Effect of Allicin and Illite Supplementation on the Methane Production and Growth Performance of the Beef Cattle

Anaerobic microorganisms in the rumen hydrolyze high molecular organic matter into low molecular organic matter and produce volatile fatty acids (VFAs), carbon dioxide (CO₂), hydrogen (H₂) and ethanol. Rumen methanogens use various substrates as electron acceptors to produce methane (CH₄) as the final metabolite (Martin et al., 2010). Of the total gas generated from ruminants, 25-30% is CH₄ (Ellis et al., 2007) and it has a negative impact on rumen energy loss and global warming (Sarkar et al., 2018).

Studies of feeds and additives that aim to reduce the CH₄ generated from ruminants, have previously been conducted (Jafari et al., 2020). However, CH₄ reduction technology has not yet been put into practical use because of problems such as reduced digestibility, increased costs, additives remaining and microbial adaptations to the additives (Jordan et al., 2006; Osita et al., 2019). Natural substances that can reduce CH₄ production under conditions that maintain or increase the existing productivity of ruminants without negative effects on livestock products or the environment are thus required. Among various other natural substances, allicin and illite have been suggested for this purpose, as previous studies have found that they are capable of reducing CH₄ released by ruminant (Liu et al., 2013). However, further research is required to verify and better understand this and there have not yet been any relevant field trials.

The objectives of this study were to evaluate the effects of allicin and illite supplementation on in vitro ruminal CH₄ production, growth performance and the carcass characteristics of the Hanwoo steers.

Study area
 
The study was conducted in the Hanwoo Research Institute and Gangwon Livestock Techcology Institute during May 2019 to April 2020.
 
Animals, treatments and management
 
For the in vitro experiment, 3 Hanwoo cows (average weight 450.4±22.6 kg) were equipped with rumen fistula and 28 late fattening Hanwoo steers (average weight: 636.3±50.7 kg) were used in the field trial. The steers were randomly assigned to one of the four dietary groups: control (CON), T1 (1.0% illite), T2 (0.1% allicin), or T3 (1.0% illite and 0.1% allicin).

Concentrate was provided three times daily using an automatic feeding system, at volumes of approximately 2.0% (as-fed basis) of the body weight (BW) for the entire experimental period. Rice straw and water could be accessed freely. The chemical compositions of experimental diets are presented in Table 1.


 
In vitro ruminal fermentable characteristics
 
Ruminal fluid was collected from the ruminal fistulas of the Hanwoo cows before feeding in the morning and filtered through four layers of cheese cloth and was then used as an inoculum for in vitro incubation. In vitro cultures were established by mixing rumen inoculum with a previously prepared artificial buffer solution in accordance. 100 mL of the prepared in vitro culture solution was placed in a 160 mL bottle containing concentrate and additives (allicin and/or illite) and O₂-free CO₂ gas was infused for 5 s to eliminate the air. The bottles were then incubated for 2, 4, 8, 12 and 24 h in a shaking incubator (HB-201SLI, Hanbaek Scientific Co., Bucheon, Korea) at 39°C.
 
Measurements and analyses
 
The in vitro ruminal pH was measured using a pH meter (Corning Glass Works, Medfield, MA, USA). The ammonia concentration was calculated by measuring the absorbance at 630 nm using a spectrophotometer (Spectronics 21D, B&L, NY, USA).

To analyze the VFAs concentrations, 10 mL of the culture solution was collected in a 160 mL bottle for each incubation time, followed by the addition of 1 mL of 20% HPO₃ and 0.5 mL of saturated HgCl₂ and then centrifuged at 4°C at 1,000 × g for 15 min. The supernatant was discarded and the VFA concentration was measured via gas chromatography (Shimadzu-17A, Shimadzu, Kyoto, Japan).

Total gas production (TGP) was measured using a pressure sensor gauge (EA-6, SunBee Instrument, Seoul, Korea). To measure CH₄ and CO₂ production after TGP, the gas generated using a vacuum tube was collected and analyzed using gas chromatography (Agilent Technologies HP 5890, Agilent, CA, USA). CH4 production was estimated using the formula of Qrskov and McDonald (1979) with the CH₄ obtained from the TGP for each incubation time.

Average daily gain (ADG) was calculated by measuring BW at 10 am every 2 months. Dry matter intake (DMI) was measured weekly and the amount of DMI was determined by measuring the orts present before the morning feeding. The feed conversion ratio (FCR) was calculated using the DMI and ADG.

At the end of the experimental period, all steers were slaughtered at the local slaughterhouse. Yield and quality grades were determined as the evaluation criteria of the Korean carcass grading system (MAFRA, 2018).
 
Statistical analyses
 
All data were analyzed using the generalized linear model procedure in the SAS package 9.1 software program (SAS Institute Inc.; Carey, NC, USA). Significant differences between the treatments were analyzed using Tukey’s test at the 95% significance level.

Ruminant pH and ammonia concentration
 
The effects of the allicin and illite supplementations on the in vitro ruminal pH and ammonia concentrations are summarized in Table 2. Ruminal pH was significantly lower in T2 and T3 compared to control at 4 h of incubation (P<0.02), but overall there was little difference among the treatment groups. Ammonia concentrations were not affected by the addition of allicin and illite.

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Similar to our study, Soriano (2014) reported a transient decrease in rumen pH in the 0.5% garlic powder treatment only at the early incubation (2 h). However, in this study, as reported by Wanapat et al. (2008) and Biswas et al., (2018), illite and allicin do not appear to affect rumen pH and ammonia production.
 
VFA concentrations
 
The effects of allicin and illite supplementation on the in vitro ruminal VFA concentrations are shown in Table 3. Acetate concentration was lowest in T3 after 4 and 8 h of incubation, but the acetate concentration after 24 h was significantly higher in T3 than in the control and T1 (P<0.05). Propionate concentrations tended to increase in all the treatment groups as the incubation time increased and were higher in the control than in the T1 and T3 after 4 h of incubation (P<0.01). The total VFAs concentration was significantly (P<0.05) higher in the control than the T3 after 4 h of incubation, but there was no difference between the treatments at different incubation times.

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When CH₄ production is reduced in the rumen, it is converted into reactions that consume H₂, such as the production of propionate and butyrate due to the H₂ sink (Busquet et al., 2005). In the present study, allicin supplementation increased the concentrations of propionate and butyrate at the partial incubation time and therefore, allicin supplementation was considered to affect the reduction in CH₄. This finding is concurrent with a previous investigation (Kongmun et al., 2011). Biswas et al. (2018) reported that the concentrations of propionate and butyrate were increased by the supplementation of illite because the trace minerals contained in illite acted as enzyme components that could increase the VFAs by accelerating the metabolic pathway. However, further research is required to better understand the exact metabolic pathways and interactions.
 
Gas production
 
The effects of the allicin and illite supplementations on in vitro ruminal gas production are shown in Table 4. TGP did not differ among treatments after 12 h of incubation but was significantly lower in T2 than in the control and T1 after 24 h (P<0.05). CH₄ production was lower in all the treatments compared to the control after 24 h of incubation (P<0.05). The production of CO₂ was significantly lower in the control and T3 treatment than in T1 and T2 after 12 h of incubation (P<0.01).

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Allicin been reported to reduce the production of CH₄ by reducing the number of methanogens (Kongmun et al., 2011). Busquet et al. (2005) reported that CH₄ production was significantly reduced by allicin supplementation. They also found that the supplementation of allicin reduced the deoxyribonucleic acid (DNA) of methanogens. Meanwhile, Liu et al. (2013) suggested that illite had a high CH₄ adsorption capacity, which reduced CH₄ production in the intestine and Biswas et al. (2018) found that CH₄ production was reduced by 13% with 1% illite supplementations. As a result, it was presumed that allicin affected the methanogens, reduced CH₄ production and thereby increased the concentration of CO₂. In addition, it was also thought that illite may reduce the production of CH₄ gas through adsorption.
 
Growth performance
 
The effects of the allicin and illite supplementation on the growth performance of the late fattening Hanwoo steers are shown in Table 5. There was little difference among the treatments for BW and ADG and the supplementation of allicin and illite had no effect on DMI or FCR.

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Allicin has antibacterial activity and is known to improve the digestibility of organic matter in the rumen (Yang et al., 2007). The supplementation of clay minerals such as illite has been reported to have a positive effect on ADG, feed efficiency and the prevention of rumen acidosis in cattle (Humer et al., 2019). However, in this study, allicin and illite had no effect on growth performance. This may be because rumen or rumen microorganisms overcome and adapt to newly introduced compounds (Castillejos et al., 2007).
 
Carcass characteristics
 
Table 6 shows the effect of allicin and illite supplementation on the carcass characteristics of the late fattening Hanwoo steers. In this study, carcass weight, Back fat thickness and Rib eye area were not affected by the addition of illite and allicin and there was no difference among treatments in quality traits.

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Sung (2001) reported that, supplementation with 0.1% garlic powder reduced back fat thickness, but increased rib eye area and marbling score in Hanwoo steers. Kang et al. (2002) reported that the supplementation of 2% illite had the effect of increasing meat yield, meat quality and the farmer’s income for Hanwoo steers. However, in this study, there was no difference in conductor characteristics and this might be due to the effect of the addition period and amount added.

Thus, allicin 0.1% and/or illite 1.0% could be used as natural feed additives to reduce CH4 production without negatively affecting the in vitro ruminal fermentation, growth performance, or carcass characteristics of Hanwoo steers. In addition, we recommend that the supplementation of allicin alone is more efficient than the mixed supplementation of allicin and illite.

All authors declare that they have no conflict of interest.