Effect of different doses of several mixtures of three essential oils of peppermint, thyme and rosemary on rumen fermentation parameters of fattening diet by in vitro method

The use of food additives such as antibiotics is a useful tool in reducing energy loss (methane) and nitrogen (ammonia). However, the use of antibiotics in animal feed has been banned in the EU since January 2006 due to their residual risk in milk and meat and its subsequent effects on human health (Aminipour et al. 2017). Essential oils of herbals are used because of their antimicrobial effect against bacteria, protozoa and fungi, their ability to manipulate rumen fermentation, and their potential to reduce methane production in ruminant diets (Gunal et al. 2017). The results of research show that peppermint essential oil reduces methane, ammonia nitrogen concentration, number of protozoa and changes the molar ratio of short-chain fatty acids (Ahmad et al. 2014). Some experiments show that thyme essential oil reduces the production of gas (Roy et al. 2015), methane (Baraz et al. 2018). Although there is useful information about the effects of peppermint, thyme and rosemary essential oils on their positive effect on rumen fermentation, a study examining the effect of a mixture of these essential oils is not available. Therefore, this study was conducted to evaluate the effect of different doses of three mixes of peppermint, thyme and rosemary essential oils (6, 12 and 18 µl) on in vitro gas production parameters.

The essential oils were mixed in three levels of 150, 300, 450 µl, with three different ratio including A=(1: 3: 2), B=(2: 1: 3) and C=(3: 2: 1); thereafter, the effect of different doses (6, 12, 18 μl) of these three combinations on fermentation parameters was studied. Rumen fluid was obtained from three sheep before morning feeding via rumen fistula. The effect of the mixture of essential oils on gas production and kinetics of gas production was investigated by incubating 125 mg of each sample at 2, 4, 6, 8, 12, 24, 36, 48, 72 and 96 hours. Also, in another experiment, by incubating the samples for 24 hours, gas production and methane production were estimated. Also, different mixtures were compared in terms of their effect on reducing the amount of methane production.

Adding a mixture of essential oils in different doses reduced gas production compared to the control group (P<0.05). Comparison of the three mixtures of essential oils tested showed that during the incubation period of 2 to 12 hours, mixtures B and C showed a decreasing trend in gas production, meanwhile mixture C had the lowest value compared to the other two mixtures (P<0.05). There was no significant difference between mixtures A and B during 24 to 48 hours of incubation, but mixture C showed the lowest value compared to the other two mixtures (P<0.05). At 72 to 96 hours of incubation, mixtures A, B and C did not show significant differences in gas production (P<0.05). The results of this experiment showed that mixture C caused a further reduction in gas production than other mixtures. Decreased gas production can be related to a decrease in the fermentation activities of microorganisms and a decrease in dry matter digestibility (Tan et al. 2011). On the other hand, reduction in gas production by essential oils may indicate more efficient use of energy due to inhibition of energy loss in the form of methane (Aminipour et al. 2017). In the present experiment, different doses of essential oil mixtures reduced gas production, so that at 2 to 96 hours of incubation, doses of 6 and 12 did not differ significantly in terms of gas production (P <0.05); but the level of 18 µl compared to other doses showed a significant decrease in gas production (P <0.05). The mixture of essential oils at the level of 6 μl showed that mixture A, which contained the highest amount of thyme and rosemary essential oils, had no effect on gas production during incubation compared to control, while mixture C, which contained higher amounts of peppermint and Thyme essential oils had the greatest impact on gas production. The gas production potential (B) was not significantly different between the three mixtures (P>0.05), although numerically it was the lowest in mixture C (19.53 versus 11.82). Gas production rate (c) had a similar trend so that despite the lack of significant differences between the three mixtures (P>0.05), numerically C mixture showed the lowest value in terms of this parameter. A decreasing trend in methane production at the level of 18 μl of essential oil of a mixture of three herbals was observed, so that in mixture B was the lowest value compared to the control group (P <0.05). Essential oils may directly inhibit the growth and activity of methanogenic microbes, or indirectly by reducing the number of methanogen-related protozoa (Gonal et al. 2017) or by indirect reduction in some processes of methanogenic microbial metabolism (Cobellis et al. 2015) affect methane production.Due to the fact that the proportion of two thirds of mixture B is peppermint essential oil, the percentage of methane production in mixture B compared to the control showed a significant decrease. It has been reported that the decrease in methane production may be due to the reduction of total protozoan by menthol as the main active ingredient of peppermint essential oil (Roy et al. 2015).

The results of this study showed that in the gas production test at 2 to 96 hours, all three levels of 6, 12 and 18 microliters significantly reduced gas production at all incubation times, even 24 hours, so At the level of 18 microliters, the largest decrease in gas production was observed. Of the three mixtures used, mixture B showed the greatest effect in reducing methane production. Therefore, a dose of 18 μl with a decrease in total gas production and mixture B with a dose of 18 μl with a decrease in methane production has the potential to change ruminal fermentation.