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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.54 No.3 pp.55-61
DOI : https://doi.org/10.14397/jals.2020.54.3.55

Effects of Garlic Powder Allicin Mixture on the in Vitro and in Vivo Rumen Fermentation of Hanwoo Steers

Seon-Ho Kim, Ashraf Ali Biswas, Mahfuzul Islam, Sang-Suk Lee*
Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, Sunchon National University, Suncheon 57922, Republic of Korea
*Corresponding author: Sang-Suk Lee Tel: +82-61-750-3237 Fax: +82-61-750-3237 E-mail: rumen@sunchon.ac.kr
May 8, 2020 ; May 30, 2020 ; June 1, 2020

Abstract


This study evaluated effects of the addition of garlic powder allicin (GPA) mixture on rumen fermentation with methane in Hanwoo steer. On in vitro trial, two experimental groups were used: control (without GPA) and treatment group (addition of 0.1% GPA mixed with the basal concentrate). Similar to in vitro trial, two experimental groups were used in vivo trial. Five Hanwoo steers (3 steers in one group and 2 in another group; average body weight = 500 ± 43 kg) were assigned by crossover design for 20 d consists of 15 d diet adaptation and 5 d data collection in each experimental period. Daily feed intake and enteric methane production were recorded by an automated head chamber system. The results of in vitro study showed that the GPA treatment group had higher acetic acid (24.30 vs 23.45 mmol/L) and butyric acid (16.55 vs 15.47 mmol/L) concentrations, but lower CH4 production (1.40 vs 2.71 mmol/ml) after 24 h of incubation compared to the control (p<0.05). Total gas, propionic acid, total volatile fatty acid (VFA), and acetic acid: propionic acid ratio were not affected by treatment after 24 h incubation. In the in vivo experiment, rumen pH and VFA were not significantly different between treatments (p>0.05), except acetic acid, which was significantly higher in GPA mixture group (60.97 vs 53.94 mM) than in the control group (p<0.05). Furthermore, no significant differences were recorded in CH4 production (g/d) and CH4 yield (g/kg DMI) between the two groups (p>0.05). In conclusion, the addition of 0.1% GPA mixture reduced CH4 proudcition on in vitro trial, but no effect on in vivo trial.



초록


    Introduction

    Garlic (Allium sativum) is one of the plant herb having a variety of functions such as antimicrobial, antioxidant, anti-inflammatory, and immunostimulatory functions, as well as maintaining the microbial ecosystem of the digestive tract (Mirzaei- Aghsaghali et al., 2012). Garlic contains different plant secondary derivatives, including diallyl sulfide (C6H10S), allicin (C6H10S2O), allyl mercaptan (C3H6S), and diallyl disulfide (C6H10S2) (Lawson et al., 1991). Allicin (allyl 2-propene thiosulfate), the main bioactive antimicrobial agent of garlic, is an intermediate volatile component that decomposes rapidly into other compounds such as diallyl sulfide, diallyl disulfide and diallyl trisulfide, dithins, and ajoene (Amagase et al., 2001).

    Methane (CH4), one of the important greenhouse gas, is produced by ruminants after feed fermentation (Islam and & Lee, 2019) that represents a gross dietary energy loss of 2-12% (Johnson and & Johnson, 1995) from an animal. So, prevention of dietary energy loss is one of the key elements to enhances animal productivity by reducing enteric methane production by the animal. As a management strategy, feed additives are commonly used in ruminant feeds to improve animal health and performance. Recently, natural feed additives especially plant secondary metabolites (PSMs) such as tannins, saponins, organosulphur compounds, flavonoids, and essential oils play significant role on rumen fermentation, rumen bacterial community composition and CH4 production (Teferedegne, 2000;Patra et al., 2006;Wanapat et al., 2008;Islam &and Lee, 2018) in ruminants which ensure improved productivity.

    Garlic powder is making through a series of process including slicing, drying and then pulverizing of garlic cloves and finally, allicin is produced after addition of water. These compounds are capable of changing in vitro rumen fermentation parameters, such as decreasing the acetate concentration and increasing pro-pionate and butyrate concentrations, and decreasing methane (CH4) production as well as the CH4:volatile fatty acid ratio (VFA) (Busquet et al., 2005a;Busquet et al., 2005b). Garlic powder has the potential to decrease CH4 production in both in vitro (Mirzaei-Aghsaghali et al., 2011) and in vivo rumen fermentation (Kongmun et al., 2011). However, some studies have stated that supplementation of garlic had no effect on total VFA and ruminal pH (Yang et al., 2007), and failed to decrease methane (Klevenhusen et al., 2011a). Controversy exists regarding the exact dose required for ruminant diets to advance rumen fermentation, as well as to reduce enteric methane emission. For instance, Busquet et al. (Busquet et al., 2005a) stated that garlic oil at a rate of 312 mg/l of culture fluid (standardized at 0.7% allicin) had a positive effect on rumen fermentation in vitro by reducing CH4; however, the supplementation of allicin at 2 g/head/d effectively increased the diet digestibility and daily CH4 emission (l/kg BW0.75) of ewe in vivo (Ma et al., 2016). In addition to these contradictory results, there is a lack of information about the effect of the addition of a minute quantity of garlic powder allicin (GPA) mixture on in vitro and in vivo rumen fermentation as well as methane mitigation from an economic point of view. Therefore, the study was conducted to evaluate the effects of the addition of 0.1% GPA mixture to the diets of Hanwoo steers through an in vitro fermentation technique and in vivo experiment, with a focus on methane mitigation.

    Materials and Methods

    The study was conducted at the animal farms and the Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology of Sunchon National University (SCNU), Jeonnam, South Korea. The in vitro and in vivo digestion trials were conducted with the addition of GPA mixture to the pelleted concentrate feed.

    1. In vitro experiment

    The in vitro batch culture included two different groups: the control and the 0.1% GPA mixture treated groups. The commercial concentrate pellets (Nonghyup Co., Haman, Korea) were used as a basal substrate (control), while the addition of the 0.1% GPA mixture to the basal substrate on a dry matter (DM) basis was considered as the treatment group. The GPA mixture was purchased from Nonghyup Company, Haman, Korea. Holstein cows (650 kg; 48 months old), which was cannulated ruminally, served as the rumen fluid donor for in vitro fermentation trial. The cow was fed the commercial concentrate pellet and rice straw at a ratio of 2:8 twice daily. The crude protein (CP) and total digestible nutrients (TDN) of rice straw were 4.45% and 38.29%, respectively, and the concentrate pellet had 12% CP and 72% TDN on a DM basis. The whole ruminal contents were collected through a cannula before the morning feeding at 07:00 h and strained through four-layer cheesecloth. The rumen fluid was placed in a glass container with a cap and transferred immediately to the laboratory, with the temperature maintained at 39 ℃. The bottle was shaken vigorously before its contents were mixed with a buffer solution. The final inoculant was prepared through the mixing of rumen fluid and a buffer medium with maintained pH 6.9, according to Asanuma et al. (1999), at a ratio of 1:3. The composition of the buffer solution is 0.45g K2HPO4 , 0.45g K2HPO4, 0.9g (NH4)2SO4, 0.12g CaCl22H2O, 0.19g MgSO47H2O, 1.0g trypticase peptone, 1.0g yeast extract and 0.6g cysteine. Under a constant flow of N2 gas, 100 mL of the inoculant was transferred anaerobically into 160 mL serum bottles that contained 1 g of the basal substrate (particle size = 1 mm), which were supplemented with or without 0.1% GPA (equivalent to 0.01mg/L of rumen fluid). Immediately after inoculation, all serum bottles were sealed by rubber stoppers and aluminum caps, and finally incubated in a shaking incubator (HB-201 SF; Han Baek Scientific Co., Buchon, Korea) for 4, 12 and 24 h at 100 rpm and 39 ℃. The serum bottles were prepared in quintuplets for each treatment and time points. At each incubation time point, total gas (TG) amount was measured, and subsequently sampled into evacuated tubes and preserved in a refrigerator until the CH4 was determined. Then, in vitro rumen fluid was collected to determine the volatile fatty acids (VFAs) and ammonia nitrogen (NH3-N) from individual bottles at each incubation time point.

    2. In vitro sample analyses

    At each incubation time point, the in vitro fermentation parameters were measured. The TG production was measured by a pressure sensor meter (EA-6; Sun Bee Instrument Inc., Seoul, Korea). Two separate aliquots of 1 mL samples from each serum bottle were collected and centrifuged at 13,000 × g for 10 min at 4 ℃ (Micro 17TR centrifuge; Hanil Science Industrial Co. Ltd., Gimpo, Korea). The obtained supernatants were collected into new 1.5 mL micro tubes and stored at -80 ℃ until the NH3-N and VFA analysis. The NH3-N was determined following the protocol of Chaney and & Marbach (1962) using a Libra S22 spectrophotometer (CB40FJ; Biochrom Ltd., Cambourne, UK) and the VFAs were measured according to the methods stated by Han et al. (2005) and Tabaru et al. (1988) using high performance liquid chromatography (HPLC; Agilent Technologies 1200 series, Waldbronn, Baden-Wurttemberg, Germany). The concentration of CH4 in the gas samples was analyzed by using gas chromatography (GC; HP 5890; Agilent Technologies, San Diego, CA, USA). Before the analysis, the vacuum tubes containing the refrigerated gas samples were warmed by stabling it on the GC machine for at least 30 min.

    3. In vivo experiment

    Five Hanwoo steers (average body weight of 500 ± 43 kg) were randomly devided into two groups (3 steers in group and 2 in another group) and used on in vivo experiment for 2 periods under crossover design. The experiment consisted of a total of 20 d, including 15 d adaptation followed by 5 d for sample collection per period. The control diet included a basal pelleted feed (Table 1) and the treatment diet included mixing of 0.1% GPA mixture (DM basis) with the basal diet. All steers were housed in same environmental conditions with free movement and access to eat feed and drink water. The GreenFeed automatic feeding system (C-Lock Inc., Rapid City, SD) was used to supply the pelleted concentrate to the experimental steers. The GreenFeed system allowed only one steer at a time to eat pelleted concentrate feed. During visit to the GreenFeed, one steer could consume 50 g of pelleted concentrate by one drop from the machine at 30 s intervals and a radio frequency identification (RFID) tag number for individual animal controlled the maximum number of drops per animal per day. The animals used in this experiment and all experimental methods were reviewed and approved by the Animal Research Ethics Committee of Sunchon National University (SCNU IACUC approval number: SCNU IACUC-2017-03).

    4. In vivo data collection and sample analyses

    The CH4 production was checked by the GreenFeed system for individual steers over a period of 5 d. The GreenFeed system constantly measured the airflow rates and the gas concentrations. The daily feed intake and the CH4 production of individual animals were calculated by the GreenFeed software system based on their RFID tag number. The rumen fluid was collected through stomach tubing at day 21 (at 09:00 h). Immediately after sample collection, the pH was measured by a pH meter (Pinnacle series M530p; Schott Instruments, Mainz, Germany). An aliquot of the rumen sample was separated and analyzed for the VFA concentration using HPLC (Agilent Technologies 1200 series, Waldbronn, Baden-Wurttemberg, Germany).

    5. Statistical analysis

    Analysis of two groups of experimental data was performed by using t-test of Statistical Analysis System (version 9.4) (SAS, 2013). The significant differences between groups was accepted while p<0.05.

    Results and Discussion

    1. In vitro experiment

    The total gas production was not significantly different between control and treatment at all incubation time points (Table 2). In this in vitro trial, CH4 production was not significantly different at the 4 h incubation periods (Table 2); however, significantly lower CH4 production was observed at 12 h in the GPA group compared to the control (1.15 vs 2.72 mmol/ml for GPA vs the control, respectively; p<0.05). Likewise, lower CH4 production was recorded in the GPA group than in the control after 24 h at 1.40 mmol/ml and 2.71 mmol/ml, respectively (Table 2). After 24 h of incubation, no significant differences were detected between groups in the case of propionic acid, the total VFA and the A:P ratio. The GPA group produced significantly more acetic acid (24.30 vs 23.45 mM for the GPA treatment compared to the control, respectively) and butyric acid (16.55 vs 15.47 mM for the GPA treatment compared to the control, respectively) at the 24 h time point (p<0.05; Table 2).

    From the in vitro experiment, GPA mixture significantly increased acetate and butyrate production, and numerically increased the total VFA and propionate production after 24 h of incubation, which may be indicated the potential of GPA in ruminant feed fermentation in vivo. In the in vitro experiment, the total gas production were not affected by GPA mixture. However, significantly lower methane production was observed in the GPA group over the incubation time, which indicated that the addition of 0.1% GPA mixture had a potential effect on methane reduction in vitro. This finding was in strong agreement with that of Hart et al. (2006), who investigated whether commercially available aqueous allicin product has any effect in the rumen simulation technique (RUSITEC). A decrease in methane production was observed (up 94% in the 20 mg/L allicin addition) at 2 and 20 mg/l of allicin, The results of the in vitro experiment were also strongly supported by Kongmun et al. (2010), who reported that garlic powder could increase the propionate and butyrate in ruminal fluid and decrease CH4 production. In contrast, Busquet et al. (2005b) reported that allicin could only significantly increase propionate at an inclusion level of 3,000 mg/L of culture fluid after 24 h of in vitro incubation; however, no effect was observed at 300 mg/L and below this level of inclusion. In addition, the acetate, butyrate, and total VFA were not significantly affected up to 3,000 mg/L of culture fluid. These findings indicated that the effect of the garlic mixture on methane mitigation and rumen fermentation depend on diet and dose.

    2. In vivo experiment

    The values of pH, propionic acid, butyric acid, the total VFA and the A:P ratio were not differ significantly (p>0.05). Acetic acid concentration was significantly higher in the GPA group compared to the control group (60.97 vs 53.94 mM; p<0.05; Table 3). No significant difference was recorded in CH4 production (g/d) and CH4 yield (g/kg DMI) between the control and the treatment group (p>0.05). CH4 production was 142.73 g/d and 164.10 g/d, while CH4 yield was 23.73 and 25.22 g/kg DMI in the control and GPA group, respectively (Table 3).

    The result of the in vivo experiment showed that acetate production was significantly higher in the GPA mixture group than in the control group. Kamel et al. (2008) reported similar results when allicin was added to an alfalfa hay:concentrate (1:1) diet, which resulted in increased acetate production. Propionate, butyrate and total VFA were numerically higher in the GPA mixture group than in the control group, which was in agreement with the findings of Kongmun et al. (2011), who reported significantly higher propionate and butyrate concentrations with the garlic powder supplemented ration. In the present study, the significantly higher acetate production compared to propionate can be attributed to the diversion of H2 and CO2 utilization from methanogenesis to reductive acetogenesis, or due to a higher pH, which provided more suitable conditions for rumen fermentation (Dijkstra et al., 2012) by cellulolytic microorganism (Church, 1988). In terms of the TVFA concentration, similar non-significant changes were observed in other studies, viz. when Panthee et al. (2017) fed sheep with freeze dried garlic leaves at 2.5 g/kg BW0.75, Parta et al. (2007) fed buffalo with garlic bulbs at 1% of their dry matter intake, and Wanapat et al. (2008) fed steers with garlic powder at 80–120 g/d along with urea treated rice straw. These findings clearly suggest that the addition of 0.1% GPA mixture in ruminant feed has no negative effect on rumen fermentation; however, it does improve individual VFAs, which is also desirable to farmers. Enteric methane (CH4), one of the important greenhouse gases, is produced by ruminants after feed fermentation (Islam and & Lee, 2019) and represents a gross dietary energy loss of 2%–12% (Johnson &and Johnson, 1995) from an animal. The prevention of dietary energy loss is one of the key elements of enhancing animal productivity by reducing enteric CH4 production by the animal. Garlic, a potential feed additive, is able to manipulate rumen fermentation with minimum CH4 emissions (Kamra et al., 2012). Kongmun et al. (2011) reported that CH4 production was reduced by 9% with the supplementation of garlic powder (100 g/day) in buffalo bulls. Similarly, Zafarian &and Manafi (2013) stated that CH4 production was reduced by 31% when lactating Murrah buffaloes were supplied garlic powder as 2% of their DMI. An 11% reduction of CH4 emissions was also observed in sheep fed with raw garlic at a rate of 1% DMI (Ferme et al., 2007). The reason for CH4 reduction is linked with the reduction of methanogen population in the rumen by the garlic extract (Ma et al., 2016). However, significant difference in CH4 production between the two groups of Hanwoo steers was not observed in the present study. Likewise, Klevenhusen et al. (2011b) observed that dietary garlic oil and its main compound diallyl disulfide had little to no effect on CH4 mitigation in sheep. The non-significant result in the present study regarding CH4 reduction may be due to the use of a smaller amount (0.1%) of GPA mixture, or that the concentration required to achieve significant CH4 reduction may vary for different species or breeds. Therefore, more in vivo studies are required to establish an appropriate dose of GPA mixture for methane mitigation in Hanwoo steers.

    The results of both in vitro and in vivo studies showed that the addition of 0.1% GPA mixture in ruminant feed had no significant effect on propionate, total VFA and A/P ratio; however, it significantly increased acetic acid and butyric acid production at the 24 h time point. The in vitro experiment showed that the addition of the 0.1% GPA mixture significantly reduced CH4 production after 12 h as well as 24 h of incubation. However, there was no significant difference observed in the in vivo CH4 production. Therefore, we conclude that 0.1% GPA mixture has some effect on in vitro rumen fermentation and CH4 reduction in Hanwoo steers, but has little and no effect on in vivo fermentation and CH4 mitigation, respectively.

    Acknowledgement

    This work was carried out with the support of the Rural Development Administration, Korea (PJ015039032020).

    Figures

    Tables

    Feed ingredients and chemical composition of basal feed (as-fed basis) used on in-vivo experiment

    In vitro effect of addition of GPA mixture on total gas, CH4 and VFA production

    In vivo effect of addition of GPA mixture on DMI, CH4 production, and rumen fermentation

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