Calf diarrhea is a major cause of mortality and economic loss in the beef industry. Infectious agents (viruses, bacteria, protozoa) and non-infectious factors (stress, diet, humidity) contribute to its occurrence. Antibiotics have been widely used for treatment and growth promotion, but their overuse leads to antibiotic resistance, residues in meat, and disruption of the gut microbiome, often resulting in diarrhea recurrence. The disruption of the intestinal microbial community triggers immune responses, inflammation, and increased peristalsis, exacerbating diarrhea. Long-term antibiotic abuse impairs rumen and intestinal microbiota development, negatively impacting digestion and absorption. Therefore, alternatives to antibiotics are urgently needed. Fecal microbiota transplantation (FMT), the transfer of fecal material from a healthy donor to a recipient, has proven effective in treating various gastrointestinal disorders in humans and other monogastric animals. Studies have shown the potential of FMT to prevent necrotizing enterocolitis in piglets and the use of microbiota-derived bacteriocins to combat diarrhea. Rumen transformation, a historical practice involving transferring rumen fluid from healthy to sick cattle, also demonstrates the principle of microbial reconstitution. This study aimed to evaluate the efficacy of FMT in ameliorating calf diarrhea and improving growth performance in ruminants, addressing the limitations of antibiotic use and exploring microbiome-based therapeutic approaches.

Extensive research highlights the significant challenges posed by calf diarrhea, emphasizing the need for effective and safe treatment alternatives to antibiotics. The use of antibiotics has been linked to various detrimental effects, including the development of antibiotic resistance and the disruption of the beneficial gut microbiota. Previous studies in other animal models have explored the potential of fecal microbiota transplantation (FMT) and microbiota-derived therapeutics in treating gastrointestinal disorders. These studies provided a rationale for investigating the efficacy of FMT in treating calf diarrhea and improving growth performance, particularly considering the historical success of rumen transformation in ruminants.

The study involved two phases: a preliminary trial and a validation trial. **Preliminary Trial:** Seven calves were used to assess the safety and efficacy of oral FMT. One calf served as a healthy donor, and six calves received fecal suspension twice daily for 16 days. 16S rRNA gene amplicon sequencing was used to analyze the temporal changes in the gut microbiota. SourceTracker analysis assessed the origin of the recipient's gut microbiota. **Validation Trial:** This larger trial involved 57 calves with diarrhea, randomized into three groups: FMT (n=20), saline control (CON, n=14), and antibiotic treatment (ABX, n=23). The FMT group received a fecal bolus five times. Diarrhea severity was monitored daily using the Bristol Stool Scale (BSS). Fecal samples were collected at various time points for metataxonomic analysis using 16S rRNA gene amplicon sequencing. SourceTracker analysis was used to assess the microbial community structure. LEfSe was used to identify discriminating taxa. Fecal metabolomes were analyzed using gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). Fecal and serum amino acid and branched-chain amino acid (BCAA) concentrations were quantified using enzyme-based assays. Body mass and dressed mass were measured at 6, 12, and 24 months. The 12-month-old cattle gut microbiota was analyzed by 16S rRNA sequencing, and functional profiles were predicted using PICRUSt. Serum metabolome of 12-month-old cattle was analyzed using GC-TOF-MS. Statistical analyses were performed using Mann-Whitney U tests, PERMANOVA, and Pearson correlations.

The preliminary trial demonstrated the safety and efficacy of oral FMT in calves, showing a shift in gut microbiota composition towards that of the donor. The validation trial showed that FMT significantly reduced the incidence and mortality associated with calf diarrhea compared to the control and antibiotic groups (Fig. 1). FMT calves showed a gradual decrease in microbial dissimilarity to healthy donors, unlike the control and antibiotic groups (Fig. 2). SourceTracker analysis indicated that FMT successfully introduced the donor's microbiota into the recipient calves (Fig. 2l). LEfSe analysis revealed that *Verrucomicrobia* was enriched in the control group, *Proteobacteria* in the antibiotic group, and *Bacteroidetes* in the FMT group (Fig. 3a). FMT was associated with increased *Porphyromonadaceae* and decreased *Enterobacteriaceae* abundance (Fig. 3). A strong positive correlation was found between *Enterobacteriaceae* abundance and diarrhea severity, while a negative correlation existed between *Porphyromonadaceae* abundance and diarrhea severity in the FMT group only (Fig. 3f). GC-TOF-MS analysis revealed distinct fecal metabolomic profiles between the groups after treatment, with lower fecal amino acid and BCAA concentrations in the FMT group (Fig. 4). Importantly, FMT-treated cattle showed significantly improved growth performance (body mass and dressed mass) at 12 and 24 months of age compared to the control and antibiotic groups (Fig. 5). Analysis of the 12-month-old cattle's gut microbiota showed distinct microbial composition in the FMT group, enriched in families associated with growth (Fig. 5f-i). PICRUSt analysis suggested enrichment of gene families related to growth and BCAA biosynthesis in the FMT group (Supplementary Fig. 8b). Furthermore, serum metabolomic analysis of the 12-month-old cattle showed significantly higher serum BCAA concentrations in the FMT group (Fig. 6).

This study provides compelling evidence that FMT is a viable alternative to antibiotics for treating calf diarrhea and improving growth performance. The findings highlight the crucial role of the gut microbiome in diarrhea pathogenesis and recovery. The increase in *Porphyromonadaceae* in the FMT group and its inverse correlation with diarrhea severity suggest its potential beneficial role. The decrease in fecal amino acids in the FMT group suggests improved microbial fermentation efficiency. The long-term improvements in growth performance observed in the FMT group highlight the lasting positive effects of gut microbiota modulation. This study's findings are consistent with previous research demonstrating the efficacy of FMT in other animal models and support the growing understanding of the microbiome's impact on host health and productivity. Further research is needed to identify the specific mechanisms by which *Porphyromonadaceae* contribute to diarrhea remission and to optimize FMT protocols for broader application.

This study demonstrates the efficacy of FMT as a treatment for calf diarrhea and a promoter of improved growth in cattle. FMT effectively alters the gut microbiota composition, leading to lower fecal amino acid concentrations and improved growth performance. This approach offers a promising alternative to antibiotics, addressing concerns about antibiotic resistance and promoting animal health and welfare. Future studies should focus on identifying the specific microbial mechanisms involved and optimizing FMT protocols for wider application in livestock production.

The study's relatively small sample size, particularly in the preliminary trial, could limit the generalizability of findings. The study was conducted on a specific breed of cattle (*Bos taurus coreanae*), and results might not be directly transferable to other breeds or species. While the study controlled for dietary factors, variations in other environmental factors could have influenced the results. Further research with larger, more diverse populations is necessary to fully validate these results.