Potential of walnut (Juglans regia) leave ethanolic extract to modify ruminal fermentation, microbial populations and mitigate methane emission

Series of in vitro trials were conducted to evaluate dose–response effects of walnut leaf ethanolic extract (WLEE) on ruminal fermentation, microbial populations, mitigation of methane emission and acidosis prevention. The treatments were conducted according to a 5 × 3 factorial arrangement in a completely randomised design formulated to contain corn (corn-based diet, CBD) and barley grain (barley-based diet, BBD), or equal amounts of barley and corn (barley and corn diet, BCD), consisting of either basal diets alone (0) or basal diets with 250, 500, 750 or 1000 µL of WLEE (W0, W250, W500, W750 and W1000 respectively) per litre of buffered rumen fluid. Three fistulated cows fed diets containing alfalfa hay and concentrate mixes (same as the control diet) plus minerals and vitamins were used for collection of ruminal fluid. The asymptote of gas production and methane emission was decreased and lag time increased in a linear and quadratic manner with an increasing dose of WLEE (P < 0.001). However, gas production rate reduced linearly as WLEE dose increased (P < 0.001). Methane production was significantly reduced linearly (L) and quadratically (Q) when walnut ethanolic extract was increased from 250 to 1000 μL/L (L and Q; P < 0.001). The addition of WLEE significantly altered the volatile fatty acid profile in comparison to control, reducing the molar proportion of acetate and increasing that of propionate (P < 0.001), and also decreased the ammonia-N concentration (L, P < 0.001). Dry-matter and organic-matter in vitro digestibility coefficients were negatively affected by WLEE supplementation (L and Q; P < 0.001). Although anti-acidosis potential of WLEE was significantly lower than that of monensin, W1000 increased medium culture pH compared with uncontrolled acidosis and the lower doses of WLEE. The populations of Fibrobacter succinogenes, Ruminococcus flavefaciens and R. albus were significantly reduced by WLEE, although to different magnitudes, depending on the corn and barley grain proportions in the diet. Results of the present study indicated that increasing addition levels of WLEE have noticeable effects on rumen microbial population and fermentation characteristics. It can be concluded that WLEE can potentially be used to manipulate ruminal fermentation patterns.