Freezing preservation procedure of caecal inoculum for microbial fermentation studies in pigs

Abstract

Aim of study: To define freezing conditions that preserve fermentative capacity of microbial inoculum for in vitro studies in pigs. Material and methods: Caecal contents from three slaughtered pigs were obtained for being used as inoculum. Part of it was immediately frozen in liquid N and stored at -80ºC, whereas the rest was directly used as fermentation inocula. Incubation substrate was pre-digested in pepsin and pancreatin to simulate the processes occurring before the caecum. Pre-digested substrate was incubated alone or supplemented by three additives consisting of two commercial additives based on essential oils mixtures (CRINA-TEP and CRINA-TMEC) and riboflavin. Gas production at 2, 4, 6, 8, 10, and 12 h, and methane, short chain fatty acids (SCFA), and ammonia concentration at 6 h and 12 h were recorded. Main results: No differences (p>0.05) were recorded along the 12 h incubation between both preservation methods of inocula regarding gas production, methane or total SCFA or their molar proportions. Only a trend for a higher ammonia concentration was detected with frozen than fresh inocula (p=0.062). Although not a main objective of the paper, gas production from the substrate alone (control) was lower than with riboflavin from 8 h onwards, and with CRINA-TEP from 4 to 10 h incubation (p>0.05). Research highlights: Caecal inoculum from pigs for in vitro fermentation studies can be preserved by freezing, provided that freezing and thawing processes are carried out under favorable conditions, especially in terms of time and temperature.