoroquinolones were
analyzed by the analysis of variance (Table 4.128). Drugs, days and their interaction did
differ significantly. Ciprofloxacin did increase (P≤0.01) muscle paraoxonase on day 4,
while norfloxacin did increase (P≤0.01) muscle paraoxonase on day1and decrease
(P≤0.01) on day 4 of experimental period (Table 4.129).
Table 4.128: Analysis of variance of paraoxonase concentration of muscles from layer
fed different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 1247.105 623.552 13.196**
Groups 3 469.754 156.585 3.314*
Drugs x Groups 6 8541.107 1423.518 30.126**
Error 24 1134.054 47.252
Total 35 11392.021
* = Significant at P≤0.05 ** = Significant at P≤0.01 Table 4.129: Mean concentration of muscle paraoxonase (PON1; U/L±SE) from layers
fed different fluoroquinolones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 79.68
±2.16bc
64.71
±0.00cd
68.27
±3.35cd
93.27
±0.49ab
76.48
±3.47B
Ofloxacin 76.82
±2.06bc
75.30
±4.07bc
66.24
±2.17cd
77.30
±3.20bc
74.63
±1.99B
Norfloxacin 75.67
±2.66bc
111.77
±8.49a
108.83
±5.10a
51.47
±5.94d
87.93
±7.80A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-d, similar alphabets on means do not different significantly at P≤0.01
125
4.9.4.3 Paraoxonase in layer liver:
Liver paraoxonase concentration was measured from layers fed different
fluoroquinolones for observing the significant differences between drugs, days and into their
interaction by statistical analysis (Table 4.130). Drug, days and their interaction were
significantly different. Ciprofloxacin did increase (P≤0.0) on day 4 of experimental period
as compared to day0, day 3 and day 4. Overall mean concentration of paraoxonase in the
liver of layer was present in the ciprofloxacin treated birds (Table 4.131).
Table 4.130: Analysis of variance of paraoxonase concentration of liver from layer fed
different fluoroquinolones
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 2527.712 1263.856 9.510**
Groups 3 3001.234 1000.411 7.528**
Drugs x Groups 6 4367.774 727.962 5.478**
Error 24 3189.524 132.897
Total 35 13086.244
** = Significant at P≤0.01 Table 4.131: Mean paraoxonase (PON1; U/L±SE) concentration of liver from layer fed
different fluoroquinolones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 151.47
±5.94bc
127.60
±6.00bc
160.30
±4.25ab
188.24
±5.10a
156.90
±6.93A
Ofloxacin 150.87
±5.33bc
144.41
±6.96bc
152.20
±2.12bc
141.77
±1.70bc
147.46
±2.44AB
Norfloxacin 151.43
±3.94bc
126.47
±13.59bc
125.00
±4.24c
142.65
±9.34bc
136.40
±5.10B
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
126
4.9.4.4 Paraoxonase in layer kidney:
Kidney paraoxonase concentration of layers was analyzed by analysis of variance
(Table 4.132). Drugs, days and their interaction were found to be significantly decreased
different. Mean kidney paraoxonase concentration did decrease on day 1 after drug
therapy of ciprofloxacin, ofloxacin and norfloxacin (Table 4.133). In ciprofloxacin,
kidney paraoxonase did increase on day 3 while norfloxacin and did increase the
paraoxonase level on day 4 of experimental period.
Table 4.132: Analysis of variance of paraoxonase concentration of kidney from layer fed different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 6089.200 3044.600 19.557**
Groups 3 22528.585 7509.528 48.238**
Drugs x Groups 6 5680.138 946.690 6.081**
Error 24 3736.223 155.676
Total 35 38034.147
** = Significant at P≤0.01 Table 4.133: Mean paraoxonase concentration (PON1; U/L±SE) of kidney from layer
fed different fluoroquinolones at different days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 136.08
±6.88a
81.83
±4.48bc
126.41
±1.73a
138.24
±13.58a
120.64
±7.68A
Ofloxacin 146.28
±5.89a
72.06
±4.27c
83.53
±2.38bc
82.06
±3.23bc
93.43
±7.78B
Norfloxacin 136.98
±6.10a
50.00
±15.28c
68.79
±1.85c
115.85
±1.44ab
92.68
±11.06B
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 abc, similar alphabets on means do not different significantly at P≤0.01
127
4.9.4.5 Paraoxonase in layer heart:
Analysis of variance of paraoxonase concentration of heart from layer fed different
fluoroquinolones did show significant differences between drugs, days and into their
interaction (Table 4.134). Mean heart paraoxonase concentration of layers did show a
significant decrease for all three fluoroquinolones under trial, however, on day 3 paraoxonase
concentration did decrease (P≤0.01) and on day 4 when these birds were treated for
ofloxacin (Table 4.135).
Table 4.134: Analysis of variance of paraoxonase concentration of heart from layer fed different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 2234.534 1117.267 20.996**
Groups 3 7003.553 2334.518 43.870**
Drugs x Groups 6 1419.126 236.521 4.445**
Error 24 1277.138 53.214
Total 35 11934.352
** = Significant at P≤0.01 Table 4.135: Mean heart paraoxonase concentration (PON1; U/L±SE) of layers fed
different fluoroquinolones at different days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 118.47
±3.60ab
83.00
±2.75cd
103.97
±4.84abc
125.00
±7.64a
107.61
±5.32A
Ofloxacin 121.40
±3.60ab
70.00
±1.36d
98.53
±2.87bc
90.71
±2.31cd
94.43
±5.36B
Norfloxacin 115.76
±3.60ab
92.97
±1.90c
118.24
±5.10ab
123.23
±6.28acd
113.23
±4.06A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.01 a-d, similar alphabets on means do not different significantly at P≤0.01
128
4.9.5 Catalase Concentration (KU/L±SE) in Layers:
4.9.5.1 Catalase in serum of layers:
Serum catalase concentration from layers fed different fluoroquinolones was analyzed
by the analysis of variance (Table 4.136). Mean serum catalase concentration of layers fed
ciprofloxacin, ofloxacin and norfloxacin at different days has been given in table 4.137.
Table 4.136: Analysis of variance of catalase concentration in serum from layers fed
different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 923.805 461.903 2.309NS
Groups 3 1464.481 488.160 2.441NS
Drugs x Groups 6 1491.649 248.608 1.243NS
Error 24 4800.292 200.012
Total 35 8680.227
NS = Non-significant
Table 4.137: Mean concentration of serum catalase (KU/L±SE) from layers fed
different fluoroquinolones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 244.62
±10.92
237.46
±4.14
246.41
±13.32
241.40
±4.45
242.47
±4.29
Ofloxacin 240.82
±11.92
232.95
±2.50
216.45
±1.53
246.58
±1.88
235.15
±4.61
Norfloxacin 254.22
±14.92
217.95
±5.55
227.67
±5.19
230.30
±3.84
230.14
±4.38
129
4.9.5.2 Catalase in layer muscle:
Muscle catalase concentration of layers fed different fluoroquinolones at various
days did show the drug and drugs × days did not show any significant difference (Table
4.138). Mean catalase in muscles did decrease on day 1 after fluoroquinolones treatment and
then almost similar on day 3 and 4 of experimental period (Table 4.139).
Table 4.138: Analysis of variance of catalase concentration of muscles from layers fed
different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 1365.440 682.720 1.672NS
Groups 3 6806.079 2268.693 5.557**
Drugs x Groups 6 2502.753 417.125 1.022NS
Error 24 9798.911 408.288
Total 35 20473.182
NS = Non-significant ** = Significant at P≤0.01 Table 4.139: Mean muscle catalase concentration (KU/L±SE) of layers fed different
fluoroquinolones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 279.13
±5.86
263.25
±20.37
269.31
±10.68
260.42
±6.78
268.02
±5.69
Ofloxacin 270.53
±5.06
241.66
±12.51
262.45
±15.31
232.31
±2.76
253.88
±7.06
Norfloxacin 287.43
±5.76
224.62
±5.18
262.45
±15.98
259.42
±16.57
256.40
±7.91
130
4.9.5.3 Catalase in layer liver:
Days and drugs×days interaction for catalase concentration of liver was significantly
different as determined by the statistical analysis (Table 4.140). Mean catalase concentration
of liver from ciprofloxacin fed layers did decrease significantly on day1 and than increased
on day 4 of sampling. Ofloxacin fed birds did decrease (P≤0.05) drugs liver catalase
concentration on day 3 and than it did increase significantly on day 4 of sampling. On the
other hand norfloxacin fed layer did decrease (P≤0.05) catalase concentration on day 1
increased P≤0.05 on day 3and then decrease P≤0.05 on day 4 of sampling period (Table
4.141).
Table 4.140: Analysis of variance of catalase concentration of liver from layers fed different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 1478.898 739.449 1.767NS
Groups 3 4369.744 1456.581 3.480*
Drugs x Groups 6 8042.215 1340.369 3.202*
Error 24 10045.867 418.578
Total 35 23936.725
NS = Non-significant * = Significant at P≤0.05 Table 4.141: Mean concentration of catalase (KU/L±SE) from liver of layers fed
different fluoroquinolones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 270.57
±9.07aba
230.44
±18.39c
233.60
±9.20c
257.09
±16.57ab
247.92
±7.83
Ofloxacin 272.07
±8.78aba
268.39
±2.18ab
232.01
±10.12c
282.31
±0.33a
263.32
±6.45
Norfloxacin 270.67
±9.80aba
228.39
±9.20c
274.77
±13.22ab
238.11
±17.82
252.96
±8.20
abc, similar alphabets on means do not different significantly at P≤0.01
131
4.9.5.4 Catalase in layer kidney:
Analysis of variance of catalase concentration of kidney from layers fed different
fluoroquinolones by statistical analysis (Table 4.142). Drugs were found to be significantly
different in this study. Mean kidney catalase concentration of layer has been presented in
table 4.143. Ciprofloxacin and ofloxacin did decrease the kidney. Catalase concentration of
layer on day 1 of experimental condition, however, these differences were not significantly
different.
Table 4.142: Analysis of variance of catalase concentration of kidney from layers fed
different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 2675.790 1337.895 3.483*
Groups 3 2049.385 683.128 1.779NS
Drugs x Groups 6 3079.626 513.271 1.336NS
Error 24 9217.801 384.075
Total 35 17022.602
NS = Non-significant * = Significant at P≤0.05 Table 4.143: Mean concentration (KU/L±SE) of catalase from kidney of layers fed
different fluoroquinolones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 249.85
±14.73
222.45
±7.03
236.58
±7.66
252.45
±7.70
240.33
±5.52B
Ofloxacin 245.50
±12.71
235.35
±3.26
232.31
±2.85
230.29
±8.95
236.95
±4.44B
Norfloxacin 242.80
±15.37
258.83
±5.18
238.54
±8.87
279.55
±22.67
256.69
±7.65A
AB, similar alphabets on overall means in a column do not differ significantly at P≤0.05
132
4.9.5.5 Catalase in layer heart:
Analysis of variance of catalase concentration from heart of layers did show
significant difference between days and drugs×days interaction in the present study (Table
4.144). Catalase concentration did decrease on day 4 in ciprofloxacin treated groups while in
norfloxacin group, catalase concentration did decrease (P≤0.01) on day 1 and then increased
(P≤0.01) on day 4 after treatment (Table 4.145).
Table 4.144: Analysis of variance of catalase concentration of heart from layers fed different fluoroquinolones.
Source of Variations
Degree of Freedom
Sum of Squares
Means Squares F-Value
Drugs 2 204.996 102.498 0.497NS
Groups 3 17154.186 5718.062 27.701**
Drugs x Groups 6 14735.966 2455.994 11.898**
Error 24 4954.127 206.422
Total 35 37049.275
NS = Non-significant ** = Significant at P≤0.01 Table 4.145: Mean concentration of catalase (KU/L±SE) from heart of layers fed
different fluoroquinolones at various days after therapy.
Drugs Day 0 Day 1 Day 3 Day 4 Overall Mean
Ciprofloxacin 262.89
±8.37bc
245.34
±0.58c
235.20
±7.61cd
299.70
±3.68ab
260.78
±7.63
Ofloxacin 251.99
±8.57bc
253.10
±8.41c
258.75
±8.24bc
247.60
±1.30c
255.58
±3.55
Norfloxacin 267.88
±9.70bc
200.86
±1.84d
235.93
±5.52cd
323.78
±19.23a
255.86
±14.33
abc, similar alphabets on means do not different significantly at P≤0.01
133
4.10 Concentration of Fluoroquinolones in Layers before and after
Cooking
4.10.1 In muscle of layer:
Analysis of variance concentration of different fluoroquinolones did reveal a
significant difference between cooking methods, drugs, days as well as in their interaction in
the present study (Table 4.146). Mean concentration of different fluoroquinolones in the
muscles of layers before cooking did show a significant decrease on day 1, day 3 as well as
day 4 (Table 4.147).
Table 4.146: Analysis of variance fluoroquinolones concentration of muscle of layer before and after cooking
Source of variations Degree of Freedom
Sum of Squares
Means Squares
F-Value
Groups 1 0.608 0.608 262.103**
Drugs 2 0.117 0.059 25.310**
Days 3 1.407 0.469 202.298**
Groups x Drugs 2 0.099 0.050 21.438**
Groups x Days 3 1.012 0.337 145.442**
Drug x Days 6 0.152 0.025 10.931**
Groups x Drug x Days 6 0.141 0.024 10.164**
Error 72 0.167 0.002
Total 95 3.703
** = Significant at P≤0.01 Table 4.147: Mean muscle concentration (ppm±SE) of different fluoroquinolones of
layer before and after cooking
Drugs Before Cooking After Cooking
Day 0 Day 1 Day 3 Day 4 Day 0 Day 1 Day 3 Day 4
Ciprofloxacin 0.00
±0.00f
0.49
±0.07b
0.13
±0.03d
0.00
±0.00f
0.00
±0.00f
0.10
±0.00d
0.00
±0.00f
0.00
±0.00f
Ofloxacin 0.00
±0.00f
0.35
±0.03bc
0.50
±0.03b
0.00
±0.00f
0.00
±0.00f
0.10
±0.00d
0.00
±0.00f
0.00
±0.00f
Norfloxacin 0.00
±0.00f
0.80
±0.07a
0.22
±0.05c
0.02
±0.004ef
0.00
±0.00f
0.03
±0.005e
0.01
±0.003ef
0.00
±0.00f
a-f, similar alphabets on means do not different significantly at P≤0.01
134
4.10.2. In Liver of layer:
Samples of liver that was positive for different fluoroquinolones were subjected to
cooking and their residual concentration between groups, drugs, days and in their interaction
were significantly different. Mean concentration of fluoroquinolones before cooking and
after cooking at various days of experimental period has been given in table 4.148. It has
been observed that cooking did decrease the concentration of individual groups and in their
own group, significantly (P≤0.01) in the liver of layers during the experimental period (Table
4.149).
Table 4.148: Analysis of variance fluoroquinolones concentration of layers liver before and after cooking
Source of variations Degree of Freedom
Sum of Squares
Means Squares
F-Value
Groups 1 5.020 5.020 163.849**
Drugs 2 6.571 3.285 107.234**
Days 3 7.715 2.572 83.943**
Groups x Drugs 2 6.655 3.328 108.612**
Groups x Days 3 6.986 2.329 76.009**
Drug x Days 6 8.182 1.364 44.510**
Groups x Drug x Days 6 8.351 1.392 45.431**
Error 72 2.206 0.031
Total 95 51.686
** = Significant at P≤0.01 Table 4.149: Mean liver concentration (ppm± SE) of different fluoroquinolones of
layer before and after cooking
Drugs Before Cooking After Cooking
Day 0 Day 1 Day 3 Day 4 Day 0 Day 1 Day 3 Day 4
Ciprofloxacin 0.00
±0.00e
0.72
±0.17c
0.04
±0.008d
0.00
±0.00e
0.00
±0.00e
0.02
±0.007d
0.00
±0.00e
0.00
±0.00e
Ofloxacin 0.00
±0.00e
0.03
±0.005d
0.00
±0.00e
0.00
±0.00e
0.00
±0.00e
0.06
±0.005d
0.00
±0.00e
0.00
±0.00e
Norfloxacin 0.00
±0.00e
3.32
±0.33a
1.48
±0.22b
0.01
±0.005e
0.00
±0.00e
0.03
±0.002d
0.00
±0.00e
0.00
±0.00e
a-e, similar alphabets on means do not different significantly at P≤0.01
135
4.11 Concentration of Fluoroquinolones after Cooking in Electric and
Microwave Ovens
4.11.1. In muscle of layers:
Residue containing muscles samples from layers were cooked in electric oven and
microwave oven for further analysis to assess the effect of cooking methods on the
availability of concentration of residue in these samples. Cooking methods, drugs, days as
well as their subsequent interaction did show a significant difference (Table 4.150). Mean
concentration of fluoroquinolones in the muscles of layers did show a significant difference
between oven and microwave cooking in ciprofloxacin group having low value (Table 4.51).
Table 4.150: Analysis of variance fluoroquinolones concentration of layers muscles after two methods of cooking
Source of variations Degree of Freedom
Sum of Squares
Means Squares
F-Value
Methods 1 0.000 0.000 2.000NS
Drugs 2 0.008 0.004 1352.000**
Days 3 0.025 0.008 3018.667**
Methods x Drugs 2 0.000 0.000 2.000NS
Methods x Days 3 0.000 0.000 12.667**
Drug x Days 6 0.024 0.004 1458.667**
Methods x Drug x Days 6 0.000 0.000 12.667**
Error 48 0.000 0.000
Total 71 0.057
** = Significant at P≤0.01 Table 4.151: Mean concentration of fluoroquinolones in the muscle layer two
different methods of cooking at various days
Drugs Oven Microwave
Day 0 Day 1 Day 3 Day 4 Day 0 Day 1 Day 3 Day 4
Ciprofloxacin 0.00
±0.00c
0.10
±0.00a
0.00
±0.00c
0.00
±0.00c
0.00
±0.00c
0.01
±0.00b
0.00
±0.00c
0.00
±0.00c
Ofloxacin 0.00
±0.00c
0.00
±0.00c
0.00
±0.00c
0.00
±0.00c
0.00
±0.00c
0.00
±0.00c
0.00
±0.00c
0.00
±0.00c
Norfloxacin 0.00
±0.00c
0.03
±0.003b
0.02
±0.003b
0.00
±0.00c
0.00
±0.00c
0.04
±0.00b
0.01
±0.00bc
0.00
±0.00c a-c, similar alphabets on means do not different significantly at P≤0.01
136
4.11.2 In Liver of layers:
Analysis of variance of concentration of different fluoroquinolones in the liver of
layers by two different cooking methods did show a significant different between cooking
methods, drugs, days and in their interaction (Table 4.152). Oven and microwave cooking
methods did reveal a significant decrease in the concentration of fluoroquinolones in the
livers of layers at different days of experimental period (Table 4.153).
Table 4.152: Analysis of variance fluoroquinolones concentration of layers liver after two methods of cooking
Source of variations Degree of Freedom
Sum of Squares
Means Squares
F-Value
Methods 1 0.000 0.000 1.471NS
Drugs 2 0.001 0.000 16.412**
Days 3 0.014 0.005 191.118**
Methods x Drugs 2 0.000 0.000 0.412NS
Methods x Days 3 0.000 0.000 1.471NS
Drug x Days 6 0.002 0.000 16.412**
Methods x Drug x Days 6 0.000 0.000 0.412NS
Error 48 0.001 0.000
Total 71 0.018
** = Significant at P≤0.01 Table 4.153: Mean concentration of fluoroquinolones in the liver layer two different
methods of cooking at various days
Drugs Oven Microwave
Day 0 Day 1 Day 3 Day 4 Day 0 Day 1 Day 3 Day 4
Ciprofloxacin 0.00
±0.00
0.02
±0.01
0.00
±0.00
0.00
±0.00
0.00
±0.00
0.02
±0.00
0.00
±0.00
0.00
±0.00
Ofloxacin 0.00
±0.00
0.05
±0.007
0.00
±0.00
0.00
±0.00
0.00
±0.00
0.05
±0.007
0.00
±0.00
0.00
±0.00
Norfloxacin 0.00
±0.00
0.03
±0.00
0.00
±0.00
0.00
±0.00
0.00
±0.00
0.02
±0.00
0.00
±0.00
0.00
±0.00
137
Chapter 5
DISCUSSION
In developing countries, particularly in Pakistan, commercial system of chicken
production is dependent upon improved breeds of layers and broilers. Poultry production in
Pakistan is increasing due to high demand for eggs and meat, however, per capita
consumption of chicken meat in Pakistan is still very low. Commercial chicken rose for food
in Pakistan, depending heavily on the use of pharmacologically active compounds. The use
of drugs in food animals is fundamental to animal health and well being as well as important
in term of economics of the industry. However, use of drugs is also associated with human
health effects. Antibiotics in poultry industry are used for prevention and treatment of disease
as well as to increase efficiency of use of feed by chicken for growth, product outputs, and
for increased production performance by modifying or reducing that portion of nutrition
requirement with fighting subclinical diseases and enhancing health defence processes.
The drugs fed to the birds for treatment and prevention of diseases are not related to
their mechanism of action as drugs concern over a wide spectrum use of antibiotics is too
permissive that food production is in Jeopardy if drugs use in chicken is restricted. Most of
the questions raised before remain mostly unanswerable because of the problems associated
with the valid information collection, data, experimental design and incomplete studies that
are impossible to control and forces scientist to stay away rather to address the future
prospective of this issue.
To overcome the high level of stress, poor nutrition and disease onset, farmer uses
excessively antibiotics which might result as drug residue in meat and egg of chicken.
Because of lack of Government Control to sell antimicrobials or other drugs in the market, as
well as lack of interest in the country, farmers do buy any antibiotics without consultation of
veterinary doctors and treat birds by themselves. Mostly farmer are not knowledgeable and
non-professional therefore correct drugs as well as choice of particular drug are unlikely to
be observed. Above all their knowledge of withdrawal time of drugs from meat and egg is
also poor, thus continuous misuse of different drugs causing a potential hazard to human
health.
138
In the present study experimental work was conducted into different phases, like first
phase designated for surveys of the antibiotics in meat, liver, heart of broilers and also in
meat, liver, heart and eggs of layers from different areas of Faisalabad and was spread over a
year. In Second phase, antibiotics (fluoroquionolones) were fed in therapeutic doses to
broilers and layers and their product were analyzed for drug residues by the most sensitive
method of high performance liquid chromatography with fluorescence detection. This phase
included the washout time of a residue in the final products. During third phase, effect of
cooking (by two different methods; electric oven and microwave oven) on the residual
concentration of different fluoroquinolones, was determined. Results of all studies are
presented in each section.
Surveys
Recently, use of antibiotics in food producing animals has attained a very important
public health issue as stated by Jafari et al. (2007). High rate of residue prevalence in
different farms in and around Faisalabad may be due to fact that antibiotics are increasingly
used on poultry farm to promote chicken growth and help in the improvement in
productivity. Gustafson and Bowen (1997) observed similar findings when raising chickens
under intensive husbandry methods of production. Soggard (1973) and Roberts (1996)
indicated that microbial residue to antibiotics and possible their transfer to human pathogens.
In addition, Linton (1977) did report that human exposure to animal products
containing significant level of antibiotic residue may alter immunological response in
susceptible individuals and in turn causes disorder of flora of intestine. World Health
Organization (WHO) and Food and Agriculture Organization (FAO) has suggested the
Maximum Residue Limits (MRLs) acceptable for daily intake for human and for withholding
times for different antimicrobial drugs before they are sold in the market.
Overall surveys of antibiotics did show that our farmers are using different
antimicrobial agent and this practice did reveals a significant number of positive samples,
particularly meat of broilers, layers and liver of both chicken for drug residue. There were
higher concentration of antimicrobial agents in milk, meat and egg as reported by Mmbando
(2004), Karimuribo et al. (2005), Kurwijila et al. (2006) and Simon (2007).
139
As use of antibiotics in the feed of chicken and/or for treatment went along with non-
compliance to withdrawal period while even though it is known that a significant number of
farmers are knowledgeable on the withdrawal period. However, in reality, none of them
comply with the recommendion of the use and drug withdrawal period. The non-compliance
of the farmer could be related to their fear of losses, losing subsistence from the government
and may be due to more losses. Lack of awareness of farmers towards the knowledge of
withdrawal period for antibiotics can be visualized as they themselves are using the same
chicken for their family that was under treatment or being fed antimicrobials or other drugs at
that time. Human exposure to chicken meat and eggs containing a reasonable concentration
of antibiotics residue could alter immunological profiles and causes disorders of intestinal
flora in human.
Residue and Washout Time for Fluroquinolones
Following oral administration of ofloxacin in chicken, the mean residual time was
reported to be 7.43 h (Kalaiselvi et al., 2006) on the other hand; MRL is much higher for
norfloxacin (Laczay et al., 1998) and ciprofloxacin (Anadon et al. 2001) in chickens. In our
study fluroquinolone residues were significantly high on day one after therapy and did
significantly decrease on day 3 in muscles. Reyes-Herrera et al. (2005) indicated that at least
for enrofloxacin concentration that not all muscle tissues did incorporate residues at the same
concentration. The FDA has established the muscle as target tissue for drug residue
monitoring particularly in turkeys and chickens. Reyes-Herrera et al. (2005) reported that
enrofloxacin concentration was much higher in breast tissue than thigh tissue and its
dependent on the dose regime during treatment. It could be speculated that possibilities of
having the presence of different residual concentration in wing, breast and leg muscles may
depends on age, seasons sex and the availability of water and nutrients in the feed. Similarly,
Schneider and Donoghue (2004) reported that in pooled muscles, norfloxacin concentration
was high in breast versus thigh muscle tissue from treated group.
Norfloxacin concentration was significantly high in tissue in the beginning and then
decreased overtime, however, in skin and fat, residue of this drug was present even a day 10
after the last dose (Pant et al., 2005).
140
Amjad et al. (2006) did observed the effects of enrofloxacin and ciprofloxacin on
liver and kidney of chicken during summer and detected 100 percent deviation from the
internationally accepted MRL’s and recommended that these organs require longer washing
out time and may be dose sophistication. They also suggested that range sizes of the
antibiotics residue depend mostly on the water intake, its metallic contents, nature, and pH of
water as well as on feed. The higher concentration of residues of ciprofloxacin and
norfloxacin in the chicken meat in the present study could be due to dosing time (5 days),
decreased renal secretion of lipophilic drugs (Yorke and Froc, 2000). It also demonstrates
that higher concentration of residues in liver and kidney may also be due to the chemical
properties (basic nature) unfavorable pH at kidney and its elimination that have to be in
ionized form. If liver, kidney and muscle of the chicken have to be considered as a single
unit, then the washing out time should be increased along with some antioxidant which
would render the kidney and muscles to work in a way to increase the secretion, while
changing the pH medium for norfloxacin, ciprofloxacin and enrofloxacin
Eggs
In the present study, fluroquinolone in eggs (albumen plus yolk) collected after 48
hours of drug withdrawl did show drug residues, which were much lower than that of
muscles, liver and kidney concentrations. Brown (1996) and Toutain et al. (2002) suggested
that fluroquinolone get in concentration-dependent manner, and its plasma concentration is
maximum initially to minimize the development of bacterial resistance. Huang et al. (2006)
detected enrofloxacin and ciprofloxacin concentration that reached high in whole egg and
albumen in 2 days after the start with the treatment. In the present study, enrofloxacin
concentration was high as compared to ciprofloxacin and norfloxacin in the whole egg.
Similar observation was reported by Huang et al. (2006) for whole eggs. However, they
further observed that enrofloxacin was higher in egg albumen than in egg yolk, whereas,
opposite was true for ciprofloxacin residues. In the present study, all fluroquinolone were
tested for validation purpose to recover the known standard by adding them in whole eggs
during spiking. Recoveries were found to be 85 to 103 percent with relative standard
deviation (RSD’s) of 1-8 %. Herranz et al. (2007) concluded enrofloxacin residue was high
on day 2 after the beginning with the treatment while highest concentration was detected two
141
days after the last drug administration. They also observed that ciprofloxacin concentration
was always lower than those of enrofloxacin as measured by LC-MS technique. Chu et al.
(2000) determined fluroquinolone in egg albumen and yolk by fluorometric method and
reported that they analyzed 27 eggs and none was found to be positive for residue. They
concluded that use of fluroquinolone in laying hens is not widespread. It is important to know
that water-soluble proteins of albumen are formed by the magnum protein of the oviduct
within first 1-2 days of egg production while the lipoproteins of the yolk portion of the egg is
synthesized by the liver, therefore, for a drug residue, to it requires generally 8-10 days to
reach the maximum level (Kan and Petz, 2000).
Oxidants and Antioxidants
Tissue damage in response to stress or by chemical or nonchemical agents attributed
by oxidative stress by formation of reactive oxygen species (ROS) that contribute to
pathogenesis in many diseases (Camkerten et al., 2009 and Dimri et al., 2010). Oxidative
stress damage can be determined by the intensity of product of ROS. Lipid peroxidation is
one of the known parameters to determine the status of oxidative mechanism. Body in a
homeostatic way keeps the balance between the oxidative and antioxidative systems for a
normal biological mechanism of an organ or any system within the body. Glutathione (GSH)
is an endogenous-derived peptide produced by liver and act as an antioxidant to protect host
cells against ROS. This notion is also supported by Akkas (1995); Kurt et al. (2002) and
Amanvermez and Celik (2004).
In the present study, enhanced oxidative status and reduced antioxidant capacity were
observed on day 1 to 3 after fluroquinolone treated. On day 4, after treatment, antioxidant
capacity becomes almost normal by reducing the oxidative status of muscles. Glutathione can
effectively neutralize free radicals either directly or indirectly through various enzymes
activation (Fang et al., 2002). Fluroquinolone and particularly ciprofloxacin are associated
either adverse effect pertaining to gastrointestinal, skin, hepatic and central nervous system
functions (Hopper and Wolfson, 1985).It has also been reported that oxidative stress also
play a significant role in the pathogenesis of Fo’s induced cartilage defects ( Hayem et al.,
1994). Gurbay et al. (2001) observed that free radical production was induced by
ciprofloxacin in dose and time dependant manner. If oxidative stress stays for much longer
142
time may indicate that oxidative stress may involve a long lasting chain. A similar
mechanism has been reported by Barclay and Ingold (1981) suggesting that a free radical
chain process that involve formation as well as propagation is responsible for a long lasting
radical stability. Gurbay et al. (2001) also explained that formation of free radicals by
ciprofloxacin is in the microsomal system. Enrofloxacin areoxidized by liver mirosomal
enzymes of cytochrome P- 450 family and can cause oxidative stress (Carreras et al., 2004).
Benzer et al. (2009) did indicate that catalase activity decreased in antibiotic treated groups.
In the present study, on day 01 catalase activity decrease and than increased on day 03 after
therapy. Thus indicating a role of antioxidant for fluroquinolone particularly involving
catalase. Using enterocyte in a study by Benzer et al. (2009) did indicate that increasing lipid
peroxidation decreases a live cells and it was attributed to the phenomena of balance between
oxidant and antioxidant (Gorowara et al., 1998). Therefore, for catalase activity in organs, it
could be an indicator of an increase followed by a decrease in oxidative status in parallel to
decrease followed by an increase catalase activity in muscles, liver, kidney and heart. Similar
argument was indicated by Altinorduly and Eraslan (2009) that ciprofloxacin, norfloxacin
and enrofloxacin generated oxidative stress physiologically when given a therapeutic dosage.
Residues after Cooking
In the present study residues of fluoroquinolone in a raw meat and liver using HPLC
fluorescent technique were elucidated during the residues wash time for individual drugs
under study. There are maximal residual limits for fluroquinolone which are legally
permitted (Table 2.1) in food under various developed countries for various organs. The raw
food in our culture is mostly cooked before we eat them. Residual effects after cooking or by
various mechanisms to resolve this issue is scanty. Few studies have been reported to
determine the residues in food after cooking or may have established how heat or other
treatment could have affected the stability of residues in cooked food (Rose, 1995a, 1995b,
1995c, 1996, 1997a, 1997b, 1997c, 1998a, 1999 and Moats, 1999)
In the present study, during residual washout study, the raw muscles and liver who
was positive and showing residue on day 1, 3 and 4 were cooked by oven and microwave
method, and then their residues were measured by the same techniques. It was observed that
on both types of cooking, a reasonable but significant decrease in residues occurs. These
143
results also indicate the residues were almost to a negligible amount after both types of
cooking methods. Electric and microwave methods might have inactivated the drugs thus
showing lost amount of analyte from tissues exudates. However, roasting and grilling did
increase the amount of residues, particularly for enrofloxacin. It was concluded that residues
data from raw tissue is a valid estimation of residual volume. Inglis and Katz (1978) and
O’Brien et al. (1980) studied the streptomycin in raw, boiled and fried egg that did not alter
the residual volume however; on the other hand, oxytetracycline concentration was reduced
but not significantly after roasting and frying of egg. In addition, there are many drugs like
sulphamethazine (Rose et al., 1995c), oxytetracycline (Rose et al., 1996) and ivermectin
(Rose et al., 1998a) which remained stable after cooking. It is worthy speculating that animal
products which contain antibiotic residues above MRL may alter immunological responses
and causes disorders of intestinal microflora (Linton, 1977; Holmberg et al., 1984 and
Woodward, 1991). Until now MRL values are yet to be fixed for antibiotic in this country.
Conclusion
Present study indicates that there is a wide spread use of antibiotics in the feed as well
as for treatment. Birds treated or fed with fluoroquinolones must be given a washout time of
two days. Antioxidants capacity did show a decrease thus indicating deterioration in the
quality of meat. Our results also indicate that a decrease in antioxidant status of meat and
organs may be due to utilization of antioxidants enzymes in the removal of ROS from meat.
It will be interesting to examine changes in antioxidants enzymes at the mRNA level that
may correlate with oxidative stress induced and enhanced by the presence and concentration
of antibiotics alone or in combination with other drugs. It could also be speculated that
results could have been different based on the organtype as well as on oxidative stress level.
Cooking of edible portion of meat before it is used for eating is strogly recommended.
144
Chapter 6
SUMMARY
Present study was conducted on broilers and layers on three phases:
During phase I, surveillance study from different areas of Faisalabad was executed.
Mean frequency percentage for surveillance data was calculated. Broiler leg muscle (30%),
breast muscle (32%), liver (99%) and heart (85%) were found to be positive for drug residue.
The inhibition zone as measured by microbiological assay did show zone of inhibition which
ranged from 2.56-9.52 mm for leg muscle, 2.5-7.72 mm for breast muscle, 9.96-14.77 for
liver and 5.56-10.04 mm for heart muscle. During different months, residues were
significantly high during the month of August. In layers, leg muscles (38%), breast muscle
(40%), liver (100%), heart (72%), egg white (72%) and egg yolk (19%) were positive for
residues. Inhibition zone in different organs ranged from 3.14 mm in egg yolk to 16.57 in
liver. Significantly high percentage of residues was determined during the month of August
and September except in egg yolk (March and April).
During phase II of this experiment, washout time for fluoroquinolones was
conducted in broilers and layers by measuring the residue in muscles on day 1, 3 and 4 after
therapy. The data was analyzed by two way analysis of variance for comparing the
fluoroquinolones residue at various time intervals. In case of significant difference Duncan
Multiple Range Test was applied. For Cooking methods, three way Analysis of Variance was
applied and then DMR. In broilers and layers, after therapy, ciprofloxacin, norfloxacin and
ofloxacin did show a significant amount of residues in muscles, liver and heart while eggs
did show residues that are much lower in concentration than edible tissue. All experimental
drugs were below MRL’s limits on day 3, however, liver did take more time to clear the
residues (day 4). Residues concentration was significantly high in broilers as compared to
layers. Serum was also obtained during this phase of experiment for biological markers. A
significant increase on day 1 in ROS in serum and muscles was determined after
fluoroquinolones treatment. Antioxidant capacity did decrease significantly in serum and
muscles indicating determination in the quality of meat.
145
In phase III of this experiment, muscles and liver which were positive for their
residues on different days of the washout time were subjected to two different cooking
methods. On each experimental day, muscle and liver residues concentration did decrease
significantly with microwave and electric oven cooked samples, and were below the MRL’s
value.
.
RECOMMENDATIONS
This study is comprehensive but on a small scale to provide the monitoring data and
most accurate assessment of the safety for using poultry products in Pakistan.
Drug residues particularly of antibiotics/pesticides must be monitored in poultry products
regularly by using state of the art techniques.
Residual effects using all organs (liver, kidney, heart and different muscles)
considering them as a single unite must be considered for washing out period to
satisfactory limits.
Washing out time for each drug must be determined in edible parts of chicken
separately.
Residual effects in broiler, layers, and their eggs must be ensured during different
seasons, considering their therapeutic dose and days of treatment as well as type of
feed, and water quality.
Along with antibiotics, antioxidant and other Ethano- pharmacological treatments
may be tried to eliminate the drug faster from muscle, heart, liver and kidneys to
decrease the oxidative stress from the organs.
Different cooking methods or treatments may be tried to make the residue in the
poultry product eatable tissue by eliminated and/or inactivated the residue.
146
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