Assessing the Impact of the Methane Inhibitors 3-Nitrooxypropanol (3-NOP) and Canola Oil on the Rumen Anaerobic Fungi

Reducing enteric methane emissions is critical for improving the sustainability of ruminant livestock production. In this study, we investigated the impact of the methane inhibitors 3-nitrooxypropanol (3-NOP) and canola oil, fed both individually and in combination, on the anaerobic gut fungi (AGF) of the rumen. Eight ruminally cannulated Angus heifers were used in a replicated double 4 × 4 Latin square over 28-day periods with a 2 (control, 3-NOP) × 2 (control, canola oil) factorial arrangement. Rumen samples were collected after 13 d dietary adaptation, and AGF communities were evaluated using amplicon sequencing of the D1/D2 region of the 28S rRNA (LSU) gene. Although 3-NOP reduced methane yield by approximately 32%, it did not substantially alter the diversity, composition, or overall abundance of the AGF community. In contrast, canola oil supplementation, either alone or combined with 3-NOP, markedly disrupted the fungal community. These treatments reduced overall fungal diversity and the abundance of key fiber-degrading taxa, such as <i>Neocallimastix</i> and <i>Piromyces</i>, while eliciting variable responses among less abundant genera. Furthermore, resilience analyses using control-diet-fed samples indicated that repeated perturbation impaired the recovery of some AGF taxa, leading to a shift in the composition of the fungal community. Overall, our findings suggest that 3-NOP offers a targeted methane mitigation strategy and does not alter the rumen AGF. In contrast, the addition of canola oil at levels that inhibit enteric methane emissions has a disruptive impact on the AGF community, contributing to reduced feed digestibility.