Livestock Research for Rural Development 31 (4) 2019 Guide for preparation of papers LRRD Newsletter

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Supplementing sainfoin (Onobrychis viciifolia) hay with garlic oil; effects on rumen in vitro gas production, digestibility and ammonia production

E Kirisci and A Kamalak

Department of Animal Science, Faculty of Agriculture, University of Kahramanmaras Sutcu Imam, Kahramanmaras, Turkey


In an in vitro incubation of sainfoin hay, the addition of garlic oil over the range 0 to 1.2 g/liter of rumen in vitro fluid volume, decreased linearly the in vitro rumen digestibility of DM and NDF of sainfoin hay decreased the production of rumen ammonia and slightly increased the rumen pH. The results imply that garlic oil inhibited the overall fermentation by the rumen microbiota.

Key words: bypass protein, cecal fermentation, deamination, essential oils, protein degradation, rumen pH


After the ban of antibiotics as growth promoters in animal feed in the European Union in 2006, researchers have directed attention to use of “essential” or “therapeutic” oils as alternatives to antibiotics, due to their perceived antimicrobial, antifulgal and antioxidant properties (Cowan 1999). These oils have been used to modulate ruminal microbial activities, reduce protein degradation and increase rumen bypass protein (McEwan et al 2002). Essential oils exert their effects on nitrogen metabolism through the inhibition of deamination and prevention of attachment and colonization of feed by proteolytic bacteria (Molero et al 2004). A number of in vitro studies showed that garlic oil decreased rumen digestion and ammonia production (Chaves et al 2008; Pawar et al 2014; Roy et al 2014); by contrast, in one study, the effect was mainly on rumen gas production (Patra and Zhongtang 2012).

The aim of the current experiment was to provide specific information on the effects of garlic oil on rumen digestion of sainfoin hay.

Materials and Methods

Chemical analysis of sainfoin hay

Chemical composition of sainfoin hay was determined by methods of AOAC (1990) and Van Soest et al (1991).

In Vitro Gas Production

Sainfoin hay samples were milled through a 1 mm sieve and incubated in vitro with rumen fluid in calibrated glass syringes following the procedures of Menke and Steingass (1988). Rumen fluid was obtained from three fistulated sheep fed twice daily with a diet containing alfalfa hay (60%) and concentrate (40%). Samples of sainfoin hay (200mg) were put in triplicate glass syringes of 100 mL in the presence garlic oil (Sigma-Aldrich) at concentrations of 0, 50, 100, 200, 400, 800 and 1200 mg/L. The syringes were prewarmed at 39 oC before the injection of 30 mL rumen fluid-buffer mixture into each syringe followed by incubation in a water bath at 39 oC. Gas production was recorded before incubation and at 3, 6, 12, 24, 48, 72 and 96 h after incubation. Total gas values were corrected for blank incubations. At the end of each incubation, the pH and ammonia in the culture fluid were determined, the latter by the spectrophotometric method described by Broderick and Kang (1980).

In Vitro Digestibility

In vitro digestibility was carried out with the DAISY Incubator using rumen fluid obtained from the same fistulated sheep used in the gas production experiment. Rumen fluid (400 ml) was transferred to a digestion jar containing 1600 ml buffer solution (pH 6.8) and heat-sealed bags containing sainfoin hay. The bags were incubated in triplicate at 39.5C for 48 h in the presence (100, 200, 400, 800 and 1200 mg/L) and in the absence of orange oil (Sigma-Aldrich). At the end of the 48h incubation period, the bags were rinsed with cold water and dried at 105oC to determine DM digestibility of the hay. The dried bags were then placed in the ANKOM200/220 Fiber Analyzer and subjected to the normal procedure for determining NDF content of the residue in the bags.

Statistical analysis

One-way analysis of variance (ANOVA) was carried out using the General Linear Model (GLM) of Statistica for windows (1993).

Result and discussion

The sainfoin hay had 92.8 % organic matter, 16.4 % crude protein, 3.29% ether extract, 52.8% NDF and 39.0% ADF (expressed as % DM basis).

Gas production

Addition of garlic oil decreased the gas production at all incubation times ((Table 1; Figure 1), in line with the findings of Kilic et al (2011), Sallam and Abdalla (2011), Patra and Zhongtang (2012) and Roy et al (2014). The greater part of the effect was found in the first 24h of incubation.

Figure 1. Effect of increasing levels of garlic oil on gas production over 96h

This result agrees with the findings of Sallam and Abdalla (2011) and Roy et al (2014).

Table 1. The effect of garlic oil on in vitro gas production after 24h incubation, and on % digestibility of DM and NDF, ammonia (mg/liter) and pH after 48h incubation
Doses (mg/L) Gas, ml DMD NDF NH3-N pH
0 60.4a 72.1a 64.7a 36.6a 5.9f
50 56.2b 70.5ab 65.0a 35.0a 6.0e
100 54.7bc 68.0b 58.3b 30.3a 6.0e
200 53.0c 62.0c 51.5c 26.4c 6.2d
400 52.5c 56.6d 47.5d 24.0d 6.2c
800 38.8d 50.4e 42.6e 20.6d 6.3b
1200 33.7e 44.8f 41.0e 17.7e 6.4a
SEM 0.714 0.943 1.036 0.808 0.020
p <0.001 <0.001 <0.001 <0.001 <0.001
abcdef Column means with common superscript, digestibility do not differ at p>0.05

There were linear decreases in gas production, in DM and NDF in vitro digestibility and in ammonia concentration with increasing level of garlic oil addition (Table 1 and Figures 2-4).

Figure 2. The relationship between garlic oil supplementation and gas production at 24 h

Figure 3. The relationship between garlic oil supplementation and NDF digestibility

Figure 4. The relationship between garlic oil supplementation and ammonia production

The decreases in gas production and in digestibility of DMM and reduction in ammonia indicate the antimicrobial activity of garlic oil causing an inhibition of the overall fermentation process.

The fact that NDF and ammonia production were reduced by garlic oil would suggest that fibrolytic and proteolytic bacteria seem to be very sensitive to this additive. It was reported that essential oil, or their active components, may conserve amino acids from ruminal degradation by inhibiting microbial deamination (McIntosh et al 2003; Newbold et al 2004). However, Molero et al (2004) suggested that the essential oils exert their effect on nitrogen metabolism through the inhibition of proteolytic activity and prevention of attachment and colonization of feed by proteolytic bacteria. McEwan et al (2002) also suggested that essential oil supplementation resulted in a reduction in the number and diversity of hyper-ammonia producing bacteria, resulting in reduced rate of ammonia production from amino acids. The increase in pH with supplementation of garlic essential oil is consistent with earlier studies of Patra et al (2012) and Mbiriri et al (2016).

The reduction in rumen DM digestibility and in protein deamination with garlic oil supplementation may have several consequences in practice. Reduction in rumen digestibility may be compensated by increase in cecal digestibility with concomitant benefits from reduced methane production, as proposed by Phonethiep et al (2016). On the other hand, reduced deamination may result in more rumen bypass of protein. It is important that these studies are repeated with animal feeding trials.


Garlic oil over the range 0 to 1.2 g/liter of rumen fluid decreased linearly the in vitro rumen digestibility of DM and NDF, decreased the production of rumen ammonia and slightly increased the rumen pH.


This study was supported by the Scientific Research Project Committee of Kahramanmaras Sutcu Imam University, Turkey (Project No: 2016/6-10 YLS).


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Received 7 February 2019; Accepted 25 February 2019; Published 1 April 2019

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