Effects of cryopreservation on the recovery of viable porcine intestinal microbiota

The pig gut microbiota plays an important role in host nutrition, intestinal physiology, and health, and cultivation remains essential for isolating viable bacteria for functional studies and microbial resource development. However, practical guidance for preserving region-specific porcine intestinal contents prior to cultivation remains limited, and most previous evidence has been derived from human stool samples or DNA-focused preservation studies. Here, we compared fresh processing under three cryopreservation conditions to assess the recovery of the culturable bacterial fraction and the composition of cultured bacterial communities from the ileum, cecum, colon, and rectum of a healthy Berkshire boar after one month of storage at − 80 °C. Combining anaerobic cultivation, colony counting, and 16 S rRNA gene sequencing of cultured biomass, we found that fresh samples consistently yielded the highest recovery of culturable bacteria across intestinal regions. Cryopreservation reduced colony counts across intestinal regions, with unprotected freezing (P0) yielding the lowest recovery. Counts decreased from 1.19 × 10 9 to 2.73 × 10 8 CFU/g in the ileum, from 8.26 × 10 8 to 3.33 × 10 8 CFU/g in the cecum, from 4.87 × 10 8 to 2.10 × 10 8 CFU/g in the colon, and from 4.37 × 10 8 to 1.24 × 10 8 CFU/g in the rectum, with the largest absolute reduction observed in the ileum. Compared with freezing without cryoprotection, glycerol-containing formulations generally retained broader cultured communities and better preserved taxonomic representation. These results suggest that immediate processing is the most favorable approach for culture-based studies of the pig gut microbiota, while cryoprotectant-assisted freezing represents a practical alternative for short-term storage when fresh cultivation is not possible. Overall, this pilot study provides region-specific evidence to guide short-term preservation of porcine intestinal samples for bacterial isolation and future culture-based microbiome research.