| Summary: | ABSTRACT: Effective preservation of rumen microbial populations for downstream use is highly sought after, yet potential biases introduced by storage time and repeated freeze-thaw cycles (FTC) remain poorly understood. This study investigates the effects of storage time and multiple FTC on the total (DNA-based) and metabolically active (cDNA-based) bacterial populations in both the rumen solid and liquid fractions. Rumen contents were collected from 4 primiparous and 4 multiparous cows and filtered through 2 layers of cheesecloth to separate ruminal solids and liquids. Each fraction was aliquoted into 6 batches and stored at −80°C. The DNA and RNA extractions were performed on batches after 30, 60, and 90 d. The remaining 3 batches were subjected to a second or third FTC before nucleic acid extractions were performed. Parity had no significant effect on microbial populations, but storage time and FTC had a significant effect on DNA and cDNA populations in the solid and liquid fractions. The ruminal solids had a lower gram-negative to gram-positive bacteria ratio (1.48:1) than the liquid fraction (3.90:1), which comprises ∼75% gram-negative bacteria in the DNA-based population. The DNA-based solid fraction demonstrated greater resilience to freezing and freeze-thaw stress, likely due to the higher proportion of gram-positive bacteria. In contrast, the liquid fraction was more sensitive to sample handling conditions, likely due to the higher proportion of fragile gram-negative taxa. The metabolically active (cDNA-based) populations showed better stability to storage time and FTC than the DNA-based populations, except for 60 d time points. Members of the Proteobacteria phyla were enriched in the liquid fraction and showed a 2-fold increase in cDNA absolute abundance after 60 d of storage and 2 FTC in both fractions (P < 0.05). This study underscores the effect of storage duration and freeze-thaw cycles on rumen microbial communities, with particular emphasis on the opportunistic response of Proteobacteria under preservation related stress.
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