Effects of dietary supplementation with Pinus Taeda Hydrolized Lignin on Rumen microbiome in Beef

The aim of this study was to evaluate the effect of a commercially available herbal preparation (Oxyphenol®, I-Green, Padua, Italy) of Pinus taeda hydrolyzed lignin (PTHL) on fattening beef in vitro rumen fermentation, and in particular its effect on rumen microbiota. Forty Limousine steers were randomly divided into two groups of twenty individuals each and reared in two separated pens. The control (CON) group without PTHL and the experimental group supplemented with PTHL (35g/day for each for the first 90 days of the trial and 70g/day for each for other 30 days). All the operations of rumen fluid sampling were performed at day 0, before starting the dietary inclusion of PTHL and then at 60 and 120 days from the inclusion of this supplement. Rumen fluid was obtained, at each sampling time, from 10 animals for each group, randomly chosen, using an esophageal tube under mild vacuum from the reticulum near the reticulo-omasal orifice. Samples of ruminal contents (filtered through eight layers of gauze cloth) were collected in thermos flasks (previously filled with distilled water at 39°C to avoid thermal shock to rumen fluid), insufflating in the headspace CO2 to ensure anaerobiosis environment, and taken within 30 min to the laboratory. Analysis of rumen microbiota was carried out by 16S, 18S and ITS1 sequencing. After quality filtering and chimera detection, reads from 16S and 18S sequencing were aligned against SILVA ribosomal RNA sequence database (https://www.arb-silva.de/) using MALT, while pre-processed ITS1 reads were aligned against UNITE fungal ITS sequencedatabase (https://unite.ut.ee/) using BLAST. Taxonomic binning was performed with MEGAN Community Edition software v6.12. The data set was subjected to two-way ANOVA, where feeding time and feeding group were set as independent variables. Values were given as mean values and standard error of the means (SEM). When a significant effect (P<0.05) was detected, means were compared using the Tukey´s test for repeated measures. Yeasts were not affected by the PTHL addition in the feed (P > 0.05). Differently, protozoa and methanogenic organisms showed a reduction in their number (P < 0.001). These observations need to be implemented with results about in vitro fermentation, gas prodction and apparent digestibility, in order to understand if there is a variation in animal performance and greenhouses gas production.