Running head: Oxalic acid, ruminal fermentation and microbiota Impact of oxalic acid on rumen function and bacterial community in sheep

Oxalic acid (OA) is a secondary compound occurring in a wide range of plants consumed by ruminants, especially in saline lands or in arid and semi-arid regions. However, its impact on the rumen microbial community and its changes over time, as well as the potential consequences on ruminal function remains unknown. To examine this impact, five ewes fitted with a ruminal cannula and fed low quality grass hay, were daily dosed 0.6 mmol of OA/kg body weight, through the cannula, for 14 days. On days 0 (before the start), 4, 7 and 14 of the administration period, samples of ruminal digesta were collected throughout the day (0, 3, 6 and 9 h after the morning feeding) for analysis of the bacterial community and fermentation parameters (pH, and ammonia and volatile fatty acid, VFA, concentrations). In addition, two feedstuffs were incubated in situ, using the nylon bag technique, to estimate ruminal degradation. Terminal restriction fragment length polymorphism (T-RFLP) was employed to monitor the dynamics of total bacteria, and quantitative real-time PCR to investigate the abundance of the oxalate-degrading Oxalobacter formigenes. Neither pH nor total VFA concentrations were affected. Nevertheless, OA dosing altered molar proportions of most individual VFA and ammonia concentrations (P<0.001). The dry matter disappearance of alfalfa hay was reduced on days 7 and 14 and that of barley straw only on day 7 (P<0.01). These slight changes were related with others observed in the relative frequency of a number of terminal restriction fragments. Variations in the ruminal microbiota occurred rapidly with OA administration, which did not significantly modify the bacterial diversity but altered the structure of the community. However, many of these changes were reversed by the end of the experiment, with no significant differences between days 0 and 14 of dosing. These results suggest a rapid adaptation of the rumen bacterial 3 community linked to the estimated increase in the abundance of O. formigenes (from 0.002 to 0.007% of oxc gene in relation to the total bacteria 16S rDNA; P<0.01), which is assumed to be responsible for oxalate breakdown. Implications This study shows that the rumen bacterial community adapts rapidly to the consumption of oxalic acid, a potentially toxic secondary compound occurring in a wide range of plants. Oxalate-rich plants may become important forage resources for ruminants when other feedstuffs are in short supply, especially in saline lands or in arid and semi-arid regions. In such occasions, the …