## 4 Notes  
* When the concentration of glycerol in the sample storage buffer is 50%, add the sample storage buffer to the resuspended solution at a ratio of 1:1 to prepare the transplantation solution and obtain a final glycerol concentration of 25%. The final concentration glycerol can be adjusted within the range of 10-30% according to different experimental purposes and conditions.
* Use a placebo as a control for the transplantation solution. The final glycerol concentration in the placebo should be equal to that of the transplantation solution.
* Metronidazole can be added to the broad-spectrum antibiotic mix, but it must be used with caution. This can achieve better bacterial depletion results, but may cause weight loss in mice [42; 43]. You can try to gradually introduce metronidazoleole to the solution [42].
* The procedure is suitable for commonly used 6-to-8-week-old mice (25-30 g). If using other target animals, refer to Tables 1 and 2.
* Compared to a tapered-bottom tube, using a round-bottom tube can achieve better homogenization effects.
* A 1 mL syringe is suitable for commonly used 6-to-8-week-old mice (25-30 g). If using other target animals, refer to Table 2. The volume received by each recipient (200-300 \(\mu\)L) is calculated for approximately 10-20 mg feces/mouse. Generally, the  
\begin{table}
\begin{tabular}{c c c} \hline \hline \multicolumn{1}{c}{**Animal Species**} & **Weight (g)** & **Gastric Volume (mL)** \\ \hline \multirow{3}{*}{Mice} & 20–24 & 0.8 \\  & 25–30 & 0.9 \\  & \(\geq\)30 & 1.0 \\ \hline \multirow{3}{*}{Rats} & 100–199 & 3.0 \\  & 200–249 & 4–5 \\  & 250–300 & 6.0 \\  & \(\geq\)300 & 8.0 \\ \hline \multirow{3}{*}{Guinea pigs} & 250–300 & 4–5 \\  & \(\geq\)300 & 6.0 \\ \hline \multirow{3}{*}{Rabbits} & 2000–2400 & 100 \\  & 2500–3500 & 150 \\  & \(\geq\)3500 & 200 \\ \hline \multirow{3}{*}{Cats} & 2500–3000 & 50–80 \\  & \(\geq\)3000 & 100–150 \\ \hline \multirow{3}{*}{Dogs} & 10,000–15,000 & 200–500 \\ \cline{1-1}  & Pigs & - & 500 \\ \hline \hline \end{tabular}
\end{table}
Table 2: Maximum intragastric intake of commonly used laboratory animals at one time [41].  
ideal effect can be obtained from a feces concentration of 50-100 mg/mL in 200 \(\mu\)L of transplantation solution. To determine the optimum conditions for a particular model, a pilot experiment is required.
* Generally, each mouse can provide 50-100 mg of fresh feces (6-to-8-week-old mice, 25-30 g). However, if the donors are enteritis-mouse models, there will be less feces. Therefore, the stool from mice of matched weight and sex can be mixed depending on the experimental design. The number of animals used can be customized to the experiment. Fresh feces should be used for transplantation within 6 h [22, 36, 44], as oxygen exposure degrades the fecal bacterial communities [45]. If frozen feces are required for subsequent use, aim to complete the freezing operation within 15 min as much as possible [46].
* The duration of the broad-spectrum antibiotic mix treatment of mice can be customized, but, generally, it lasts for at least 14 days.
* Before FMT, a \"rest\" period of 12-48 h is required [22, 36, 42, 44].
* Fasting should be started for at least 4-8 h before gavage to avoid the gastric contents hindering gavage injection and affecting the drug absorption rate.
* The transplanting of recipients once a day for 5 consecutive days is suitable for commonly used 6-to-8-week-old mice (25-30 g). To determine the optimum conditions for a particular model, a pilot experiment is required. For the solutions to other common problems, see Table 3.