Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Effects of Detoxified Sulfur as a Feed Supplement on in Vitro Rumen Fermentation and Methane Mitigation

제독 유황의 반추위 발효성상 및 메탄 저감 효과 연구

  • Kim, Seon-Ho (Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, Sunchon National University) ;
  • Islam, Mahfuzul (Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, Sunchon National University) ;
  • Biswas, Ashraf Ali (Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, Sunchon National University) ;
  • Cho, Kwang-Keun (Department of Animal Resources Technology, Gyeongnam National University of Science and Technology) ;
  • Lee, Sang-Suk (Ruminant Nutrition and Anaerobe Laboratory, Department of Animal Science and Technology, Sunchon National University)
  • 김선호 (순천대학교 동물자원과학과 반추영양 및 혐기미생물연구실) ;
  • 이스람 마푸줄 (순천대학교 동물자원과학과 반추영양 및 혐기미생물연구실) ;
  • 비스와스 아쉬라프 알리 (순천대학교 동물자원과학과 반추영양 및 혐기미생물연구실) ;
  • 조광근 (경남과학기술대학교 동물소재공학과) ;
  • 이상석 (순천대학교 동물자원과학과 반추영양 및 혐기미생물연구실)
  • Received : 2020.04.27
  • Accepted : 2020.08.04
  • Published : 2020.09.30
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Abstract

Sulfate is a reductant that competes for electrons and may lower CH4 production in the rumen. This study was designed to evaluate the beneficial effect of detoxified sulfur powder supplementation on in vitro rumen fermentation and methane mitigation. A ruminally cannulated Holstein Friesian cow was used as a rumen fluid source, and commercial pelleted concentrate was used as a substrate at 1 g dry matter. Treatments included the addition of detoxified sulfur powder at the rate of 0% (Control), 0.2% (T1), 0.4% (T2), 0.6% (T3), 0.8% (T4), and 1.0% (T5) as dry matter (DM) basis. The pH, total gas (TG), methane (CH4) production, DM digestibility, organic matter (OM) digestibility, and volatile fatty acids (VFA) production were analyzed after 12 hr of incubation. The results showed that CH4 production was significantly lowest in T1 (13.78 ml) but highest in the control (20.16 ml). Insignificantly higher total VFA was observed in control and T1 (64.99 and 64.28 mM, respectively) compared to other treatments after 12 hr of incubation. After 12 hr of incubation, the significantly lowest acetate:propionate was observed in T1 (1.90) while the highest was observed in T4 (2.44). However, no significant differences were recorded for pH, TG, DM digestibility, OM digestibility, acetate, propionate, and butyrate between the control and T1. Total number of bacterial DNA copies was significantly lower in the treatment group than the control. Therefore, it can be concluded from this study that detoxified sulfur at 0.2% inclusion level is optimal for production performance and ruminal CH4 mitigation.

제독유황은 황산염의 환원제로서 황화수소로 환원되면서 메탄생성과정에서 에너지 이용 경쟁을 통해 저감 할 수 있다. 본 연구는 유독성분이 제거된 유황을 in vitro를 통해 반추위 발효 성상 및 메탄 저감 효과를 평가하였다. 반추 위액은 케뉼라가 부착되어 있는 홀스타인 젖소에서 채취하여 이용하였으며, 기질 사료는 농후사료 1 g (DM)을 사용하였다. 처리구는 0% (대조구), 0.2% (처리구 1), 0.4% (처리구 2), 0.6% (처리구 3), 0.8% (처리구 4) 및 1.0% (처리구 5)로 실시하였다. pH, 총가스발생량 (TG), 메탄, 건물 소화율, 유기물 소화율 및 휘발성지방산을 분석하였다. 메탄생성량은 0.2% 처리구에서(13.78 ml) 가장 낮았으며, 대조구에서(20.16 ml)로 가장 높았다(p<0.05). 휘발성 지방산은 0.2% 처리구에서 64.28 mM 높았다. A/P 비율은 0.2% 처리구에서 1.90으로 가장 낮았으며, 0.8%에서 2.44로 유의성이 있었지만, pH, 총가스발생량, DM 소화율, OM 소화율, acetate, propionate 및 butyrate에서는 차이가 없었다. 총 세균의 DNA는 대조구보다 모든 처리구에서 낮았다. 이 연구를 통해 0.2% 처리구에서 반추위 발효 성상 및 메탄 저감에 효과가 있었다.

Keywords

Introduction

Methane (CH4) is an important greenhouse gas, that imposes momentous environmental troubles related to global warming. Ruminal methane constitutes up to 15% of global CH4 emission also contribute a loss of dietary energy (2 to 12%) in ruminants [12]. The average CH4 emissions are from 161 to 323 g/day in beef cattle and 151 to 497 g/day in dairy cows [4]. Therefore, developing feedings strategies to suppress methane production is deemed necessary. Many factors such as feed intake, feed composition, feed additives, and rumen microflora influence CH4 emissions from ruminants. The CH4 emissions can reduce from cattle through manipulation of these factors.

Sulfate is a reductant, reduce to hydrogen sulfide as well as consumes 8 electrons, which compete for the electrons and which may lower CH4 production [25]. Both synthetic and chemical sulfur can reduce methane production from ruminants [23,26]. However, excessive dosage (>0.3-0.4%) of dietary sulfur from chemical or synthetic sources either as sulfate or elemental sulfur may lead to toxic effect, even fatal in the extreme case. For a long history of sulfur on the disease producing effect, applying sulfur was limited to animals and humans. The naturally occurring sulfur compounds, as sulfide minerals are mercury sulfide, lead sulfide, zinc sulfide, antimony sulfide, and iron sulfide [20], containing toxic mineral. Detoxification is the process of heating and cooling with water and additives with the repetition of this process in several times. These detoxified compounds can be used as safe feed additives. Thus, researchers are encouraged to develop detoxified sulfur (nontoxic sulfur), which doesn’t contain the toxic mineral. Detoxified sulfur can be used as feed additives and influence animal performance [10]. However, detoxified sulfur has not yet tried in in vitro rumen fermentation and methane mitigation. Therefore, the present study was conducted to evaluate the effects of detoxified sulfur on in vitro rumen fermentation parameters using pelleted concentrate as a substrate as well as identify the inclusion rate of detoxified sulfur for reducing in-vitro CH4 production.

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 recommendations of “The Guide for the Care and Use of Laboratory Animals,” published by the Institutional Animal Care and Use Committee (IACUC) of the National Institute of Animal Science (2012-C-037) in Korea, were followed during animals’ experiment.

In-vitro experiment

An in vitro fermentation technique was performed using pelleted concentrate as a substrate. Sulfur powder which has used in this experiment contains 54.42% sulfur and its pH was 8.33. Treatments included 0% (without), 0.2%, 0.4%, 0.6%, 0.8%, and 1.0% of detoxified sulfur as DM basis and, hereafter referred to as Control, T1, T2, T3, T4, and T5, respectively. Ruminal contents were obtained from rumencannulated Holstein cow (48-month). The collected ruminal fluid was squeezed and the extracted fluids were strained through 4 layered cheesecloths and were placed in a glass bottle, subsequently capped and immediately transported to the laboratory maintaining the temperature at 39°C. Bottle containing rumen fluid was shaken vigorously by hand before mixing with the buffer. The pooled and particle-free rumen fluid was then transferred to a buffer medium with pH 6.9 [9] at a ratio of 1:3 (rumen fluid: buffer) which was prepared earlier according to Asanuma et al. [2]. One hundred milliliter (ml) of buffer mixed rumen fluid was transferred anaerobically, under a constant flow of N2 gas, into 160 ml serum bottles containing the 1 g pellet substrate (1 mm particle size) (Table 1) and added with detoxified sulfur (Coms. Nazu, Jeonnam, South Korea) at different concentrations. Then, bottles were sealed with rubber septum stopper and aluminum cap [1], and incubated for 12 hr in a shaking incubator with 120 rpm at 39°C [8]. Three replicates and 1 blank per experimental treatment were used in this experiment.

Table 1. Chemical composition of pelleted concentrate feed used for in vitro experiment as substrate

SMGHBM_2020_v30n9_743_t0001.png 이미지

Analyses of in vitro fermentation parameters

Total gas (TG) production was measured by a pressure sensor meter (EA-6; Sun Bee Instrument Inc., Seoul, Korea) and pH of the fermented rumen fluids were measured by a pH meter (Pinnacle series M530p; Schott Instruments, Mainz, Germany) at 12 hr incubation time point. One aliquot 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 microtubes and stored at -80°C until volatile fatty acid (VFA) analysis. A separate aliquot of 1 ml samples from each serum bottle was collected and stored at -80°C until microbial population analysis. The gas produced inside the bottle was sampled and trapped inside a vacuum tube for CH4 determination.

The VFAs were measured according to the methods stated by Han et al. [7] and Tabaru et al. [24] using high-performance liquid chromatography (HPLC; Agilent Technologies 1200 series, Waldbronn, Baden-Wurttemberg, Germany). The CH4 in the gas samples was measured by using gas chromatography (GC; HP 5890; Agilent Technologies, San Diego, CA, USA) according to the formula described by Ørskov and McDonald [15]. 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. The total CH4 production per bottle was calculated as parts per million (ppm) multiplied by the TG produced [27].

Determination of in vitro dry matter and organic matter digestibility

Before starting the in vitro rumen fermentation, Dry matter (DM) and organic matter (OM) content of the pellet substrate were analyzed by drying at 105℃ for 16 hr and ashing at 550℃ for 12 hr, respectively. The percents of DM and OM before incubation were used as the initial DM (DMi) and initial OM (OMi), respectively for calculation. After 12 hr incubation, the fermented samples from each serum bottle were poured in dried and pre-weighed nylon bags and knotted using nylon thread, then splashed with water flow for 15 minutes or until the disappearance of turbidity of washed water. The final DM (DMf) and OM (OMf) of the substrate were determined by the same protocol followed for the initial values (DMi and OMi). The DM and OM digestibility (%) were calculated as ({[DMi－DMf]/DMi} ×100) and ({[OMi －OMf]/OMi} ×100), respectively.

Quantitative real-time PCR (qPCR)

Total bacteria, protozoa, methanogens, Fibrobacter succinogenes, Ruminococcus flavefaciens and fungal DNA copies were estimated by using the protocol of Christophersen [5]. The concentration of gDNA extracted from each sample was determined by using Epoch Microplate Spectrophotometer (BiotekR). PCR forward and reverse primers were used for an individual organism. Total bacterial primers Bac 1114F (5ˊ-CGG CAA CGA GCG CAA CCC-3ˊ) and Bac 1275R (3ˊ-CCA TTG TAG CAC GTG TGT AGC C-5ˊ), methanogens primer qmcraA-F (5ˊ-GGA TTA GAT ACC CSG GTA GT-3ˊ) and qmcrA-R (3ˊ-GTT GAR TCC AAT TAA ACC GCA-5ˊ), protozoal forward primer F (5ˊ-GCT TTC GWT GGT AGT GTA TT-3ˊ) and reverse primer R (3ˊ-CTT GCC CTC YAA TCG TWC T-5 ˊ), F. succinogenes forward primer F (5ˊ-GTT CGG AAT TAC TGG GCG TAA A-3ˊ) and reverse primer R (3ˊ-CGC CTG CCC CTG AAC TAT C-5ˊ), R. flavefaciens forward primer F (5ˊ-CGA ACG GAG ATA ATI TGA GTI TAC TTA GG-3ˊ) and reverse primer R (3ˊ-CGC TCT CTG TAT GTT ATG AGG TAT TAC C-5ˊ), fungal forward primer F (5ˊ-GAG GAA GTA AAA GTC GTA ACA AGG TTT C-3ˊ) and reverse primer R (3ˊ-CAA ATT CAC AAA GGG TAG GAT GAT T-5ˊ) were used. Amplification was done by Eco Real-Time PCR (Illumina, Inc) with a total volume of 20 μl per reaction mixture (10 μl of 2x qPCRBIO SyGreen Mix, 0.8 μl each forward and reverse PCR primers, and 8.4 μl template DNA of 50 ng/μl in sterile distilled water).

Statistical analysis

All the data were analyzed using the general linear model (GLM) under a completely randOMized design. The significant (p<0.05) differences between and among treatments and control were identified by Duncan’s Multiple Range Test and Orthogonal PolynOMial Contrast. The Statistical Analysis System (SAS) (version 9.1; SAS Inst. Inc., Cary, NC) was used to carry out the above analysis [21].

Results

In vitro fermentation, Methane (CH4) production, and digestibility

The pH, TG, acetate, and total VFA were not different among treatments (p>0.05) (Table 2). However, a significant difference (p<0.05) was observed on propionate. The propionate was significantly highest in control (18.81 mM) and lowest in T3 and T4 (15.32 and 14.20 mM) after 12 hr of incubation. In case of butyrate, significant difference (p[0.05) was observed after 12 hr of incubation which the highest value was in T5 (13.23 mM) and the lowest was in control(11.74 mM). There was a significant difference in acetate: propionate (A: P) value was observed after 12 hr of incubation. Moreover, the lowest A: P value was observed in control (1.83) followed by T1 (1.90), and the highest was in T4 (2.44) treatment. After 12 hr of incubation, the lowest CH4 production was observed in T1 (13.78 ml) followed by T5 (14.81 ml) and the highest in the Control (20.16 ml) (Fig. 1). The DM and OM digestibility were not varied significantly among control and treatments (p]0.05) (Table 2).

Table 2. The pH, total gas and Volatile Fatty Acid production from in vitro rumen fermentation by the addition of sulfur

SMGHBM_2020_v30n9_743_t0002.png 이미지

DM: Dry matter, OM: Organic matter.

Control: Without sulfur, T1: 0.2% sulfur, T2:0.4% sulfur, T3:0.6% sulfur, T4:0.8% sulfur, T5:1.0% sulfur.

C. vs T. is the contrast between control and treatment.

a,b,c Means with different superscripts in the same row are significantly different (p<0.05).

SMGHBM_2020_v30n9_743_f0001.png 이미지

Fig. 1. Methane production from in vitro rumen fermentation after 12 hr incubation by the addition of Sulfur. Control: Without Sulfur; T1: 0.2% Sulfur; T2:0.4% Sulfur; T3:0.6% Sulfur; T4:0.8% Sulfur; T5:1.0% Sulfur; a,b,c Means with different superscripts in the same row are significantly different (p<0.05).

Microbial DNA copies by qPCR

The result of total bacteria from the qPCR revealed that there was a significant difference (p<0.05) between control and treatments. The highest (DNA copies) value of total bacteria were observed in control (9.27×108) compared to treatments where T1 was 3.51×108, T2 was 3.86×108, T3 was 3.38×107, T4 was 1.83×108 and T5 was 3.09×108 (Table 3). Protozoal DNA copies were significantly different (p<0.05) among control and treatments where the lowest DNA copies were in T4 (4.47×104) within the treatments. There was no significant difference in the case of methanogens DNA copies, but numerically lower methanogen was observed in treatment groups than the control. Higher methanogen (DNA copies) were in control (1.04×106 ) than the treatment group which were 9.13×105, 4.82×105, 4.96×105, 2.16×105, 2.50×105 in T1, T2. T3, T4, and T5, respectively. Fibrobacter succinogenes DNA copies were significantly different (p<0.05) among treatments and control group. Higher DNA copies were observed in control and T1 (5.96×105 and 5.40×105) than in T2, T3 and T4 (3.00×105, 2.88×105 and 1.71×105, respectively). Likewise, Ruminococcus flavefaciens DNA copies were significantly different (p<0.05) among the treatments and control groups. Higher DNA copies were observed in T4 (7.67×104) than the others treatments and control groups (p<0.05). There was no significant difference in fungal DNA copies among the control and treatments group.

Table 3. Microbial DNA copies by qPCR after 12 hr incubation from in vitro fermentation by the addition of sulfur

SMGHBM_2020_v30n9_743_t0003.png 이미지

Control: Without sulfur, T1: 0.2% sulfur, T2:0.4% sulfur, T3:0.6% sulfur, T4:0.8% sulfur, T5:1.0% sulfur.

C. vs T. is the contrast between control and treatment.

a,b,c Means with different superscripts in the same row are significantly different (p<0.05).

Discussion

Feed additives play a significant role in rumen fermentation as well as CH4 emission. The CH4, a greenhouse gas, contributes 20-50 times more than that of CO2 to exert the greenhouse effect [3,6]. Sulfur can be used to improve rumen fermentation as well as methane mitigation in ruminants as a feed additive. The NRC [14] suggested that the maximum tolerable amount of sulfur is 0.3% (DM basis) for beef cattle with high-concentrate diets and 0.5% for beef cattle with high forage diets. Moreover, researchers are struggled to find out the optimum dose of sulfur for a ruminant.

In the current experiment, CH4 production was significantly lowest at 0.2% detoxified sulfur supplemented group. Lower CH4 production noted with 0.2% sulfur supplementation agrees with the findings of Pakmaluek et al. [16], who stated 0.2% sulfur supplementation significantly lower CH4 production while using rice straw as substrate. This result also supported by the Ungerfield and Kohn [25] who revealed that sulfate reduction is more favorable than methanogenesis. Isa et al. [11] confirmed that low sulfate concentration direct relatively more electrons toward sulfate reduction compare with a high sulfate concentration.

The present study of the experiment stated that there was no significant difference in pH after 12 hr of incubation period that was supported by Kung et al. [13]. After 12 hr of incubation, the lack of effect of sulfur on total gas is consistent with the findings of Smith et al. [22] and Kung et al. [13], who also reported non-significant TG production with sulfur supplementation. The non-significant acetate concentration observed on our study is similar to the findings of Kung et al. [13], who described lack of effect of sulfur concentration at a rate of 0.29% and 1.09% (DM basis) on VFA production with a medium-concentrate substrate in vitro. Rahman et al. [19] revealed similar findings when sodium lauryl sulphate, fumaric acid, and their coupled addition were used as treatments. Significantly lower propionate concentration was observed in treatments than control in our experiment. The result of this experiment is tended to similar to the findings of Promkot et al. [17], where they reported that lower propionate concentration was recorded along with the increasing of sulfur supplementation.

After 12 hr of incubation, the lack of effect of sulfur on DM and OM digestibility, observed in this study, is consistent with the findings of Quinn et al. [18], who stated the DM digestibility was not differed with sulfur supplementation at 0.17 or 0.42% of DM. Similarly, Zinn et al. [28] revealed no effects of sulfur supplementation in diet ranging from 0.15% to 0.25% (DM basis) on OM digestibility in vivo. In the current study, significantly (p<0.05) lower total bacterial DNA copies and tended to lower methanogens DNA copies were revealed in the treatment groups than the control, which is related to the CH4 reducing tendency in sulfur supplemented group.

In conclusion, detoxified sulfur at 0.2% inclusion level is competent to decrease CH4 production. Consequently, detoxified sulfur at lower inclusion level (0.2% DM) is a prospective feed supplement for ruminal CH4 mitigation and increasing productivity of ruminant animal.

Acknowledgement

This research was supported by Sunchon National University Research Fund (2020-0214).

The Conflict of Interest Statement

The authors declare that they have no conflicts of interest with the contents of this article

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