| Livestock Research for Rural Development 31 (4) 2019 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
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.
Chemical composition of sainfoin hay was determined by methods of AOAC (1990) and Van Soest et al (1991).
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 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 39.5°C 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.
One-way analysis of variance (ANOVA) was carried out using the General Linear Model (GLM) of Statistica for windows (1993).
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).
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).
AOAC 1990 Official Method of Analysis. Association of Official Analytical Chemists, 15th Edition, Washington, DC. USA.
Broderick G A and Kang J H 1980 Automated simultaneous determination of ammonia and total amino acids in ruminal fluid and in vitro media. Journal of Dairy Science, 63, 64-75. https://www.researchgate.net/profile/Glen_Broderick/publication/15825999_Automated_Simultaneous_Determination_of_Ammonia_and_Total_Amino_Acids_in_Ruminal_Fluid_and_In_Vitro_Media/links/5621ea3808ae93a5c927e392/Automated-Simultaneous-Determination-of-Ammonia-and-Total-Amino-Acids-in-Ruminal-Fluid-and-In-Vitro-Media.pdf
Chaves A V, He M L, Yang W Z, Hristov A N, McAllister T A and Benchaar C 2008 Effects of essential oils on proteolitic, deaminative and methanogenic activities of mixed ruminal bacteria. Canadian Journal of Animal Science, 88, 117-122. http://www.nrcresearchpress.com/doi/pdf/10.4141/CJAS07061
Cowan M M 1999 Plant products as antimicrobial agents. Clinical Microbiology Review, 12, 564-582. https://cmr.asm.org/content/cmr/12/4/564.full.pdf
Kilic U, Boga M, Gorgulu M and Sahan Z 2011 The effects of different compounds in some oils on in vitro gas production. Journal of Animal and Feed Science, 20, 626-636. http://www.jafs.com.pl/The-effects-of-different-compounds-in-some-essential-oils-on-in-vitro-gas-production,66221,0,2.html
Mbiriri D T, Cho S, Mamvura S and Choi N J 2016 Assessment of rumen microbial adaptation to garlic oil, carvacrol and thymol using the consecutive bath culture system. Journal of Veterinary Science and Animal Husbandry, 4(1), 1-7. http://www.annexpublishers.co/articles/JVSAH/4101-Assessment-of-Rumen-Microbial-Adaptation-to-Garlic-Oil,-Carvacrol-and-Thymol-Using-the-Consecutive-Batch-Culture-System.pdf
McEwan N R, Graham R C, Wallace R J, Losa R, Williams P and Newbold C J 2002 Effect of essential oils on ammonia production by ruminant microbes. Reproduction Nutrition Development, 42:S65 https://rnd.edpsciences.org/articles/rnd/pdf/2002/01/rnd200242supl1.pdf
McIntosh F M, Williams P, Losa R, Wallace R J, Beever D A and Newbold C J 2003 Effects of essential oils on rumen microorganism and their protein metabolism. Applied Environmental Microbiology, 69, 5011-5014. https://w ww.ncbi.nlm.nih.gov/pmc/articles/PMC169102/pdf/0337.pdf
Menke H H and Steingass H 1988 Estimation of the energetic feed value obtained from chemical analysis and in vitro gas production using rumen fluid. Animal Research and Development, 28, 7-55.
Molero R, Ibara M, Calsamiglia S, Ferret A and Losa R 2004 Effects of a specific blend of essential oil compounds on voluntary on dry matter crude protein degradability in heifers fed diets with different forage to concentrate ratios. Animal Feed Science and Technology 114, 91-104. https://ac.els-cdn.com/S0377840103003857/1-s2.0-S0377840103003857-main.pdf?_tid=b5c621b8-74be-4ddc-94ee-2dde762751d7&acdnat=1548737202_694aa5d9fddba8b7bf544d62061888ab
Newbold C,J, McIntosh F,M, Williams P, Losa R and Wallace R J 2004 Effects of specific blend of essential oil compounds on rumen fermentation. Animal Feed Science and Technology, 114.105-112. https://ac.els-cdn.com/S0377840103003845/1-s2.0-S0377840103003845-main.pdf?_tid=34905d8d-4913-4841-8bc5-edb2a43d07c6&acdnat=1548738329_48b38da03391e7e353047be0d233cdee
Patra A K and Zhongtang Y 2012 Effects of essential oils on methane production and fermentation by and abundance and diversity of microbial populations. Applied and Environmental Microbiology, 78(12), 4271-4280. https://aem.asm.org/content/aem/78/12/4271.full.pdf
Pawar M M, Kamra D N, Agarwal N and Chaudhary L C 2014 Effects of essential oils on in vitro methanogenesis and feed fermentation with buffalo rumen liquor. Agricultural Research, 31(1), 67-74. https://link.springer.com/content/pdf/10.1007%2Fs40003-014-0092-z.pdf
Phonethep P, Preston T R and Leng R A 2016 Effect on feed intake, digestibility, N retention and methane emissions in goats of supplementing foliages of cassava (Manihot esculenta Crantz) and Tithonia diversifolia with water spinach (Ipomoea aquatica). Livestock Research for Rural Development. Volume 28, Article #72. http://www.lrrd.org/lrrd28/5/phon28072.html
Roy D, Tomar S K, Sirohi S K, Kumar V and Kumar M 2014 Efficacy of different essential oils in modulating rumen fermentation in vitro using buffalo rumen liquor. Veterinary World, 7(5), 213-218. http://www.veterinaryworld.org/Vol.7/April-2014/5.pdf
Sallam S M A and Abdalla A I 2011 Antimethanogenic and antiprotozoal effect of some essential oils in vitro. Egyptian Journal of Animal Production, 48(2), 203-215. https://pdfs.semanticscholar.org/33ed/f6e0159617576a8275bcce033899dbe0770c.pdf?_ga=2.65215278.1613242863.1548675214-899502509.1545134815
Statistica 1993 Statistica for Windows release 4.3, StatSoft, Inc. Tulsa, Oklahoma, USA
Van Soest P J, Robertson J B and Lewis B A 1991 Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74(10), 3583-3597. http://www.journalofdairyscience.org/article/S0022-0302(91)78551-2/pdf.
Received 7 February 2019; Accepted 25 February 2019; Published 1 April 2019