Introduction
Methane (CH4) production by ruminants, a major source of greenhouse gas emissions, is generally recognized as a fermentative inefficiency resulting in 2–12% loss of the gross energy consumed by the host (Johnson and Johnson, Reference Johnson and Johnson1995; Thauer et al., Reference Thauer, Kaster, Goenrich, Schick, Hiromoto and Shima2010). During the past few decades, researchers have put a great deal of effort into mitigating CH4 emission in ruminant animals (Patra, Reference Patra2012; Vyas et al., Reference Vyas, Alemu, McGinn, Duval, Kindermann and Beauchemin2018; Ochoa-García et al., Reference Ochoa-García, Arevalos-Sánchez, Ruiz-Barrera, Anderson, Maynez-Pérez, Rodríguez-Almeida, Chávez-Martínez, Gutiérrez-Bañuelos and Corral-Luna2019). Among these mitigating strategies, nitroethane (NE), 2-nitroethanol (NEOH), 2-nitro-1-propanol (NPOH) and 3-nitro-1-propionic acid (NPA) have been found to inhibit ruminal methanogenesis by as much as 90% in vitro through inhibiting biochemical conversions involved in methanogenesis (Anderson et al., Reference Anderson, Krueger, Stanton, Callaway, Edrington, Harvey, Jung and Nisbet2008; Smith and Anderson, Reference Smith and Anderson2013; Latham et al., Reference Latham, Anderson, Pinchak and Nisbet2016; Correa et al., Reference Correa, Trachsel, Allen, Corral-Luna, Gutierrez-Bañuelos, Ochoa-Garcia, Ruiz-Barrera, Hume, Callaway, Harvey, Beier, Anderson and Nisbet2017; Ochoa-García et al., Reference Ochoa-García, Arevalos-Sánchez, Ruiz-Barrera, Anderson, Maynez-Pérez, Rodríguez-Almeida, Chávez-Martínez, Gutiérrez-Bañuelos and Corral-Luna2019), and NE and NPOH have been shown to reduce CH4-producing activity by more than 69% in vivo (Anderson et al., Reference Anderson, Carstens, Miller, Callaway, Schultz, Edrington, Harvey and Nisbet2006; Gutierrez-Bañuelos et al., Reference Gutierrez-Bañuelos, Anderson, Carstens, Slay, Ramlachan, Horrocks, Callaway, Edrington and Nisbet2007; Latham et al., Reference Latham, Anderson, Pinchak and Nisbet2016). However, it is not clear if the aforementioned inhibition can also be achieved directly by decreasing the population and/or carbon dioxide (CO2)-reducing activity of methanogens. It is well known that methylcoenzyme M reductase (MCR) catalyses the final step in methanogenesis, converting the coenzyme M-bound methyl group to CH4 (Thauer, Reference Thauer1998), and the prosthetic group of the MCR is coenzyme F 430, which is a nickel porphinoid (Ankel-Fuchs et al., Reference Ankel-Fuchs, Jaenchen, Gebhardt and Thauer1984). Additionally, coenzyme F 420 shows strong fluorescence in the oxidized form and was believed to exist only in methanogenic bacteria (Schulze et al., Reference Schulze, Menkhaus, Fiebig and Dellweg1988). However, it is unknown how nitrocompounds could affect these coenzymes' activities during inhibition of ruminal methanogenesis.
Zhang and Yang (Reference Zhang and Yang2011) noted that the optimal combination of 15 mm NE, 10 mm NEOH, 5 mm NPOH, 0.07 mm pyromellitic diimide and 0.01 mm 2-bromoethanesulphonate in cultures with an orthogonal experiment led to >95% CH4 inhibition of a hay-rich substrate, and the combination of these inhibitors shifted ruminal fermentation from acetate towards propionate production. However, it was not clear what difference existed among NE, NEOH and NPOH at same dosage in terms of CH4 inhibition as well as the shift of the methanogen community. In the present study, methyl-coenzyme M reductase activity as well as related coenzymes (e.g. F 420 and F 430) was determined, and the objective was to explore the inhibition action mode of nitrocompound on in vitro rumen methanogenesis through a comparison of NE, NEOH and NOPH under a fixed dosage of 10 mm. The obtained outcomes were expected to provide a scientific reference for future in vivo investigation of these nitrocompounds in reducing methane emission in ruminant animals.
Materials and methods
Nitrocompound chemicals
The nitrocompound products were purchased commercially from Sigma Aldrich (St. Louis, MO, USA) and stored at 4 °C. Among these nitrocompounds, the NE product is a colourless oily liquid and almost insoluble in water. Both NEOH and NPOH products are light yellow liquids with a low boiling point. Their analytical grades were 99, 90 and 98%, respectively.
In vitro batch cultures and sampling
Alfalfa hay, harvested at the early bloom stage, was chopped into 2–5 mm strips with a paper cutter and oven dried at 65 °C for 48 h. The dried hay samples were then ground in a Wiley mill to pass through a 2.0 mm sieve, and then mixed with maize meal (4:1, w/w) to prepare a hay-rich substrate for subsequent in vitro batch culture experiments.
Five rumen-cannulated lactating Holstein dairy cows, fed in a free stall, served as donor animals for rumen fluids. The cows had free access to water and were fed a total mixed ration of 18.0 kg maize silage, 4.0 kg alfalfa hay and 14.5 kg concentrate daily. On the day before starting in vitro batch cultures, the animals were driven away from the lactating herds: rumen fluid was collected from each animal through rumen fistula 3 h after the morning feed and kept in pre-warmed vacuum flasks.
Glass bottles (volume capacity of 120 ml) with Hungate stoppers and screw caps were used as incubators. A completely randomized design was applied to three runs of in vitro batch cultures, and 0.5 g hay-rich substrate was weighed into 80 bottles/run with 20 bottles for each treatment. The treatment included a nitrocompound-free control, 10 mm of NE, 10 mm of NEOH and 10 mm of NPOH, respectively.
Following the experimental design, 80 bottles in each run were incubated anaerobically with 25 ml of rumen fluids strained through four layers of cheesecloth and 50 ml of 39 °C pre-warmed media buffer (pH 6.85; Menke and Steingass, Reference Menke and Steingass1988). In addition, four fermentations without substrate and nitrocompounds were used as blanks. The batch cultures were carried out at 39 °C in both automated and manual systems. In the automated system, cumulative gas production (GP) was recorded continuously by connecting treated bottles (five bottles/treatment) to the gas inlets of an automated gas recording system and incubating continuously for 72 h. In the manual system, fermentation gas samples were collected from the treated bottles (three bottles/treatment/incubation time) by connecting them to pre-emptied air bags which were then removed at 6, 12, 24, 48 and 72 h of incubation. A 1.0 ml gas sample was taken from the airbags, and CH4, CO2 and H2 contents in fermentation gas samples were determined using a gas chromatographic method (Zhang and Yang, Reference Zhang and Yang2011).
The biomass content of each bottle was filtered through a nylon bag (8 × 12 cm, 42 µm pore size) to determine in vitro dry matter disappearance (IVDMD) at 6, 12, 24, 48 and 72 h. Then the filtered culture fluid (5 × 1.0 ml) was sampled into DNase-free polypropylene tubes and stored at −80 °C for the analysis of volatile fatty acid (VFA), methanogen population, mcrA gene expression, coenzyme contents of F 420 and F 430.
Determination of in vitro dry matter digestibility, volatile fatty acids and coenzyme content
The difference between initially incubated dry matter (DM) and the residual DM in nylon bags (corrected using the blanks, after incubation) was calculated to determine the IVDMD. The culture fluid samples (1.0 ml) were treated with 0.3 ml metaphosphoric acid solution (25 mg/ml) and centrifuged at 15 000 g for 10 min at 4 °C. The concentrations of acetate, propionate, butyrate and branch-chained VFAs of iso-butyrate and iso-valerate in the supernatants were measured by a gas chromatography (GC522, Wufeng Instruments, Shanghai, China). Coenzyme F 420 content was determined as previously described by Reuter et al. (Reference Reuter, Egeler, Schneckenburger and Schoberth1986) and expressed as fluorescence intensity of the coenzyme. Following the method of Ellefson et al. (Reference Ellefson, Whitman and Wolfe1982), coenzyme F 430 content was determined via the ultraviolet/visible spectrum by measuring the loss of absorbance and expressed as the relative absorbance of coenzyme F 430 at 430 nm.
Expression analysis of mcrA gene
Total genomic RNA was extracted from a 1 ml aliquot of culture fluid samples using a RNeasy Mini kit (Tiangen® Biotech, Beijing, China) with an RNase-Free DNase Set (Qiagen) following the manufacturer's instructions. The cDNA was synthesized with a FastKing RT cDNA Kit (Tiangen® Biotech). The enumeration of cDNA of mcrA gene was measured on a Bio-Rad Multicolor Real-Time Polymerase Chain Reaction (PCR) Detection System (Bio-Rad Company, California, USA) using the RealMasterMix SYBR® Green (Tiangen® Biotech). The 2−ΔΔCt method was used for expression analysis of the mcrA gene with 16S rRNA set as the reference gene (Livak and Schmittgen, Reference Livak and Schmittgen2001). The specific primer set for 16S rRNA gene and mcrA gene (Supplementary Material Table S1) were applied as described by Denman and McSweeney (Reference Denman and McSweeney2006) and Denman et al. (Reference Denman, Tomkins and McSweeney2007), respectively.
Determination of methanogenic population with real-time polymerase chain reaction
A bead-beating method, described by Denman and McSweeney (Reference Denman and McSweeney2006) and the FastDNA kit and FastPrep instrument (Tiangen® Biotech) were used for total DNA extraction. Total genomic DNA was isolated from a 1 ml aliquot of cultural fluid samples. Following the real-time PCR method as described by Denman and McSweeney (Reference Denman and McSweeney2006) and Denman et al. (Reference Denman, Tomkins and McSweeney2007), the enumeration of total methanogens (a primer applied as described by Zhou et al., Reference Zhou, Hernandez-Sanabria and Guan2009), Methanobacteriales, Methanococcales and Methanomicrobiales (primers applied as described by Yu et al., Reference Yu, Lee, Kim and Hwang2005) was measured on a Bio-Rad Multicolor Real-Time PCR Detection System (Bio-Rad Company) using the RealMasterMix SYBR® Green (Tiangen® Biotech). Their microbial abundances are expressed as a proportion of total estimated rumen bacterial 16S rDNA (Denman and McSweeney, Reference Denman and McSweeney2006) according to the equation: relative quantification = 2–(CT target–CT total bacteria), where CT represents the threshold cycle (Guo et al., Reference Guo, Liu, Lu, Zhu, Denman and McSweeney2008).
Calculations
The Microsoft Excel data of the cumulative gas production against the different incubation time (GPt, ml/g DM) were imported into an SAS data set and fitted with the non-linear (NLIN) procedure of SAS 9.4 (Statistical Analysis for Windows, SAS Institute Inc., Cary, NC, USA) according to the France et al. (Reference France, Dijkstra, Dhanoa, Lopez and Bannink2000) model using Eqn (1):
where GPt is the cumulative gas production at time t (h); A is the estimated asymptotic gas production (ml/g DM); c is the fractional gas production rate (/h), and L is the lag time phase before GP commenced.
Following the method of García-Martínez et al. (Reference García-Martínez, Ranilla, Tejido and Carro2005), the average gas production rate (AGPR, ml/h) was calculated using Eqn (2):
The time when half of A occurred (T 1/2) was calculated using Eqn (3):
Following Demeyer and Graeve (Reference Demeyer and Graeve1991), hydrogen recovery (2Hrec) was calculated using Eqn (4):
where acetate, propionate and butyrate are given as their molar percentages in total VFA production, and CH4 and H2 as their molar percentages in the total gas production.
Statistical analysis
Data were analysed by analysis of variance using the general linear model procedure of SAS 9.4 (Statistical Analysis for Windows, SAS Institute Inc.). The model was applied as:
where Y ij is the dependent variable under examination; μ is the overall mean; N i is the fixed effect of nitrocompound treatment (i = control, NE, NEOH and NPOH); T j is the fixed effect of incubation time (6, 12, 24, 48 and 72 h); N × T is the interaction effect between nitrocompound treatment and incubation time. Least square means (LSMEANS) and standard errors of the means (s.e.m.) across 6, 12, 24, 48 and 72 h were calculated using the LSMEANS statement of SAS and tabulated in Tables 1 and 2. Overall differences among nitrocompound treatments were determined by Tukey's test. Pearson correlation analyses between variables under examination were performed using the correlation (CORR) procedure of SAS 9.4. Significance was declared at P < 0.05 unless otherwise noted.
Effect of nitroethane (NE), 2-nitroethanol (NEOH) and 2-nitro-1-propanol (NPOH) addition (10 mm) in culture fluids on kinetic gas production and fermentation gas composition during 72 h incubation
NE, nitroethane; NEOH, 2-nitroethanol; NPOH, 2-nitro-1-propanol; IVDMD72, in vitro dry matter disappearance of 72 h; GP 72, cumulative gas production at 72 h; A, the asymptotic gas production (ml/g DM); c, the fractional gas production rate (/h); T 1/2, the time when half of A occurred (h); AGPR, the average gas production rate (ml/h) between the start of the incubation and the time when half of A occurred; H2, hydrogen gas; CO2, carbon dioxide; CH4, methane.
Effect of nitroethane (NE), 2-nitroethanol (NEOH) and 2-nitro-1-propanol (NPOH) addition (10 mm) on the relative abundance of methanogenic populations, coenzyme content, mcrA gene expression and volatile fatty acid production in fermentation fluids across different incubation times of 6, 12, 24, 48 and 72 h
F 420, coenzyme content expressed in fluorescence intensity; F 430, coenzyme content expressed in ultraviolet absorbance; VFA, volatile fatty acids; BCVFA, branch-chained VFAs including iso-butyrate and iso-valerate; 2Hrec, hydrogen recovery.
Results
In vitro dry matter disappearance and kinetic gas production
After 72 h incubation, IVDMD72 did not differ among different treatments (Table 1 and Supplementary Material Fig. S1, P = 0.472). Asymptotic gas production (A) and GP72 were decreased in both NE and NEOH in comparison with the control (P < 0.001), but no difference occurred between NPOH and the control. As shown in Table 1 and Fig. S1, neither NE nor NEOH addition altered c and T 1/2, but NPOH decreased c (P = 0.026) and increased T 1/2 (P = 0.024). Consequently, AGPR was decreased by the addition of NE, NEOH and NPOH (P = 0.012).
Fermentation gas composition
The accumulation of H2 in fermentation gases was far greater in nitrocompound treatments than the control (Table 1, P < 0.001). The addition of NE, NEOH and NPOH increased the molar CO2 proportion by 11.3, 11.6 and 5.0%, respectively, in comparison with the control. Meanwhile, the addition of NE, NEOH and NPOH in comparison with the control notably decreased the molar CH4 proportion, by 97.3, 96.7 and 35.3%, respectively. Interaction did occur between the nitrocompound addition and incubation time for the fermentation gas composition as shown in Fig. 1 (P < 0.001).
Molar proportion of (a) methane (CH4), (b) hydrogen gas (H2) and (c) carbon dioxide (CO2) of a hay-rich feed incubated with rumen fluids in the presence of nitroethane (NE), 2-nitroethanol (NEOH) and 2-nitro-1-propanol (NPOH). Effect of nitrocompounds was significant at P < 0.001.
As the incubation time increased, the molar CH4 proportion increased continuously in the control and NPOH group though it was lower with NPOH than the control (P < 0.001, Fig. 1(a)). The molar CH4 proportion in NE and NEOH group was continuously far lower than that of control. In contrast, H2 accumulation in fermentation gases continuously increased in NE and NEOH group against the increase of incubation time, and the accumulation was far greater than the control and NPOH (P < 0.001, Fig. 1(b)). As the incubation time increased, molar CO2 proportion continuously decreased in all groups, and it was greater in nitrocompound groups than in the control (P < 0.001, Fig. 1(c)).
Methanogen populations
The relative abundance of total methanogens, Methanobacteriales, Methanomicrobiales and Methanococcales across different incubation time was decreased with the addition of NE, NEOH and NPOH (Table 2, P < 0.001). Interaction did occur between nitrocompounds addition and incubation time for all of the relative abundance of methanogen populations (P < 0.001). Briefly, total methanogens (Fig. 2(a)) and methanogen from Methanobacteriales (Fig. 2(b)) in nitrocompound treatments in comparison with the control presented less difference under first 24 h incubation time than subsequent incubation time. In contrast, a limited abundance of Methanomicrobiales (Fig. 2(c)) and Methanococcales (Fig. 2(d)) were detected during first 12 h incubation time, but all of them decreased almost to zero in subsequent incubation times.
Relative abundance changes of (a) total methanogens, (b) Methanobacteriales, (c) Methanomicrobiales and (d) Methanococcales of a hay-rich feed incubated with rumen fluids in the presence of nitroethane (NE), 2-nitroethanol (NEOH) and 2-nitro-1-propanol (NPOH). Effect of nitrocompounds was significant at P < 0.001.
Expression of the mcrA gene, coenzyme F 420 and coenzyme F 430 contents in cultures
The coenzyme contents of F 420 and F 430 in culture fluids across different incubation time were decreased with the addition of NE, NEOH and NPOH in comparison with the control (Table 2, P < 0.001). The mcrA gene expression relative to the control decreased remarkably (by 83.1, 79.7 and 53.5%, respectively) with the addition of NE, NEOH and NPOH (P < 0.001). Interaction did occur between nitrocompounds addition and incubation time (P < 0.001) for mcrA gene expression, coenzyme F 420 and F 430 contents (P < 0.01). Briefly, F 420 content in NE group and F 430 in NEOH continuously decreased to the lowest levels against the incubation time (Figs 3(a) and (b)). After 36 h incubation, F 420 content ranked: control > NPOH > NEOH > NE and less decline of F 430 content was observed for NE and NPOH in comparison with the control (Figs 3(a) and (b)). The mcrA gene expression in nitrocompound groups relative to the control peaked at 24 h, and thereafter it was greater in NPOH than NE and NEOH group (Fig. 3(c)).
Relative expression of (a) mcrA gene, (b) content of coenzyme F 420 and (c) F 430 of a hay-rich feed incubated with rumen fluids in the presence of nitroethane (NE), 2-nitroethanol (NEOH) and 2-nitro-1-propanol (NPOH). Effect of nitrocompounds was significant at P < 0.001.
Fermentation characteristics in culture fluids
No significant difference occurred for total VFA though its numerically highest concentration was reached in the NPOH group (Table 2 and Supplementary Material Fig. S2). However, the nitrocompound addition decreased acetate and increased the concentrations of propionate, butyrate and BCVFA (P < 0.01), in comparison with the control.
The response of methane production to mcrA gene expression and coenzyme contents
As shown in Table 3, CH4 production during the 72 h incubation period was correlated positively with the abundance of the total methanogen population (r = 0.61), Methanobacteriales population (r = 0.35), mcrA gene expression (r = 0.74), coenzyme contents of F 420 (r = 0.56) and F 430 (r = 0.31). The total methanogen population was correlated positively with the Methanobacteriales population (r = 0.79), Methanomicrobiales population (r = 0.55), mcrA gene expression (r = 0.26) and coenzyme F 430 content (r = 0.52). In addition, mcrA gene expression was correlated positively not only with coenzyme F 420 content (r = 0.69) but also the coenzyme F 430 content (r = 0.44). Meanwhile, a positive correlation was observed between coenzyme F 420 content and coenzyme F 430 content (r = 0.44).
Pearson correlation coefficients between methane production, abundance of methanogenic populations, mcrA gene expression, coenzyme F 420 content and coenzyme F 430 content regardless of the type of nitrocompound
P values for the Pearson correlation coefficient are noted in parenthesis.
Discussion
The inhibition of CH4 production sometimes results in a depression of rumen fermentative parameters associated with digestive efficiency, including gas production (Zhang and Yang, Reference Zhang and Yang2012). Similarly, the nitrocompound additions in the present study slowed down AGPR, and NE and NEOH decreased cumulative gas production notably, when compared with NPOH and control. Latham et al. (Reference Latham, Anderson, Pinchak and Nisbet2016) noted that most ruminal microbes tolerate relatively high concentrations of nitrocompounds, with only 0.10–0.20 of the population being inhibited by concentrations likely to be present in the rumen of animals exposed to nitrocompounds. The gas production profile differences presented in the current study suggested that rumen microbes were more sensitive to NE and NEOH than NPOH under the same inclusion level (10 mm).
Total amounts of VFA produced in present incubations were not significantly lower within the nitrocompound-supplemented cultures, in agreement with Anderson et al. (Reference Anderson, Callaway, Van Kessel, Jung, Edrington and Nisbet2003). The results of the current study indicate that the inclusive dose level of nitrocompounds (10 mm) may have no adverse effect on fermentative bacterial population. To compensate for the disruption of electron flow in the production of CH4, the rumen microbial ecosystem often disposes of excess reducing equivalents by increasing the production of more reduced VFA (e.g. propionate, butyrate), which results in decreased production of acetate (Van Nevel and Demeyer, Reference Van Nevel and Demeyer1996). This phenomenon also occurred in the present study, and a portion of the reduced equivalents spared from CH4 production appeared to have been used for the production of more reduced VFA under the conditions of the current experiment.
The greatest CH4 inhibition occurred with the addition of NEOH and NE (96.8 v.96.4%) in comparison with NPOH (35.0%). These results are consistent with earlier studies by Anderson et al. (Reference Anderson, Callaway, Van Kessel, Jung, Edrington and Nisbet2003, Reference Anderson, Carstens, Miller, Callaway, Schultz, Edrington, Harvey and Nisbet2006), who reported that NE and NEOH were almost equally effective in inhibiting ruminal CH4 production in vitro and that NE inhibited CH4 production more effectively than NPOH in the ovine rumen. Regarding CH4 inhibition in the rumen, these nitrocompounds generally serve as alternative electron acceptors by competitively consuming reducing equivalents and inhibiting H2 and formate oxidation (Zhang and Yang, Reference Zhang and Yang2012; Zhang et al., Reference Zhang, Cao, Wang, Wang, Yang and Li2018). A notable accumulation of H2 occurred in the present study, which might indicate an inhibition of H2 oxidation by NE, NEOH and NPOH (Božic et al., Reference Božic, Anderson, Carstens, Ricke, Callaway, Yokoyama, Wang and Nisbet2009; Latham et al., Reference Latham, Anderson, Pinchak and Nisbet2016; Ochoa-García et al., Reference Ochoa-García, Arevalos-Sánchez, Ruiz-Barrera, Anderson, Maynez-Pérez, Rodríguez-Almeida, Chávez-Martínez, Gutiérrez-Bañuelos and Corral-Luna2019). Hydrogen is usually present at approximately 1 µm (0.1 kPa) in the unperturbed rumen (Thauer et al., Reference Thauer, Jungermann and Decker1977); however, H2 concentration often increases to levels that inhibit hydrogenase activity (1 kPa) when ruminal CH4 production is inhibited due to decreased H2 consumption by methanogens (Van Nevel and Demeyer, Reference Van Nevel and Demeyer1996). Due to the remarkable inhibition of CH4 production by the nitrocompounds, H2 accumulation in cultures in the current study might have been great enough to inhibit hydrogenase activity in NE-, NEOH- and NPOH-supplemented incubations.
The addition of NE, NEOH and NPOH decreased 2Hrec; however, the accumulation of H2 did not have an adverse effect on IVDMD and the synthesis of total VFA in the present study. This could be explained by the fact that nitrocompounds have high electron-accepting characteristics (Latham et al., Reference Latham, Anderson, Pinchak and Nisbet2016). However, the significant accumulation of H2 within the incubations supplemented with NE, NEOH and NPOH implies that microbial interspecies-hydrogen transfer might not have been completely optimized. In addition, the reduction of 2Hrec by supplementation of NE, NEOH and NPOH suggested that the efficiency of H2 utilization for the synthesis of VFA and CH4 was reduced, consequently resulting in the increase of H2 accumulation. Therefore, accumulation of H2 in the current study confirmed the inhibitory effect of nitrocompounds on the H2 oxidation and thereafter inhibited the ruminal methanogenesis. The molar H2 proportion in total fermentation gas production was 5.2, 5.0 and 1.4% in NE, NEOH and NPOH groups, respectively. However, the extent to which 2Hrec decreased was far lower than that of CH4 production. The fate of the remaining H2 was not known with certainty, but possible sinks include use for anabolic processes, such as microbial cell growth, reduced products of nitrocompounds metabolism, or synthesis of intracellular polyhydroxyalkanoate or extracellular polysaccharides (Wachenheim and Patterson, Reference Wachenheim and Patterson1992; Russell, Reference Russell1998).
The inhibition of ruminal methanogenesis can also be achieved directly, by diminishing the numbers and/or activity of methanogens (Cieslak et al., Reference Cieslak, Szumacher-Strabel, Stochmal and Oleszek2013). In the current study, methanogen populations were quantified using real-time PCR and the results showed that total methanogens and methanogens from the order Methanobacteriales were all decreased significantly with the addition of NE, NEOH and NPOH. Thus, it was possible that the nitrocompounds exerted a direct inhibitory effect on rumen methanogens, with NE and NEOH showing a greater reduction of methanogens than NPOH. The greater capacities of NE and NEOH to inhibit methanogen populations compared to NPOH may explain why NE and NEOH inhibited CH4 production more effectively than NPOH in the present study.
In the rumen, most methanogens are hydrogenotrophic obligate anaerobes, involved in the reduction of CO2 to CH4 with formate or H2 as the electron donor (Saminathan et al., Reference Saminathan, Sieo, Gan, Abdullah, Wong and Ho2016). Coenzyme F 420 acts as a cofactor for formate dehydrogenase and hydrogenase, which is important for ruminal methanogenesis and believed to be present in almost all methanogens (Hendrickson and Leigh, Reference Hendrickson and Leigh2008). Dolfing and Willem (Reference Dolfing and Willem1985) reported that coenzyme F 420 was an indicator of methanogenic activity. In addition, the formation of CH4 from methyl-CoM is also a key step in ruminal methanogenesis from H 2 and CO2. Methyl-CoM reductase (MCR) is one of the components involved in the catalysis of this reaction (Thauer, Reference Thauer1998). The McrA gene, encoding the α subunit of MCR and determining mcrA gene expression, has been well-accepted as a means of measuring MCR activity (Guo et al., Reference Guo, Liu, Lu, Zhu, Denman and McSweeney2008). In the present study, a significant positive correlation between the content of these enzymes and CH4 production was observed, suggesting that MCR, coenzyme F 420 and F 430 indeed play an important role in the process of CH4 production. Furthermore, the addition of NE, NEOH and NPOH decreased mcrA gene expression and coenzyme F 420 content to different degrees, suggesting that nitrocompounds exerted a direct effect on the activity of the methanogens via deactivation of the aforementioned enzymes. In the present study, NE and NEOH presented greater inhibitory efficiency on mcrA gene expression, coenzyme contents of F 420 and F 430 than that of NPOH and corresponded a greater decrease in CH4 production by NE and NEOH treatment than that of NPOH.
Conclusion
Both NE and NEOH presented much greater capacities to inhibit CH4 production compared to NPOH, resulting in a dramatic increase of H2 accumulation during the in vitro rumen fermentation of a hay-rich feed. Although the addition of nitrocompounds did not decrease feed digestion and total VFA production, rumen fermentation shifted towards increasing propionate and decreasing acetate production. The CH4 inhibition response to the nitrocompounds was associated with the direct inhibition of methanogen and a substantial depression of not only mcrA gene expression, but also the coenzyme contents of F 420 and F 430 in rumen methanogenesis. The findings in the present study ultimately provide a scientific, concrete reference for the practical use of these nitrocompounds with the aim of reducing CH4 emissions in ruminant animals.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0021859619000868.
Financial support
The authors acknowledge financial support from the National Natural Science Foundation of China (grant no. 31572432) and the National Key Research & Development Project of China (No. 2018YFD0502104-3).
Conflict of interest
The authors declare no conflict of interest.
Ethical standards
The experiment was conducted at China Agricultural University. All animal care and experimental operations were complied with the Guidelines of the Beijing Municipal Council on Animal Care.
