This manual has been developed as a comprehensive reference for students, researchers, and professionals engaged in the field of animal nutrition and feed science. It is primarily intended for post-graduate and doctoral students of Animal Nutrition and allied disciplines, but will also be useful to undergraduates seeking practical exposure to laboratory methods. The book provides standardized protocols, stepwise procedures, and interpretation guidelines for a wide range of techniques, from the preparation of standard solutions and proximate analysis to advanced methods, such as in vitro and in sacco digestibility, energy estimation, and blood metabolite profiling. Detailed chapters on the use of modern analytical instruments— including GC, HPLC, AAS, ICP, NIR, and amino acid analyzers equip readers with the knowledge required for contemporary nutritional research. Researchers working in animal nutrition laboratories, feed testing facilities, and livestock research institutions will find the manual valuable for ensuring accuracy, reproducibility, and reliability of data. Academicians and instructors can use it as a structured guide for teaching laboratory courses, while feed industry professionals and quality control specialists may rely on it for routine analysis and standardization. In addition, selected sections on tracer methods, methane emission measurement using the SF6 technique, and the Rumen Simulation Technique (RUSITEC) make this manual relevant for addressing current challenges in sustainable livestock production and environmental research. By integrating fundamental principles with practical applications, this manual bridges the gap between classroom learning and laboratory practice. It is designed to serve as a dependable tool for academic training, research excellence, and professional advancement in the domain of animal nutrition.
UNIT - 1
1 Principles of Animal Experimentation ................................................3
a) Ethical considerations .........................................................................3
b) Experimental design principles ...........................................................3
c) Animal welfare and management ........................................................4
d) Data recording and reporting ..............................................................4
2 Common Statistical Tools for Nutritional Research ..........................7
a) Descriptive statistics in animal nutrition .............................................7
b) Inferential statistics .............................................................................9
c) Regression and correlation ..................................................................9
d) Non-parametric tests .........................................................................10
e) Experimental designs commonly used in nutrition research.............10
f) Statistical software commonly used ..................................................13
UNIT - 2
1 Preparation of Standard Solutions ....................................................17
1. Introduction .......................................................................................17
2. Types of standards .............................................................................17
3. Units and description .........................................................................18
4. Standard solutions and their preparation ...........................................18
2 Proximate Analysis of Feeds and Fodders .......................................23
1. Introduction .......................................................................................23
2. Components of Proximate Analysis ..................................................23
3. Principle and procedures ...................................................................23
3 Fiber Analysis Using Van Soest Method (Detergent System) ..........35
Neutral detergent fiber (NDF) ...............................................................39
Acid detergent fiber (ADF) ...................................................................39
Acid detergent lignin (ADL) .................................................................39
4 Use of Markers in Digestibility Studies .............................................41
1. Internal markers (naturally present in feeds).....................................41
2. External markers (added to feeds or dosed to animals) ....................41
5 In vitro and In Sacco Digestibility Techniques ..................................45
Introduction ...........................................................................................45
Procedure (Tilley and Terry method) ....................................................45
In sacco digestibility technique (nylon bag technique) .........................46
Comparative advantages and limitations ..............................................47
Actual rumen environment, reflects in vivo conditions. ........................47
Variation due to animal and rumen conditions. .....................................47
Provides detailed degradation kinetics. .................................................47
Precautions ............................................................................................47
6 Determination of Energy Content Using Bomb Calorimeter ..........49
1. Introduction .......................................................................................49
2. Apparatus ...........................................................................................50
3. Procedure (Generalized) ....................................................................50
4. Applications in Animal Nutrition ......................................................51
5. Precautions ........................................................................................51
6. Limitations ........................................................................................52
7. Typical Energy Values (approximate, dry matter basis) ....................52
7 Determination of Blood Metabolic Profile ........................................53
1. Introduction .......................................................................................53
2. Objectives ..........................................................................................53
3. Common Blood Metabolites Analyzed .............................................53
4. Blood Collection and Handling .........................................................54
5. Analytical Methods ...........................................................................54
6. Interpretation of Blood Parameters ...................................................54
7. Applications in Research and Practice ..............................................54
8. Precautions ........................................................................................55
9. References .........................................................................................55
8 Principles and Applications of Analytical Instruments ...................57
8.1. Gas Chromatography (GC) ............................................................57
1. Introduction .......................................................................................57
2. Basic Principle of Gas Chromatography ...........................................57
8.2. High-Performance Liquid Chromatography (HPLC) ...................68
1. Introduction .......................................................................................68
2. Principle of HPLC .............................................................................68
3. Basic Components of HPLC .............................................................68
4. Types of HPLC Techniques ...............................................................68
5. Common HPLC Detectors in Nutrition Studies ................................69
6. Sample Preparation in HPLC ............................................................69
7. Applications of HPLC in Nutritional Studies ....................................69
8. Example Practical Protocols .............................................................70
9. Advantages of HPLC in Nutritional Studies .....................................71
10. Limitations ......................................................................................71
8.3. Atomic Absorption Spectroscopy (AAS) ......................................72
1. Introduction .......................................................................................72
2. Principle of Atomic Absorption Spectroscopy ..................................72
3. Instrumentation of AAS ....................................................................73
4. Sample Preparation in Animal Nutrition Studies ..............................74
5. Applications of AAS in Animal Nutrition .........................................76
6. Advantages of AAS ...........................................................................77
7. Limitations ........................................................................................77
8. Safety and Quality Control Tips ........................................................77
9. Practical Exercise for Students ..........................................................77
8.4 Inductively Coupled Plasma (ICP) ..................................................81
1. Introduction .......................................................................................81
2. Principle of ICP .................................................................................81
3. Components of an ICP System ..........................................................82
4. Sample Preparation in Animal Nutrition Studies ..............................82
5. Applications in Animal Nutrition ......................................................82
6. Advantages of ICP Techniques ..........................................................83
7. Limitations ........................................................................................83
8. Summary for Practical Use ..............................................................84
8.5. Tracer Techniques (Isotopic and Non-Isotopic) ...........................84
1. Introduction .......................................................................................84
2. Isotopic Tracer Techniques ................................................................85
3. Non-Isotopic Tracer Techniques .......................................................86
4. Advantages and Limitations ..............................................................88
8.6 Flame Photometry ...........................................................................89
1. Introduction .......................................................................................89
2. Principle of flame photometry ...........................................................89
3. Emission wavelengths of common elements ....................................90
4. Instrumentation .................................................................................90
5. Applications in animal nutrition ........................................................91
6. Sample preparation and analytical procedure ...................................92
7. Advantages of flame photometry ......................................................93
8. Limitations ........................................................................................93
9. Safety precautions and quality control ..............................................93
10. Practical exercises for students ......................................................93
8.7 Near-Infrared Reflectance Spectroscopy (NIR) ..............................94
1. Introduction .......................................................................................94
2. Principle of NIRS ..............................................................................94
3. Instrumentation of NIRS ...................................................................94
4. Applications of NIRS in animal nutrition .........................................95
5. Sample preparation for NIRS ............................................................96
6. Advantages of NIRS ..........................................................................96
7. Limitations of NIRS ..........................................................................96
8. Practical exercises for students ........................................................96
8.8 Sulphur Hexafluoride (SF6) Technique ...........................................97
1. Introduction .......................................................................................97
2. Principle of the SF6
Technique ..........................................................97
3. Components and Equipment .............................................................97
4. Calibration of Permeation Tubes .......................................................98
5. Animal Preparation and Sampling ....................................................98
6. Sample Analysis ................................................................................98
7. Calculations .......................................................................................98
8. Applications in Animal Nutrition ......................................................98
9. Strengths and Limitations .................................................................99
10. Recent Advances and Modifications ...............................................99
11. Ethical and Environmental Considerations .....................................99
12. Future Perspectives .........................................................................99
13. References and Further Reading .....................................................99
8.9 Rumen Simulation Technique (RUSITEC) ...................................100
1. Feed evaluation and digestibility ....................................................100
2. Study of fermentation kinetics and microbial activity ....................100
3. Assessment of feed additives and anti-methanogenic compounds..101
4. Simulation of dietary interventions: ................................................101
5. Study of rumen microbial ecology: .................................................101
6. Understanding nitrogen metabolism: ..............................................101
8.10 Amino Acid Analyzer ..................................................................102
1. Introduction .....................................................................................102
2. Principle of Amino Acid Analyzer ..................................................102
3. Instrumentation of Amino Acid Analyzer .......................................103
4. Applications in Animal Nutrition ....................................................104
5. Advantages ......................................................................................104
6. Limitations ......................................................................................105
7. Recent Advances .............................................................................105
Appendix ..............................................................................................107
References ...........................................................................................115
