Effects of marine macroalgae on in vitro ruminal fermentation characteristics and methane emission

다국어 초록 (Multilingual Abstract)

Experiment Ⅰ. Effect of Phaeophyta Extracts on In vitro Ruminal Fermentation Characteristics, Methanogenesis and Microbial populations
Algae have become as a source of intensive research in many fields of study. Areas of application are becoming increasingly diverse with the advent of technologies particularly in the mass production of algae biomass. Algae contain complex bioactive compounds and these are gaining importance in emerging technologies with nutritional and environmental applications. The aim of this work was to investigate the effect of Phaeophyta extracts on in vitro rumen fermentation and rumen microbial diversity in view of methane emission. A cannulted Holstein cow was used as the rumen fluid donor. The 15 ml of rumen fluid:buffer (1:2) was incubated for up to 72 h with six possible treatments: control (no addition of algae) and five kinds of Phaeophyta extracts (Ecklonia stolonifera Okamura, Eisenia bicyclis (Kjellman) Setchell, Sargarssum fulvellum (Turner) C. Agardh, Undaria pinnatifida (Harvey) Suringar, Sargassum fusiformis (Harvey) Okamura), respectively. Our results indicate that Phaeophyta extracts can be used as a possible viable alternative to assist in ruminant for improved growth performance (increased total gas production and glucose concentration and decreased A/P ratio) and methane abatement as compared to control. In particular, Real-time PCR indicated that the ciliate-associated methanogens, Ruminococcus albus and Ruminococcus albus flavefaciens populations significantly decreased, while the Fibrobacter succinogens population significantly increased at 24 h, when supplemented with Phaeophyta extracts as compared with control, respectively. As expected, all species of Phaeophyta tested in the study have greater effect on gas production and microbial growth, expecially ciliate-associated methanogens. It has been demonstrated in this study that all classes of algae have candidate members with potential to assist in ruminant feeding for improved gas production, fermentation management, and methane abatement. Ecklonia stolonifera Okamura significantly reduced methane production and had a moderate effect on total gas as compared with control, suggesting that Phaeophyta extracts can mitigate variable effects on overall in vitro rumen fermentation. However, more research is required to demonstrate and elucidate what various Phaeophyta extracts can improve feed intake and utilization efficiency, growth performance and methane abatement.
Experiment Ⅱ. Effect of Rhodophyta Extracts on In vitro Ruminal Fermentation Characteristics, Methanogenesis and Microbial Populations
Research on feed additives is currently ongoing to determine their potential effect in reducing anthropogenic greenhouse gas emissions, especially from ruminants. This study investigated the effect of dietary supplementation of Rhodophyta extracts on in vitro rumen fermentation and rumen microbial diversity. A cannulated Holstein cow was used as the rumen fluid donor. The 15 ml of rumen fluid:buffer (1:2) was incubated for up to 72 h with six possible treatments: control with only timothy and five kinds of Rhodophyta extracts 5% as basis of substrate (Grateloupia lanceolate (Okamura) Kawaguchi, Hypnea japonica Tanake, Pterocladia capillacea (Gmelin) Bornet, Chondria crassicaulis Harvey, or Gelidium amansii (Lam.) Lamouroux, respectively. Our results indicated that Rhodophyta extracts increased cumulative gas production at 24 and 72 h (p = 0.0297 and p = 0.0047) as compared with control. Rhodophyta extracts reduced methane emission at 12 and 24 h (p = 0.0077 and p = 0.0008 and p = 0.0183), in particular Real-Time PCR, indicated that ciliate-associated methanogens, Ruminococcus albus and Ruminococcus flavefaciens populations decreased at 24 h (p = 0.0002, p < 0.0001 and p < 0.0001), while the Fibrobacter succinogens population significantly increased at 24 h (p = 0.0004) as compared with control, respectively. Additionally, Rhodophyta extracts improved acetate concentration at 12 and 24 h (p = 0.0766 and p = 0.0132) and A/P ratio at 6 and 12 h (p = 0.0106 and p = 0.0278) as compared with control, respectively. In conclusion, Rhodophyta extracts can be used as a possible viable alternative to assist in ruminant for improved growth performance (increased total gas production and decreased A/P ratio) and methane abatement (reduced ciliate-associated methanogens, Ruminococcus albus and Ruminococcus flavefaciens population and increased Fibrobacter succinogenes population) as compared to control.
Experiment Ⅲ. Impact of Ecklonia Stolonifera Extracts on In vitro Ruminal Fermentation Characteristics, Methanogenesis and Microbial Populations
Ecklonia stolonifera is a brown algae belonging to the Laminariaceae family and is found commonly in the sea forests, off the coasts of Korea and Japan. E. stolonifera has traditionally been utilized as an edible sea weed and contains high levels of diverse phlorotannins, which have diverse biological activities. This study investigated the effect of dietary supplementation of E. stolonifera extracts on in vitro rumen fermentation by assessing pH, total gas, volatile fatty acid production, gas profile (methane, carbon dioxide, hydrogen and ammonia) and rumenal microbial diversity as compared to basal diet with timothy. A cannulated Holstein cow was used as the rumen fluid donor. The 15ml of rumen fluid:buffer (1:2) was incubated for up to 72 h with four possible treatments and divided into four treatments with three replicates. The doses were: control with only timothy, basal diet with E. stolonifera extract 1%, basial diet with E. stolonifera extract 2% and basial diet with E. stolonifera extract 3%. Our results indicate that E. stolonifera extracts can be used as a possible viable alternative to assist in improved growth performance (significantly increased total gas production) in ruminants, suggesting that E. stolonifera extracts could mitigate variable effects throughout the whole period of in vitro rumen fermentation. Unexpectedly, E. stolonifera extracts did not appear a noticeable effect on methane and hydrogen abatement, which is different from previous observations with brown algae extracts use under in vitro fermentation conditions. Interestingly, Real-time PCR indicated that the ciliate-associated methanogen and Fibrobacter succinogenes population significantly decreased, whereas the Ruminococcus flavefaciens population increased as a result of from E. stolonifera extract supplementation as compared with control. More research is required to demonstrate and elucidate what E. stolonifera extracts can do to improve growth performance and affect methane production in ruminants.
Experiment Ⅳ. Effect of Gelidium Amansii Extracts on In vitro Ruminal Fermentation Characteristics, Methanogenesis and Microbial Populations
Gelidium amansii (Lamouroux) Lamouroux is a red algae belonging to the family Gelidaceae and is commonly found in the shallow coasts of many east Asian countries, including Korea, China and Japan. G. amansii has traditionally been utilized as an edible sea weed for a long time and has various biological activities. The objective of this study was to investigate and determine whether dietary supplementation of G. amansii could be useful for improving ruminal fermentation, as assessed by in vitro fermentation parameters, such as pH, total gas, volatile fatty acid (VFA) production, gas profile (methane, carbon dioxide, hydrogen and ammonia) and microbial growth rate as compared to basal diet with timothy. Cannulated Holstein cows were used as rumen fluid donors and 15 ml of rumen fluid:buffer (1:2) was incubated for up to 72 h with four possible treatments with three replicates. The treatments were: control (timothy only), basal diet with 1% G. amansii extract, basal diet with 2% G. amansii extract and basal diet with 3% G. amansii extract. Overall, the results of our study indicate that G. amansii supplementation is potentially useful, improving ruminant growth performance, via increased total gas and decreased methane and hydrogen emission; but does come with some undesirable effects, such as decreasing total volatile fatty acids and unaffected microbial growth performance. In particular, Real-time PCR indicated that the methanogenic archaea and Fibrobacter succinogens populations were significantly reduced, while the Ruminococcus flavefaciens population significantly increased at 24 h, when supplemented with G. amansii extracts as compared with control.

목차 (Table of Contents)

• Experiment Ⅰ Effect of Phaeophyta extracts on in vitro ruminal fermentation characteristics, methanogenesis and microbial populations 1
• 1.1. Introduction 2
• 1.2. Materials and Methods 3
• 1.2.1. Preparation of Phaeophyta extracts 3
• 1.2.2. Ruminal inoculum and in vitro incubation 3
• 1.2.3. Analysis of gas profiles and ruminal fermentation 5
• 1.2.4. Microbial growth rate 5
• 1.2.5. Quantitative PCR assays 7
• 1.2.6. Statistical analysis 10
• 1.3. Results 11
• 1.3.1. pH, gas production and DM disappearance 11
• 1.3.2. Gas profiles of methane, carbon dioxide and ammonia 16
• 1.3.3. Volatile fatty acid profiles (total VFA, acetate, propionate and A/P ratio) 20
• 1.3.4. Microbial growth rate, protein and glucose concentration 25
• 1.3.5. Ruminal microbial populations 29
• 1.4. Discussion 31
• 1.5. Conclusion 33
• 1.6. References 34
• Experiment Ⅱ Effect of Rhodophyta extracts on in vitro ruminal fermentation characteristics, methanogenesis and microbial populations 38
• 2.1. Introduction 39
• 2.2. Materials and Methods 41
• 2.2.1. Preparation of Rhodophyta extracts 41
• 2.2.2. In vitro fermentation design 43
• 2.2.3. Analysis of gas profiles and ruminal fermentation characteristics 45
• 2.2.4. Microbial growth performance 46
• 2.2.5. Quantitative Real-Time PCR 46
• 2.2.6. Statistical analysis 47
• 2.3. Results 50
• 2.3.1. pH, gas production and DM disappearance 50
• 2.3.2. Gas profiles of methane, carbon dioxide and ammonia 54
• 2.3.3. Volatile fatty acid profiles (acetate, propionate and A/P ratio) 58
• 2.3.4. Microbial growth rate, protein and glucose concentration 62
• 2.3.5. Ruminal microbial diversity 66
• 2.4. Discussion 68
• 2.4.1. In vitro fermentation characteristics 68
• 2.4.2. Microbial growth performance 70
• 2.5. Conclusion 72
• 2.6. References 73
• Experiment Ⅲ Impact of Ecklonia stolonifera extract on in vitro ruminal fermentation characteristics, methanogenesis and microbial populations 80
• 3.1. Introduction 81
• 3.2. Materials and Methods 83
• 3.2.1. Preparation of E. stolonifera extract 83
• 3.2.2. In vitro fermentation design 83
• 3.2.3. Gas profile and ruminal fermentation characteristics analysis 84
• 3.2.4. Microbial growth performance 85
• 3.2.5. Quantitative PCR assays 85
• 3.2.6. Statistical analysis 86
• 3.3. Results 88
• 3.3.1. In vitro fermentation characteristics 88
• 3.3.2. Volatile fatty acid profiles (total VFA, acetate, propionate, butyrate and A/P ratio) 92
• 3.3.3. Gas profiles of methane, carbon dioxide, hydrogen and ammonia 98
• 3.3.4. Microbial growth rate, protein and glucose concentration 103
• 3.3.5. In vitro ruminal change in microbial diversity 107
• 3.4. Discussion 109
• 3.5. Conclusion 112
• 3.6. References 113
• Experiment Ⅳ Effect of Gelidium Amansii extract on in vitro ruminal fermentation characteristics, methanogenesis and microbial populations 118
• 4.1. Introduction 119
• 4.2. Materials and Methods 121
• 4.2.1. Preparation of G. amansii extract 121
• 4.2.2. Polymerase in vitro fermentation design 121
• 4.2.3. Analysis of gas profiles and ruminal fermentation characteristics 122
• 4.2.4. Microbial growth performance 123
• 4.2.5. DNA Quantitative Real-Time PCR 123
• 4.2.6. Statistical analysis 124
• 4.3. Results 126
• 4.3.1. In vitro fermentation characteristics (cumulative pH, gas production, dry matter (DM) disappearance rate) 126
• 4.3.2. Volatile fatty acid profile and A/P ratio 130
• 4.3.3. Emission gas profile (methane, carbon dioxide and ammonia) 136
• 4.3.4. In vitro ruminal change in microbial diversity 140
• 4.4. Discussion 145
• 4.5. Conclusion 148
• 4.6. References 149