Effect of Preservation Temperature and Time on Fermentation Characteristics, Bacterial Diversity and Community Composition of Rumen Fluid Collected from High-Grain Feeding Sheep

This study aimed to explore the dynamic variations in fermentation characteristics, bacterial diversity and community composition at two preservation temperatures as preservation time extended. Six rumen fluid samples collected from high-grain feeding sheep were stored at -20 degrees C or -80 degrees C for 0 day, 7 days, 14 days, 30 days, 60 days, 120 days, and 240 days. The results showed that the current preservation temperature did not alter the fermentation characteristics, bacterial diversity and community composition (p > 0.05). The concentrations of ammonia, microbial crude protein, acetate, propionate, butyrate, valerate, and total volatile fatty acids were higher when stored at 60 days (p < 0.05). Preservation time had no influence on bacterial richness and evenness (p > 0.05), whilst the relative abundances of Bacteroidota and Prevotella were numerically higher when stored at 30 days, and the opposite results were observed regarding Firmicutes. Both principal co-ordinates analysis (PCoA) and non-metric multidimensional scaling (NMDS) showed clusters among treat-ments in terms of preservation time and preservation temperature. Analysis of similarities (ANOSIM) also revealed similarities between treatments (p > 0.05). This study indicates that most fermentation characteristics in rumen fluid were altered after a 60-day preservation, whilst the preservation time for rumen bacterial community profile alteration was 30 days. It is recommended to finish the sample determination of rumen fluid within 30 days. This study may assist decision-making regarding the practicable time for rumen fluid determination, as well as viable preservation conditions for inoculum used for in vitro fermentation testing.

Automated summary

This study investigated the dynamic variations in fermentation characteristics, bacterial diversity, and community composition during the extended preservation time at two different temperatures. The results showed that the preservation temperature did not have a significant effect on the fermentation characteristics, bacterial diversity, and community composition. However, after 60 days of preservation, there were changes in the concentrations of various fermentation products. Bacterial richness and evenness were not affected by the preservation time, but the relative abundances of certain bacterial groups changed. This study suggests that the fermentation characteristics in rumen fluid may be altered after 60 days of preservation, while the bacterial community profile may change after 30 days. It is recommended to determine rumen fluid samples within 30 days for accurate results. This study provides valuable information for determining the practical time for rumen fluid determination and appropriate preservation conditions for in vitro fermentation testing.