Livestock Research for Rural Development 31 (7) 2019 Guide for preparation of papers LRRD Newsletter

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Effect of a thymol-based additive on rumen fermentation, on methane emissions in eructed gas and on milk production in Holstein cows

Houda Hamdani, Najat Chami, Mounia Oukhouia, Imane Jabeur, Chaimae Sennouni and Adnane Remmal

Department of biology, Faculty of Science Dhar El-Mahraz, University Sidi Mohammed Ben Abdellah, PO Box 1796 Fez-Atlas, Morocco


The objective of this study was to assess the effect of a thymol-based feed additive as a means of modulating the rumen fermentation, the methane emissions and milk production in dairy cows. The additive was made by mixing 7% thymol with 48 % of bentonite clay, 30% stevensite clay, 10% Rosmarinus essential oil and 5% of olive oil. The first experiment was to evaluate in a rumen in vitro system the effect of different concentrations of the additive on gas production. In the second experiment, the additive was fed to Holstein dairy cows to test the effect on the emission of methane and milk production. Total gas production and percent methane in the gas in the in vitro rumen incubation was reduced curvi-linearly by increasing concentrations of the thymol additive. Supplementing the diet of lactating Holstein cows with 100 g/d of the additive reduced methane in eructed gas by 33% and increased milk production by 10%.

Keywords: antimicrobial, fermentation, gas production, milk production


In Morocco, most of the milk production comes from small regional farms (FAO 2019), but this production remains low knowing the expenses incurred by the breeders in the feeding of dairy cows. The low zootechnical productivity of these small cattle farms is the result of several factors related to the feeding of cows and livestock management (FAO 2019). The problem of methane emissions is neglected by farmers although if this could be reduced the benefits would be reduced greenhouse gas emissions with potential for associated improvement in ruminant production.

Current strategies to reduce methane focus primarily on understanding the microbial rumen ecosystem. Several studies have shown that essential oils can reduce enteric methane (Evans and Martin 2000; Benchaar et al 2006; Calsamiglia et al 2007) and increase milk production (Kung et al 2008).

Thymol is a common component of essential oils derived from Thymus and Origanum plants conferring on them antimicrobial properties (Burt 2004; Calsamiglia et al 2007; Marchese 2016). Thymol has been tested directly for its antibacterial activity on a broad spectrum of bacteria including rumen microorganisms (Evans and Martin 2000; Marchese 2016). Because of this antimicrobial action, thymol has bee shown to be capable of modifying the ruminal flora (Busquet et al 2006; Calsamiglia et al 2007).

Microbial digestion constitutes a major particularity in the ruminant species, characterized by the phenomenon of fermentation. This fermentation can be modified through feed and feed additives to create a better equilibrium of the microbial population in the rumen, thus optimizing growth and milk production (Benchaar et al 2008; Patra and Yu 2013).

The objective of this research was to evaluate the efficacy of a thymol-based additive to modulate the fermentation of the rumen fluid and to evaluate in vivo its effect on methane emissions and milk production in dairy cows.

Materials and methods

In vitro effect on gas production in the rumen fluid

The thymol-based additive was elaborated in the laboratory by mixing 7% of thymol with 48% bentonite clay, 30% stevensite clay, 10% of Rosmarinus essential oil and 5% of olive oil.

Ruminal content was collected by esophageal sampling from Holstein dairy cows and filtered through two layers of sterile gauze strips to remove plant debris and isolate the rumen fluid. Mueller Hinton agar (MHA) (biokar, France) and Mueller Hinton broth (MHB) (biokar, France) alone or supplemented with ruminal liquid were used for the gas production test.

In screw tubes containing 9 ml of MHB and 1 ml of ruminal liquid, the thymol additive was added at concentrations of 2.5, 5, 10 and 20 mg/ml of substrate. Three tubes were prepared for each concentration. Product activity was studied by the inverted tube technique in anaerobic jars (Durham 1898). The system consists of inverting the tube containing the fermentation mixture in the form of a bell in sterile tubes and incubating under anaerobiosis for 24h at 37C, at the end of which gas production is measured.

Effect of thymol additive on enteric methane emissions and milk production in Holstein cows

The 10 dairy cows of Holstein breed had a live weight between 360 and 420 kg. They were fed a daily ration of 3kg of alfalfa hay, 3kg wheat straw and 5kg of commercial concentrate. The ration was distributed in two meals per day.

The experiment was divided into three periods of three weeks each (2 weeks for adaptation to the feeding ration and one week for the measurement of the enteric methane emission). To test the dose response effect of the product on enteric methane production two different dose rates, 50g/cow/day in (Period 2) and 100g cow/day (Period 3) were used.

The production of methane was measured by indirect calorimetry (open circuit system) with a gas mask put on the face of the animal for several hours after the morning feeding. The gas handling system was essentially the same as that described by Kempton et al (1976). In this system air is drawn across the nose and mouth of the animal. A suction pump circulates the extracted gases in the tubes at an adjusted flow rate of 152 liters/min). A second sampling pump, connected to the system, takes exhaled air samples, which after dehumidification are returned to the methane analyzer which is previously calibrated. Calibration of the system consists of injecting nitrogen into the methane analyzer.

Effect of thymol additive on milk production

The trial was with 30 cows in lactation stage between 190 and 210 days after calving. The cows were divided into 2 groups: The experiment was carried out in two phases: a first pre-experimental phase of 30 days. A second experimental phase of 30 days. The control group received the standard ration alone; the thymol group received in addition to the standard ration, 100 g/cow/day of the additive. Individual daily milk production was measured at 8:00 am and 4:00 pm during the 60 days of the experiment.

Statistical analyses

The data were analyzed by the ANOVA program in the Sigma Stat 4.0 software.


There was a linear decline in gas production as the concentration of the thymol additive was increased to 20 mg/ml in the fermentation medium (Table1; Figure 1).

Table 1. Effect of thymol additive on gas production
in the in vitro rumen incubation
Thymol additive,
Gas production,
0 5 0.5
2.5 4.2 0.1
5 3 0.62
10 2.3 0.27
20 1.0 0.5

Figure 1. Gas production was reduced with a curvilinear trend as the
concentration of thymol additive was increased

Addition of 50 g/d thymol additive to the feed reduced the enteric methane emissions by 22% (Table 2); increasing the dose to 100 g/d reduced methane by 33%.

Table 2. Mean values with SE for methane emissions in cows fed 50 or 100g/day
of thymol additive or no additive (control)
Thymol additive, g/d
0 50 100 p
Methane, L/d 2009.8 1547.61 1346.68 <0.05

Supplementing the dairy cow diet with 100 g/d of thymol additive increased milk production by 10% (Table 3).

Table 3. Mean values for milk production prior to and during
inclusion of 100 g/d of thymol additive in the diet
Pre-experiment Experimental
Control 19.32.10 17.81.46
Thymol additive. 20.71.8 20.22.64


Thymol is a molecule that possesses high antimicrobial properties (Hart et al 2008, Calsamiglia et al 2007; Marchese et al 2016; Kokoska et al 2018). Our findings support those of many researchers (Evans and Martin 2000; Chaves et al 2008; Macheboeuf et al 2008; Rochfort et al 2008; Jahani Azizabadi et al 2011) that thymol reduces methane production in rumen in vitro incubations.

However, to our knowledge the rumen in vitro effects of thymol have not previously been projected to an in vivo production system, such as milk production in dairy cows. The increase in milk production recorded in our experiment, as a result of feeding a compound that depresses rumen gas production, in general, and methane production in particular, is similar to the improvements in N retention observed in goats supplemented with foliage from cassava varieties rich in precursors of hydrogen thiocyanate – substances known to have a depressing effect on rumen gas production and on methane in particular (Binh et al 2018; Thuy Hang et al 2019; Phuong ett al 2019). This unexpected relationship in which a depression of rumen fermentation is reflected in improved animal productivity was ascribed by Phonethep et al (2016) to be due to the more efficient utilization of protein by intestinal digestion versus rumen fermentation, and the fact that carbohydrate-rich substrate escaping the rumen would be fermented more efficiently in the cecum-colon in which the end product of fermentation is acetic acid – not methane (see Demeyer 1991; Immig 1996; Popova et al 2013).



This research is a partial achievement of Houda Hamdani’s PhD thesis. The authors thank Miss Imane Remmal and Mrs Hlne Mock for their assistance in checking the English of the manuscript.


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Received 2 May 2019; Accepted 12 June 2019; Published 2 July 2019

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