## 3 Results
### Greenhouse Gas Emission (Experiment 2)  
The total gas emissions per DMD (\(p\) < 0.001; 132.5 vs. 102.6 mL/g DMD) and OMD (\(p\) < 0.001; 112.1 vs. 90.4 mL/g DMD) decreased with increasing blanching temperatures (CON vs. LOS) (Figure 2). At each blanching temperature, the lowest total gas emissions per DMD were detected (\(p\) < 0.05) in the CON 0% and LOS 5% treatment. The lowest total gas emissions per DMD were observed in the CON treatment with 0 and 5% supplementation levels. Meanwhile, carbon dioxide showed no significant differences (\(p\) > 0.05), except for carbon dioxide per DMD by blanching temperature, where levels in the CON treatment were lower than in the LOS treatment (\(p\) = 0.001; 26.1 vs. 29.8 mL/g DMD). Methane per DMD decreased with increasing blanching temperature (\(p\) < 0.001; CON vs. LOS = 10.6 vs. 6.15 mL/g DMD) and supplementation levels (\(p\) < 0.010; 0 vs. 1 vs. 3 vs. 5% = 8.97 vs. 10.1 vs. 7.07 vs. 7.30 mL/g DMD). In particular, methane per DMD was the lowest (\(p\) < 0.001; 0 vs. 1 vs. 3 vs. 5% = 7.59 vs. 7.10 vs. 5.25 vs. 3.49 mL/g DMD) in the LOS 5% treatment, showing a 20.7% decrease compared to the 0% treatment. Methane per DMD was also the lowest (\(p\) < 0.001; 0 vs. 1 vs. 3 vs. 5% = 6.39 vs. 6.13 vs. 4.47 vs. 4.53 mL/g DMD) in the LOS 3% treatment, showing a 30.7% reduction compared to the 0% treatment. Additionally, methane decreased (\(p\) < 0.05) linearly with increasing supplementation levels.  
\begin{table}
\begin{tabular}{l c c c c c c c c c} \hline  & \multicolumn{4}{c}{**CON 1**} & \multicolumn{4}{c}{**LOS**} & \multirow{2}{*}{**SEM**} \\ \cline{2-2} \cline{4-9}  & **0\%** & **1\%** & **3\%** & **5\%** & **0\%** & **1\%** & **3\%** & **5\%** & \\ \hline pH & 5.36 & 5.38 & 5.38 & 5.36 & 5.38 & 5.33 & 5.39 & 5.37 & 0.034 \\ NH\({}_{2}\)N & 18.0 & 19.6 & 19.1 & 18.9 & 19.1 & 18.9 & 18.2 & 17.3 & 1.557 \\ TVFA, mM/L & 117.4\({}^{*}\) & 112.0\({}^{*}\) & 99.6\({}^{*}\) & 102.0\({}^{*}\) & 101.9\({}^{*}\) & 77.9\({}^{*}\) & 74.6\({}^{*}\) & 70.7\({}^{*}\) & 4.987 \\ Acetate, \% mol & 59.1 & 59.7 & 59.9 & 58.4 & 59.4 & 60.0 & 60.5 & 58.7 & 1.095 \\ Propionate, \% mol & 23.6\({}^{*}\) & 22.0\({}^{*}\) & 22.3\({}^{*}\) & 23.4\({}^{*}\) & 23.9\({}^{*}\) & 23.1\({}^{*}\) & 22.5\({}^{*}\) & 25.0\({}^{*}\) & 0.883 \\ Butyrate, \% mol & 11.3 & 11.4 & 11.5 & 11.6 & 11.3 & 11.2 & 10.9 & 11.3 & 0.865 \\ A-P ratio & 2.53 & 2.71 & 2.48 & 2.50 & 2.44 & 2.58 & 2.70 & 2.38 & 0.123 \\ Contrast & pH & NH\({}_{3}\)-N & TVFA & Acetate & Propionate & Butyrate & A:P ratio \\ \hline TEMP & 0.761 & 0.991 & \(<\)0.001 & 0.561 & 0.052 & 0.410 & 0.369 \\ LEVEL & 0.447 & 0.240 & 0.007 & 0.032 & 0.010 & 0.595 & 0.029 \\ TEMP \(\times\) LEVEL & 0.143 & 0.326 & 0.003 & 0.799 & 0.508 & 0.523 & 0.162 \\ LEVEL Linear & 0.848 & 0.428 & 0.557 & 0.142 & 0.156 & 0.478 & 0.362 \\ LEVEL Quadratic & 0.744 & 0.677 & 0.203 & 0.003 & 0.002 & 0.358 & 0.013 \\ \hline \end{tabular} \({}^{1}\) CON, _Sargassum horneri_ blanched in cool water (21 \({}^{*}\)C) for 3 min; LOS, _Sargassum horneri_ blanched in water at 70 \({}^{*}\)C for 3 min; 0, 1, 3, and 5%, supplemented levels of _Sargassum horneri_ at 0, 1, 3, and 5%, respectively; TVFA, total volatile fatty acid; A:P ratio, acetate to propionate ratio; SEM, standard error of mean; TEMP, blanching temperature effect; LEVEL, supplementation level effects; TEMP \(\times\) LEVEL, interaction effect between blanching temperature and supplementation level; LEVEL Linear, linear effect by supplementation level of SH; LEVEL. Quadratic, quadratic effect by supplementation level of SH. \({}^{*\times}\) Means in the same row with different superscripts differ significantly (\(p\) < 0.05).  
\end{table}
Table 4: The effect of blanching temperature and supplementation level on fermentation characteristics of _Sargassum horneri_ incubated with rumen buffer at 48 h in Experiment 2.