Title Page
Contents
ABSTRACT 12
Chapter 1. Literature review 15
1.1. General introduction 15
1.2. Global warming and livestock 17
1.3. Rumen methanogenesis 17
1.4. Strategies to mitigate methane emissions from ruminants 24
1.5. Seaweeds as methane mitigation strategies 25
1.5.1. Green seaweed 26
1.5.2. Brown seaweed 26
1.5.3. Red seaweed (genus Asparagopsis) 28
1.5.4. Red seaweed (except for genus Asaragopsis) 32
Chapter 2. Screening the methane-reducing potential of the red seaweed species 41
2.1. Abstract 41
2.2. Materials & Methods 41
2.2.1. Red seaweeds and substrate preparation 41
2.2.2. Experimental design 42
2.2.3. In vitro incubation procedures 42
2.2.4. Analysis of gas and rumen fermentation characteristics 43
2.2.5. Statistical analysis 44
2.3. Results 44
2.3.1. Gas profiles 44
2.3.2. Rumen fermentation characteristics 47
2.3.3. Principal component analysis 47
2.4. Discussion 52
2.5. Conclusion 54
Chapter 3. Determination of the optimal feed characteristics for Gracilaria chorda 55
Study 3.1. Effects of drying methods of Gracilaria chorda on methane production 55
3.1.1. Abstract 55
3.1.2. Materials and Methods 56
3.1.3. Results 59
3.1.4. Discussion 66
3.1.5. Conclusion 69
Study 3.2. Dose-dependent effects of Gracilaria chorda on methane production 70
3.2.1. Abstract 70
3.2.2. Materials and Methods 70
3.2.3. Results 73
3.2.4. Discussion 79
3.2.5. Conclusion 83
Chapter 4. Metagenomic analysis of microbial community, enzymes, and metabolic pathways in the rumen supplemented with Gracilaria chorda 84
4.1. Abstract 84
4.2. Materials and Methods 84
4.2.1. Sample preparation 84
4.2.2. DNA extraction, PCR, and 16S rRNA gene sequencing 85
4.2.3. Bioinformatics and statistical analysis 85
4.3. Results 86
4.3.1. Rumen microbial richness, diversity, and compositions 86
4.3.2. Microbial genes and their correlation with rumen fermentation parameters 92
4.3.3. Metabolic pathways and their correlation with rumen fermentation parameters 93
4.4. Discussion 98
4.5. Conclusion 102
Chapter 5. General conclusion 104
References 106
Appendix 120
[Appendix Ⅰ] Supplementary Tables & Figures 120
[Appendix Ⅱ] List of Abbreviations 147
Abstract (in Korean) 148
Table 1-1. Categorized reactions and involved enzymes in each methanogenesis pathway. 21
Table 1-2. Summary of rumen fermentation characteristics data from the previous in vitro experiments with genus Asparagopsis. 35
Table 1-3. Summary of methane, hydrogen, and carbon dioxide data from previous in vivo experiments with genus Asparagopsis. 36
Table 1-4. Summary of rumen fermentation characteristics data from previous in vivo experiments with genus Asparagopsis. 37
Table 1-5. Summary of productivity data from previous in vivo experiments with genus Asparagopsis. 38
Table 1-6. Summary of halogenated compounds data from previous in vivo experiments with genus Asparagopsis. 39
Table 1-7. Summary of rumen fermentation characteristics data from the previous in vitro experiments with other red seaweed species. 40
Table 2-1. Effects of thirty-eight species of red seaweeds on rumen fermentation characteristics. 48
Table 3-1. Chemical compositions of differently dried Gracilaria chorda. 61
Table 3-2. Effects of drying methods of Gracilaria chorda on rumen fermentation characteristics. 64
Table 3-3. Contents of halogenated methane analogues in differently dried Gracilaria corda and Asparagopsis taxiformis. 65
Table 3-4. Dose-dependent effects of the red seaweed on rumen fermentation characteristics. 77
Table S1. Crude ash (% dry matter) contents of thirty-eight species of red seaweed used in Chapter 1. 120
Table S2. Comparison of gas production of the control group in each batch. 121
Table S3. Effects of thirty-eight species of red seaweeds on rumen gas profiles. 122
Table S4. Effects of drying methods of Gracilaria chorda on rumen gas profiles. 124
Table S5. Dose-dependent effects of hot-air dried Gracilaria chorda on rumen gas profiles. 125
Table S6. Dose-dependent effects of Gracilaira chorda on methane production. 126
Table S7. Effects of Gracilaria chorda on rumen microbial richness and alpha-diversity. 127
Table S8. Effects of Gracilria chorda on the relative abundance of rumen microbial taxa representing 〉 0.1% of total sequences. 128
Table S9. Effects of Gracilaira chorda on the relative abundance of rumen microbial genes that account for 〉 0.01% of total sequences predicted through PICRUSt. 130
Table S10. Correlation coefficients (r) between rumen fermentation characteristics and key rumen microbial genes. 142
Table S11. Effects of Gracilaira chorda on the relative abundance of rumen metabolic pathways that account for 〉 0.01% of total sequences predicted through PICRUSt. 143
Table S12. Correlation coefficients (r) between rumen fermentation characteristics and key rumen metabolic pathways. 144
Figure 1-1. Overview of three major methanogenic pathways occurred in the rumen by methanogens. Each reaction is identified by colors and codes. Green color (code: H1 to H5) represents hydrogenotrophic pathway, orange color (code: A1 and A2) shows acetoclastic pathway,... 20
Figure 1-2. Eight metabolic hydrogen ([H]) sinks in the rumen. This figure is reconstructed with references to Ungerfeld (2020). 23
Figure 2-1. Effects of thirty-eight species of red seaweed on rumen gas production over incubation time. Gas production was represented as mL per g organic matter. Mean values are displayed in Table S3. 45
Figure 2-2. Effects of thirty-eight species of red seaweed on methane production (A) and proportion (B). Methane production was represented as mL per g organic matter. Asterisks (*) mean the significant difference compared to control group. Mean values are displayed... 46
Figure 2-3. Principal component analysis (PCA) displaying correlation among red seaweed species and rumen fermentation parameters. 51
Figure 3-1. Effects of drying methods of Gracilaria chorda on gas production. FD, freeze-dried; OV40, oven-dried at 40℃; OV60, oven-dried at 60℃; OV105, oven-dried... 62
Figure 3-2. Effects of drying methods of Gracilaria chorda on methane production (A) and proportion (B). FD, freeze-dried; OV40, oven-dried at 40℃; OV60, oven-dried at 60℃; OV105, oven-dried at 105℃. Methane production was represented as mL per g organic matter.... 63
Figure 3-3. Effects of Gracilaria chorda on gas production over incubation time. Gas production was represented as mL per g organic matter. Error bars represent the standard... 75
Figure 3-4. Effects of Gracilaria chorda on methane production (A) and proportion (B) over incubation time. Methane production was represented as mL per g organic matter. Error bars represent the standard error (± SE). Mean values are displayed in Table S5. 76
Figure 3-5. Does-dependent effects of Gracilaria chorda on methane production over incubation time. Methane production was represented as mL per g organic matter. Values... 78
Figure 4-1. Alpha diversity metrics of the rumen microbial compositions in control or treatment (5% OM of Gracilaria chorda). Mean values are displayed in Table S7. 88
Figure 4-2. Taxonomic profiles of the relative abundance at phylum-level (A) and genus-level (B) of rumen microbiome in control or treatment (5% OM of Gracilaria chorda) classified from 〉 0.1% of the total sequences. Mean values are presented in Table S8. 89
Figure 4-3. Linear discriminant analysis (LDA) effect size (LEfSe) cladogram (A) and bar-plot (B) showing taxonomic differences between control and treatment (5% OM of... 90
Figure 4-4. Principal component analysis (PCA) presenting the correlations among microbial communities between control and treatment. (5% OM of Gracilaria chorda). 91
Figure 4-5. The relative abundance of key microbial genes represented in the related metabolism. Error bars represent the standard error (± SE). Mean values are presented in Table S9. 94
Figure 4-6. Heatmap of correlation between rumen fermentation characteristics and relative abundance of key microbial genes grouped in related metabolism. The color represent the strength of correlation (Red, +1; Blue, -1) Asterisks (*) mean the significant of correlation.... 95
Figure 4-7. Differences of key metabolic pathway through Phylogenic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) based on Kyoto Encyclopedia of Genes and Genomes (KEGG) between control and treatment (5% OM of Gracilaria... 96
Figure 4-8. Heatmap of correlation between rumen fermentation characteristics and relative abundance of key metabolic pathways. The color represent the strength of correlation (Red, +1; Blue, -1) Asterisks (*) mean the significant of correlation. The correlation coefficient... 97
Figure 4-9. Schematic overview of pathways affected by the supplementation of Gracilaria chorda. The genes enriched in control or treatment are represented by blue... 98
Figure S1. Comparison of gas production of the control group in each batch. OM, organic matter; control-n, control group in batch n (n=1 to 6); SEM, standard error of... 145
Figure S2. Linear regression analysis between methane production and molar proportion of acetate, propionate, butyrate, or acetate to propionate ratio. The data were pulled from... 146