In vitro screening of plant resources for extra-nutritional attributes in ruminants: nuclear and related methodologies (eBook)

Foreword 4
Contents 6
Contributors 8
Introduction 10
1 Selecting Potential Woody Forage Plants That Contain Beneficial Bioactives 12
Introduction 12
Overview of Process 13
Defining the Project Parameters (Steps 13) 14
Step 1 15
Step 2 15
Step 3 15
Database Collation (Steps 4 and 5) 16
Step 4 16
Step 5 16
Literature Search (Step 6) 17
Working List (Step 7) 18
Prioritisation (Step 8) 18
Indices for Ranking (Step 9) 19
External Expert Review (Step 10) 21
Germplasm and Sample Acquisition (Step 11) 21
Field Evaluation (Step 12) 22
Species for Ongoing Development (Step 13) 23
Conclusions 23
References 23
2 Collecting, Processing and Storage of Plant Materials for Nutritional Analysis 25
Introduction 25
Sample Collection 25
Diversity Within a Species 26
Physiological Age of Plants and Leaves 26
Position on the Plant 27
Seasonality 27
Methodology 28
Collection Method for Leaf Material for Proximate Analysis 28
Materials Required 28
Procedure 28
Collection Method to Freeze Dry Leaf Material for Analysis of Plant Secondary Metabolites/Bioactive Compounds 28
Materials Required 28
Procedure 29
Sample Processing 29
Oven Drying of Leaf Material for Proximate Analysis 30
Materials Required 30
Procedure 30
Freeze Drying Plant Material for Analysis of Plant Secondary Metabolites/Bioactive Compounds 31
Materials Required 31
Procedure 31
Grinding Plant Samples 31
Materials Required 32
Procedure 32
Storage of Dried Plant Samples 32
Materials Required 32
Procedure 33
References 33
3 In Vitro Methods for the Primary Screening of Plant Products for Direct Activity against Ruminant Gastrointestinal Nematodes 34
Introduction 34
Preparation of Parasite Material 36
Mass Extraction of Nematode Eggs 36
Description 36
Materials 36
Procedure 36
Polyallomer Tube Method 37
Polystyrene/Glass Tube Method 37
Culture of First Stage (L 1 ) Larvae from Nematode Eggs 38
Description 38
Materials 38
Procedure 38
Culture of Third Stage (L3) Nematode Eggs from Sheep Faeces 39
Description 39
Materials 39
Procedure 39
Larval Identification 40
Isolation of Adult Nematodes 40
Description 40
Materials 40
Procedure 41
In Vitro Screening 41
Preparation of Stock Solutions of Plant Material 42
Description 42
Materials 43
Preparation of a 10 mg/mL Aqueous Plant Material Stock Solution 43
Egg Hatch Test 43
Description 43
Materials 44
Procedure 44
Larval Migration Inhibition Test 45
Description 45
Materials 46
Procedures 46
Larval Feeding Inhibition Assay (LFIA) 47
Materials 48
Procedures 48
Larval Exsheathment Assay (LEA) 49
Materials 50
Procedure 50
Larval Development Assay (LDA) 51
Materials 51
Procedure 51
Adult Motility Assay (AMA) 52
Materials 52
Procedure 53
References 53
4 Assessing Antiprotozoal Agents 55
Introduction 55
Screening of Antiprotozoal Agents Based on Their Ability to Inhibit the Breakdown of 14 C-Leucine-Labelled Selenomonas ruminantium 55
Basis of the Method 56
Preparation of Labelled Bacteria 56
Bacteriolytic Activity of Protozoa 56
Calculations 58
Screening of Antiprotozoal Agents Based on Their Ability to Inhibit the Uptake of 14 C-Choline in Mixed Rumen Fluid 59
Basis of the Method 59
Calculations 59
Visual Assessment of Protozoal Viability 59
Basis of the Method 60
References 60
5 Screening for Anti-proteolytic Compounds 62
Introduction 62
Screening for Antiproteolytic Plant Compounds Using Diazotized or Radio-Labelled Proteins 63
Diazotized Proteins 63
Preparation of Azoproteins 63
Measurement of Proteolysis 63
Calculation of Proteolytic Activity 64
Radio-Labelled Proteins 65
Preparation of 14 C-Formaldehyde-Labelled Proteins 65
Measurement of Proteolysis 66
Calculation of Proteolytic Activity 67
Screening for Antiproteolytic Plant Compounds by Short-Term Incubation with a Complex, Protein Rich Substrate 67
Incubation 67
Substrates 67
Donor Animals and Preparation of Inoculum 68
Experimental Design 69
Incubation Medium 69
Inoculation 70
Gas Reading 70
Calculation of Gas Production Rate and Cumulative Gas Production 71
Sampling 72
Optional: 24 h Digestibility 73
Analytical Procedures 73
SCFA Analysis by Gas Chromatography 73
Ammonium Determination by Phenol Hypochlorite Reaction 75
Quantitative Protein Analysis by Dot Blot Assay 76
Dot Blot Procedure 76
Evaluation of Results 79
Qualitative Protein Analysis by SDS-PAGE 79
Preparation of the Polyacrylamide Gels 80
Loading and Running the Gels 82
Fixation and Staining 83
Calibration and Evaluation 84
Conclusion 84
Inhibitor In Vitro Assay of Rate and Extent of Ruminal Protein Degradation 85
Rationale and Overview 85
Incubation 86
Materials 87
Inoculum 88
Pre-incubation 88
Sample Preparation 89
Incubation 89
Sampling 89
Analytical Procedures 90
Inoculum Dry Matter Determination 90
Determination of Ammonium and TAA 90
Calculations 90
References 91
6 Screening for Compounds Enhancing Fibre Degradation 94
Introduction 94
Fibre-Degrading Enzymes 95
Extraction of Enzyme 96
Reagents 96
Procedure 96
Cellulases 97
Carboxymethylcellulase (Endo-1,4-B-glucanase, EC 3.2.1.4) 97
Reagents 97
Procedure 97
Calculation 98
Avicelase (Exo-B-1,4-glucanase, EC 3.2.1.91) 98
B -Glucosidase (B -d -glucoside glucohydrolase, EC 3.2.1.21) 99
Reagents 99
Procedure 99
Calculations 100
Xylanases 100
Endoxylanase ( -1,4-xylan xylanohydrolase Endo-B -1,4-xylanase, EC 3.2.1.8)
Procedure 100
B-Xylosidase (B -1,4-d xylan xylohydrolase: Exo- B-1,4- d xylosidase, EC 3.2.1.37) 101
Procedure 101
Acetyl Esterases (EC 3.1.1.6) 101
Reagents 101
Procedure 102
Feruloyl and p-coumaryl Esterases 102
Reagents 102
Procedure 102
Protein Estimation 103
Estimating Protein Quantities 103
Reagents 103
Procedure 103
Precautions 104
In Vitro True Digestibility 104
Reagents 104
Neutral detergent solution 104
Procedure 105
Isotopic Labelling of Plant Cell Walls 105
14 C- and 13 C-Labelling of Plant Cell Walls as a Means of Assessing Ligno-Cellulose Degradation 106
Equipment Required 106
Reagents 106
14 C or 13 C Procedure for Whole Plant Labelling 106
14 C-Labelled Plant Substrates 109
Collection of Liberated 14 CO 2 109
Estimation of Retained 14 C in Plant Material 109
13 C-Labelled Plant Substrates 109
Indicators of Digestive Activity 110
14 C-Labelled Lignin Using l-[U- 14 C] Phenylalanine 110
Indicators of Digestive Activity 111
References 111
7 In Vitro Screening of Feed Resources for Efficiency of Microbial Protein Synthesis 113
Introduction 113
In Vitro Feed Evaluation Methods 114
Nylon Bag Technique 114
Tilley and Terry Technique 114
In Vitro Gas Method 115
General Description 115
Origin of Gas and Stoichiometry 116
A modified in vitro gas method: gas production with concomitant microbial mass measurement 118
General Description 118
Determination of Microbial Mass 119
Partitioning Factor and Efficiency of Microbial Protein Synthesis 120
The Partitioning Factor as a Reflection of Efficiency of Microbial Protein Synthesis for Tannin-Containing Feeds 121
Incubation Time and Partitioning Factor 122
Method for Measuring Partitioning Factor 123
Sample Preparation 123
Reagents 123
Weighing of Samples and Preparation of Syringes 124
Preparation of In Vitro Rumen Fermentation Buffer Solution and Incubation 124
Medium Composition 124
Incubation Procedure 125
An Example 125
Net Gas Production 126
Organic Matter Degradability 126
Reagents 126
Procedure 127
Determination of Partitioning Factor 128
An Example 128
Some Data from the Literature 129
Determination of Other Parameters 131
Methods for Measurement of Microbial Mass, Microbial-Nitrogen, Microbial Purines and 15 N Incorporation in Microbes and Determination of Efficiency of Microbial Protein Synthesis 134
Determination of Microbial Protein Production and Efficiency of Microbial Protein Synthesis Using Purines as a Marker 137
Preparation of Apparent Undegraded Residue 137
Preparation of Lyophilized Rumen Microbial Fraction 138
Spectrophotometric Method for Determination of Purines (Marker for Microbial Mass/Protein) ([ 53 ], With Some Modification as Described in [ 13 ]) 138
Reagents 138
Procedure 138
Some Data on Purines [31] 140
High Performance Liquid Chromatograph (HPLC) Method for Determination of Purine Bases (Adenine and Guanine) ([ 2 ], with some modifications as described in [ 31 ]) 140
Sample Hydrolysis 141
Some Data on Purine Bases [31] 142
Determination of Microbial Protein Production and Efficiency of Microbial Protein Synthesis Using 15 N Incorporation 144
Reagents 144
Procedure 144
Dos and Donts for the Gas Method 145
Conclusions 147
References 147
8 Screening of Plants for Inhibitory Activity Against Pathogenic Microorganisms from the Gut of Livestock 151
Introduction 151
Experimental Approach 153
Plant Extractions 153
Dissolving Plant Extracts for Testing 154
Microorganism Selection, Inoculation and Culture Conditions 155
Media Selection 155
Microbial Inoculation Culture 155
Detection of Metabolic Activity Using p-iodonitro tetrazolium Violet 156
Control Inhibitor Selection 156
Agar Plate Diffusion Assay 156
Broth Microbial Growth Assay 157
Percentage Inhibition 158
Minimum Inhibitory Concentrations 159
In Vitro Testing with Rumen Fluid Inoculum 159
Anaerobic Culture Techniques and Media Preparation 159
Enumeration of Target Bacteria 160
Conclusions 161
Appendix Basal Media Composition 161
References 163
9 Screening Plants for the Antimicrobial Control of Lactic Acidosis in Ruminant Livestock 165
Introduction 165
Overview of the Protocol 166
Methodology 166
Step 1 Initial Screening: In Vitro Batch Fermentations 167
Background 167
Material Required 168
Method 170
Analysis 177
Sample Results 177
Step 2 Agar Dilution Method 178
Background 178
Materials Required 179
Method 181
Sample Results 187
Step 3 Extent of Acidosis Protection 188
Background 188
Material Required 189
Method 189
Lactate and VFA Analysis 192
Statistical Analysis 192
Sample Results 192
References 194
10 Screening Plants and Plant Products for Methane Inhibitors 196
Introduction 196
General Considerations 196
Screening Methodology 196
Design of Screening Assays for Anti-Methanogenic Effects 197
General Characteristics of the Assay 197
Definition of Objectives 198
Plant Selection 199
Physical Form of Plant Additives 199
Dose 200
Blanks, Controls and Replication 201
The Hohenheim Syringe-Based In Vitro Gas Method 201
Screening of Plants 202
Sample Preparation 202
Reagents 202
Weighing of Samples and Preparation of Syringes 203
Preparation of In Vitro Rumen Fermentation Buffer Solution and Incubation 203
Incubation Procedure 203
Total and Net Gas Production 204
Total and Net Methane Production 204
Calculations and Expression of Results 205
Expression of Results 206
Screening of Plant Extracts or Additives in Liquid State 207
Preparation of Plant Extract 207
Reagents 208
Weighing of Samples and Preparation of Syringes 208
Preparation of In Vitro Rumen Fermentation Buffer Solution and Incubation 208
Total and Net Gas Production 208
Total and Net Methane Production 208
Calculations and Expression of Results 209
The Pressure Transducer Method 210
In Vitro Incubations 210
Preparation and Weighing of Feed Substrate to be Fermented and Plant Additive to Be Tested 211
Plant Additive 211
Preparation of Fermentation Medium 212
Collection of Rumen Fluid and Dilution with the Medium 213
Inoculation of Cultures 213
Measurements at the End of Incubation 214
Measurement of Gas Production Using a Pressure Transducer 214
Sampling of Gas 216
Analysis of Methane in Fermentation Gas 217
Fermentation Pattern 217
Calculations 218
Total and Net Production of Gas and Methane 218
Expression of Results and Statistical Evaluation 219
In Vitro Continuous Fermentation System (RUSITEC) 222
Preparation of Nylon Bags and Experimental Feed 222
Preparation of Plant Additive 223
Preparation of Incubation Medium 224
Reagents 224
Collection of Rumen Fluid and Solid Inoculum 225
Description of the RUSITEC Fermentation System 225
Setting Up of the Fermentation Units 227
Daily Feed Supply to the Fermenter 229
Measurements and Laboratory Analyses 230
Gas and Methane Production and Composition 230
Other Measurements 230
Calculations 232
Statistical Analyses 234
References 234
11 Challenges in Extrapolating In Vitro Findings to In Vivo Evaluation of Plant Resources 237
Introduction 237
Simple vs. Complex Environments: Interactions of Bioactives with Other Chemicals 237
Incubation of Plant Bioactives with Rumen Fluid: Static vs. Dynamic Fermentation Systems 238
Concentration of Bioactives in Biological and Artificial Media 239
Ruminal Adaptation: Short vs. Long Term Effects 240
Ingestive Behavior 240
Food Aversions and Willingness to Consume Bioactives 240
Pulse-Delivery of Bioactives to the Digestive Tract 242
Voluntary Intake and Sequence of Feeding Patterns 242
References 243
List of Participants 247
Index 249