Dose-response and inclusion effects of pure natural extracts and synthetic compounds on in vitro methane production

This study explored the effects of one ionophore compound (monensin) and four pure extracts (limonene, allyl sulfide, cinnamaldehyde, and eugenol) on in vitro rumen fermentation and methane (CH4) production of a commercial diet for dairy cows. Four consecutive incubations at 24 h were conducted using an automated gas production (GP) system, where gas was vented at fixed pressure. Bottles (317 ml) were filled with 1.0 ± 0.010 g of diet and 150 ml of buffered rumen fluid. Additives were added at two dosages: 0.015 (low) or 0.030 mg/g of diet (high) for monensin, 3 (low) or 30 mg/g of diet (high) for the pure extracts. In each incubation, eleven treatments were tested in 3 replications: a control (CTR; bottles without additive), plus 5 additives × 2 dosages. Twelve bottles (3/run), containing only the buffered rumen fluid, were included as blanks. At the end of each incubation, gas samples were collected from bottle headspace and analyzed for CH4. The proportion of CH4 lost with venting was calculated. Fermentation fluids were analyzed for aNDF (NDFd) and true DM (TDMd) degradability, ammonia N, and VFA. Data were analyzed using a model considering the treatment as fixed factor and run as a random factor. Additives were ineffective at the low dosage. Compared to CTR, the high dosage of monensin reduced NDFd (−17%), TDMd (−3%), and CH4 produced per g of truly degraded DM (−19%). Acetate was reduced (−13%) in favor of propionate (+25%), but the total VFA production did not change. Compared to CTR, the high dosage of limonene reduced NDFd (−66%), TDMd (−14%), GP (−25%) and CH4 production (−34%) per g of truly degraded DM, and the total production of VFA (−25%), acetate (−31%), and propionate (−13%) (P < 0.001 for all). The high dosage of allyl sulfide did not influence NDFd and TDMd, reduced GP (−14%) and CH4 production (−32%) per g of truly degraded DM, the total VFA (−12%) and acetate production (−24%), but increased that of propionate (+15%). The high dosage of cinnamaldehyde did not influence NDFd, TDMd or the total VFA production, reduced GP (−10%), CH4 production (−12%) per g of truly degraded DM and acetate production (−14%), but increased that of propionate (+9%). The high dosage of eugenol reduced only NDFd (−18%) and TDMd (−3%). In conclusion, the most promising results were observed for the high dosage of cinnamaldehyde, as in vitro CH4 production was reduced without effects on degradability and VFA production. However, these effects should be confirmed using dosages and experimental conditions proper of in vivo trials.