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Article

Rapid Screening of Methane-Reducing Compounds for Deployment via Water with a Commercial Livestock Supplement Using In Vitro and FTIR-ATR Analyses

by
Ryan J. Batley
1,*,
Elieder Prates Romanzini
1,2,
Joel B. Johnson
1,3,
William Luiz de Souza
1,4,
Mani Naiker
1,
Mark G. Trotter
1,
Simon P. Quigley
1,
Guilhermo Francklin de Souza Congio
5 and
Diogo Fleury Azevedo Costa
1
1
Institute for Future Farming Systems, Central Queensland University, Rockhampton, QLD 4701, Australia
2
DIT AgTech, Wilsonton, QLD 4350, Australia
3
Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Health and Food Sciences Precinct, Brisbane, QLD 4108, Australia
4
Faculty of Agrarian and Veterinary Sciences, São Paulo State University, Jaboticabal 14884900, SP, Brazil
5
Noble Research Institute LLC, Ardmore, OK 73401, USA
*
Author to whom correspondence should be addressed.
Methane 2024, 3(3), 437-455; https://doi.org/10.3390/methane3030025
Submission received: 31 May 2024 / Revised: 13 July 2024 / Accepted: 26 July 2024 / Published: 2 August 2024
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Abstract

The addition of methane-reducing compounds (MRCs) to livestock drinking water presents an alternative method for enteric methane mitigation in extensive systems where these compounds cannot be fed through the diet. This work evaluated several such compounds with the potential to be deployed in this manner. Methane-reducing compounds were selected based on the existing literature and likelihood of dissolution when combined with a commercially available water-based nutrient supplement (uPRO) (uPRO ORANGE®, DIT AgTech, QLD, Australia). This, in turn, would demonstrate the capacity for MRCs to be administered through animal drinking water when such supplements are in use. This technique requires the analysis of MRC solubility and stability in solution, which was completed via Fourier transform infrared-attenuated total reflectance spectroscopy. The uPRO supplement is comprised of urea, urea phosphate, and ammonium sulfate, providing nitrogen, phosphorus, and sulfur—limiting nutrients for ruminants grazing extensive systems during drier periods of the year. Accordingly, medium-quality Rhodes grass hay was used in fermentation runs to simulate a basal diet during the dry season. Methane-reducing compounds were assessed in accordance with each variable measured (gas/methane production, dry matter digestibility, stability under different environmental conditions) along with existing research in the field to determine the most suitable compound for co-administration. Whilst most compounds examined in this study appeared to retain their structure in solution with uPRO, fermentation results varied in terms of successful methane mitigation. The additive Agolin Ruminant L emerged as the most promising compound for further in vivo investigation.

1. Introduction

Currently, there is a significant challenge in meeting the growing global demand for ruminant products, such as meat and milk, due to continued expansion of the world population [1]. Intensification of ruminant production systems to meet this demand is accompanied by growing concern regarding the concomitant increase in atmospheric concentrations of greenhouse gas (GHG) and associated climate change [2], as 6% of global anthropogenic GHG emissions stem from the enteric methane emissions of ruminants [1].
Among the various strategies employed to mitigate enteric methane emissions from ruminants, one of the most utilized is the provision of additives that modulate ruminal fermentation or inhibit methane production [3,4]. This is achieved by disrupting the biological pathways for methane production or altering the population of rumen microbiota to remove methanogenic species [5]. However, the strategy of providing additives to mitigate enteric methane emissions from ruminants in extensive grazing systems is challenging, due to the difficulties in daily supplementation associated with labour shortages [6], remote locations, and inconsistent consumption of supplements containing these additives [7]. In light of these challenges, water medication emerges as an alternative for the supplementation of additives to ruminants. According to Ahlberg et al. [8], cattle exhibit a water intake of 8.0 to 9.8% of their live weight daily, facilitating the use of water supplementation to provide nutrients and additives. This technique has been utilized as a tool to provide nitrogen (N), phosphorus, sulfur and other nutrients, promoting adequate consumption to help ensure optimal health, growth, and reproduction [9]. Of particular importance is N availability, which is required for microbial protein synthesis and the ultimate supply of the majority of protein to the small intestine [10]. Where N availability is low in forages, especially prevalent conditions found in extensive production systems, supplementation is vital to ensure maintenance of live weight. If a suitable methane-reducing compound (MRC) could be incorporated as part of this existing supplementation, then water medication of ruminant drinking water in troughs would provide a novel mechanism to address enteric methane emissions from extensive systems. Although the use of animal drinking water has been posited as a potential delivery mechanism for MRCs to reduce methane emissions from grazing livestock [2,11], at the time of writing, very little research has been carried out in this area.
Such a delivery method necessitates that the compound retains its structure, solubility, and potential methane mitigation capability when combined with an aqueous solution of nutrient supplement. However, many MRCs are complex mixtures, and may even contain proprietary formulations, such that utilising analytical equipment like gas or liquid chromatography-mass spectrometry may not be possible, suitable, or require time-consuming separations and sample preparations. The use of these and other analytical methods such as colorimetric assays also requires the establishment of methods specific to each compound. Conversely, the use of Fourier transform infrared-attenuated total reflectance (FTIR) spectroscopy has been shown to capably identify compounds by establishing unique spectra quickly and cheaply [12]. Accordingly, a series of laboratory experiments were undertaken to characterize the solubility and stability of additives identified as potential methane reducers under varying temperature and pH conditions using FTIR spectroscopy.
Compounds were selected based on their demonstrated capacity for methane mitigation and low level of risk for both animal and environment as described by Beauchemin et al. [13], and likely ability to dissolve in water. The only compound with any level of animal risk was nitrate, which can potentially be toxic. During ruminant conversion of nitrate to ammonia, the intermediate nitrite can be transferred to the blood through the rumen wall and bind to haemoglobin, turning it to methemoglobin which is unable to transport oxygen [14]. The resulting hypoxia is poisonous, presenting as reduced productivity, infection, coma, or even death.
Additionally, assessments were made regarding in vitro digestibility and total gas/methane production when selected MRCs were incubated with a medium-quality Rhodes grass forage substrate and ruminal fluid to replicate extensive production systems in northern Australia. The additives were diluted in an aqueous solution of a non-protein nitrogen, phosphate, and sulfate supplement comprised of ammonium sulfate, urea, and urea phosphate (uPRO) (uPRO ORANGE®, DIT Ag Tech, QLD, Australia), simulating current commercial application of water supplementation. The results of this analysis were combined with existing knowledge around the mechanism and performance of the MRCs to suggest a suitable candidate for combination with uPRO and future in vivo research.

2. Results

2.1. FTIR Analysis of Methane-Reducing Compound Solubility/Stability in Aqueous Solution with uPRO

In the present study, FTIR analysis was based on the retention of absorbances of infrared light at conspicuous wavelengths associated with specific functional groups. The key peaks from aqueous solutions containing the MRC only were identified, then solutions combining the MRC with uPRO were analysed to see if those key peaks were retained. Following this, various treatments of time, temperature, and starting pH levels of water used to prepare the solutions were also analysed to show that the MRC retained both solubility and stability in solution with uPRO over the period and conditions it would be expected to be in a water trough for animals. Significant decreases across all key peak absorbance intensities but consistency in wavenumber location were used to indicate a decrease in concentration and therefore solubility of the MRC, whilst large variations in wavenumber locations and intensities were used to indicate alteration to the structure or concentration of individual components of the MRC.

2.1.1. uPRO

The commercial supplement uPRO contains ammonium sulfate, urea, and urea phosphate. Absorbance peaks from those components may influence spectra after uPRO has been combined with the MRC of interest. Therefore, a baseline spectrum for neat uPRO was established to identify the extent of this influence (Figure A1). Spectral analysis could not be obtained from the uPRO once it had dried on the instrument. Therefore, this spectrum was derived from the neat, undried solution. The neat compound shows broad absorbance from 3000 to 3500 cm−1 indicative of O-H groups, and there are also distinctive overlapping peaks at 3214 and 3352 cm−1 likely to be associated with N-H stretching. The peak at 1650 cm−1 with shoulder at 1622 cm−1 may be a combination of C=N stretching, N-H bending, and the distinctive H-O-H bending of water. The peaks at 1458 and 1000 cm−1 likely correspond to N-H bending/C-N stretching and S=O stretching of the sulfate [15,16,17].

2.1.2. Agolin Ruminant L (Agolin)

Agolin is a complex mixture of essential oils, an emulsifier, and stabiliser, and exhibits key peaks at around 2925, 2863, 1730, and 1105 cm−1 (Figure A2). These peaks can be associated primarily with symmetric/asymmetric C-H stretching, C-O and C=O stretching of the essential oils, polysorbate 20, and benzoic acid [18,19,20,21,22,23,24,25]. The intensity of the Agolin key peak absorbances are similar once it is combined with uPRO, apart from a slight decrease in the peak at 2860 cm−1 after the addition of uPRO, and the wavenumber of these peaks remains consistent except for a small shift of the peak at 1105 cm−1 (Table 1; Figure A2). After treatments, there is a significant increase (p≤ < 0.001, < 0.001, and p = 0.02, 0.02) in the absorbance intensity of the key Agolin peaks compared to the baseline, but they are very close with an average difference of only 0.01. This suggests that Agolin is readily retained in an aqueous uPRO solution, with a slight increase in concentration associated with treatment conditions.

2.1.3. Beeocitrix+

Beeocitrix+ is another complex mixture composed primarily of ricinoleic acid (97.3%) and green propolis (2%). The resulting spectrum is largely reflective of the ricinoleic acid, and key peaks are observed in a band between 3500 and 3000 cm−1, and at 2926, 2854, 1726, 1561, and 1404 cm−1 (Figure A3). These peaks can be associated with O-H stretching, methyl and methylene C-H stretching, C=O stretching, C=C stretching, and O-H bending of both the ricinoleic acid and propolis compounds [26,27]. Upon the addition of Beeocitrix+ to the uPRO-containing solution, a white precipitate was observed. Absorbance intensities at 1561 and 1404 cm−1 were also absent or greatly reduced, indicating either a change in MRC structure or solubility, and no further analysis was completed.

2.1.4. Nitrates

Four nitrate compounds were assessed, with the cation components consisting of either calcium, magnesium, potassium, or sodium. Asymmetric and symmetric vibrations of nitrate anions have been shown in the region of 1369 to 1338 cm−1 [28,29], and this interaction was used to avoid issues with absorbances associated with water molecules. Key peak absorbances were recorded at 1334, 1329, 1346, and 1344 cm−1 for calcium, magnesium, potassium, and sodium nitrates, respectively (Figure A4, Figure A5, Figure A6 and Figure A7). The absorbance intensities for the nitrates as listed previously are very similar across the baseline and treatments, with differences of 0.01, 0.04, 0.02, and 0.07, respectively, and a significant increase (p = 0.003, 0.046, and p < 0.001, < 0.001, respectively) in the intensity of the absorbances in the treatments was observed (Table 1). This indicates a retention of all the nitrates in solution when combined with uPRO, with the treatment conditions resulting in a slight concentration increase.

2.1.5. Choline Chloride

Analysis of FTIR data of choline chloride crystals identified prominent broad peaks between 3500 and 3100 and at 3018 and 1636 cm−1 [30,31]; however, these become masked after drying the aqueous solution, as some water molecules are retained even when it appears dry (Figure A8). Two remaining key peaks can be used for choline chloride analysis however, found at 1479 and 1084 cm−1, associated with CH2 scissoring and CH2CH2-O stretching [30]. There is an observable decrease in the intensity of absorbances at these wavenumbers when comparing solutions with and without uPRO, but intensity and wavenumbers are very similar when comparing the solutions with uPRO and before and after treatment conditions, with a difference in absorbance of only 0.025 across both key peaks. This difference translates to a significant increase (p < 0.001) in both absorbance intensities for the key peaks, indicating the apparent retention of choline chloride across the uPRO solution treatments with a slight increase in concentration (Table 1).

2.1.6. Saponin

The saponin used in this study was an extract from the bark of Quillaja saponaria. Spectra from FTIR analysis identified several key peaks associated with saponin, observed to be between 3600 and 3000 cm−1 as well as at 2936, 1722, 1602 and 1039 cm−1. These are associated with O-H stretching, C-H stretching, C=O stretching, C=C stretching, and C-O-C stretching, respectively [32]. However, the influence of uPRO in aqueous solution masks these key peaks, with the only potentially discernible peak available for analysis at the same wavenumber occurring at 2930 cm−1 (Figure A9). The variation in the absorbance intensities between the spectra when comparing solutions with and without uPRO means that it is unlikely that reliable measurements can be obtained using FTIR. Whilst the curves of the solutions containing saponin before and after treatment are very similar, a different method would be required to provide a definitive measurement of saponin content in a uPRO solution.

2.1.7. SilvaFeed

SilvaFeed contains extracts from the Quebracho tree (Schinopsis lorentzii and Schinopsis balansae). The active ingredients of interest are tannins, which exhibit distinct peaks between 3600 and 3000 cm−1, and at 1605, 1517 and 1446 cm−1. The peaks between 3600 and 3000 cm−1 relate to O-H stretching, while the remaining peaks are characteristic of C=C-C stretching in aromatic rings [33]. In this study, the broad peak between 3600 and 3000 cm−1 has become obscured by the apparent N-H stretching of uPRO, but distinctive key peaks can still be observed at 1612, 1519, and 1450 cm−1 (Figure A10). Whilst the wavenumbers remain the same, there is an increase in the intensity of absorbances between the SilvaFeed solutions with and without uPRO. There was no significant difference (p = 0.684 and 0.885) between the baseline SilvaFeed/uPRO solution and treatments for absorbances at 1519 and 1450 cm−1, and a significant (p = 0.044) increase for the peak at 1612 cm−1 (Table 1). The difference in absorbances across the key peaks is only 0.003, and the similarity of the key peak absorbances suggests that Silvafeed is retained in a solution containing uPRO.

2.1.8. Tartaric Acid

Tartaric acid is an organic acid, and as such it features distinctive carboxylic acid peaks between 3600 and 2600 and at 1739 cm−1, as well as sharp peaks between 1132 and 1066 cm−1 associated with C-O stretching [34]. Once combined with water, however, the carboxylic acid peaks between 3600 and 2600 cm−1 are masked by those associated with water, even when dry (Figure A11). However, key peaks at 1714, 1124, and 1078 cm−1 associated with the aforementioned absorbances were visible and used for analysis. There were no significant differences (p = 0.358, 0.272, and 0.184, respectively) in these key peaks between the baseline tartaric acid/uPRO solutions and the treatments, indicating that tartaric acid is likely retained in the uPRO solution under those conditions.

2.2. pH Values of Prepared Solutions

Solutions containing combinations of MRCs tested and uPRO resulted in considerably lower pH values compared to those without uPRO (Table 2).

2.3. In Vitro Apparent Dry Matter Digestibility and Gas Production

Fermentations containing choline chloride, Agolin, and tartaric acid exhibited slight increases in the amount of dry matter (DM) digested when compared to the control fermentation (3.8 to 2.55%) (Table 3). Conversely, there was a significant (p = 0.0015) reduction observed when calcium nitrate was added (31.49%).
There was a significant (p = 0.0029) increase in the total gas production when choline chloride was added, representing a 92.9 mL/g DM increase when compared to the control. Conversely, significant reductions (p = 0.0291 and 0.0012) in total gas production were observed for the sodium and calcium nitrates, reflective of variances of −30.5 and −104 mL/g DM, respectively (Table 4). A significant reduction (p = 0.0086) in total gas production was also found to come from Agolin (19.9 mL/g DM).
There were no significant reductions in the total methane production or ratio of methane to total gas produced (Table 5 and Table 6). This is largely attributable to the large standard deviation of the control fermentations. However, there were large negative variances of (10.0, 4.6, and 1.5 mL/g DM) methane produced when adding calcium nitrate, sodium nitrate, and Agolin, respectively. Similar reductions of 10.3 and 2.3 in the methane to total gas production ratio were also observed for calcium and sodium nitrate. Conversely, when choline chloride was added to the fermentation there were large increases of 22.3 mL/g DM and 6.8 in methane production and methane to total gas ratio, respectively.

3. Discussion

3.1. FTIR Analysis

With the exception of saponin and Beeocitrix+, the FTIR method demonstrated the solubility and stability of the MRCs in uPRO solutions under varying temperature and pH conditions, either at the same or slightly increased concentrations compared to their baseline solution. Increases in concentration are likely due to evaporative loss of water from, but retention of the compound in, solution. That said, there are limitations in the technique. There is the potential for inconsistencies in the drying of the solution and/or compound crystallization on the ATR crystal, leading to inaccurate absorbance measurements [12]. This can be mitigated by taking the measurements before compounds crystallise and increasing replicates. Furthermore, because FTIR is not able to discern specific compounds in complex mixtures, and instead establishes a unique fingerprint for the whole, there is the potential that volatile active compounds related to methane mitigation may be lost from the solution before it has dried on the instrument, and resulting spectra are largely reflective only of emulsifying agents/preservatives that support the active ingredients. The only way to overcome these issues is to utilize more powerful but expensive and time-consuming analytical instrumentation such as gas and liquid chromatography-mass spectrometry. Finally, small-scale analysis using test tubes may not necessarily be an accurate representation of water trough conditions in the field, which may see greater evaporative loss. Despite these limitations, FTIR spectroscopy appears to be effective as a rapid and cheap initial screening tool for MRCs in aqueous solutions.

3.2. Water Supplementation of MRCs and In Vitro Analysis

Supplementing diets is a common practice in beef cattle systems to enhance productivity and efficiency. In extensive grazing systems, where animals roam over large areas, water supplementation emerges as an efficient method to deliver essential nutrients. Between 1984 and 1987, McLennan et al. [35] evaluated various supplementation methods for weaner heifers during the dry season in extensive grazing systems, and highlighted drinking water supplementation as a potential alternative for providing nutrients to livestock. Fundamental to this was an understanding of whether water supplementation would impact an animal’s water consumption, aiming to avoid health issues associated with low water intake. Previous studies suggest that animals exhibit similar water consumption patterns regardless of whether they receive water supplemented with nutritional plans [9,35], indicating that similar results may be achievable when medicating the water with MRCs.
However, recognizing that combining methane mitigants with water supplementation may be an effective greenhouse gas reduction strategy in extensive production systems is just the initial step. Methane-reducing compounds can be classified based on their mode of action within the rumen as either inhibitors of methanogenesis or modifiers of the rumen environment [5]. Rumen inhibitors act directly on the biological methanogenesis pathway, disrupting the process and reducing methane production. In contrast, rumen modifiers alter the rumen environment, limiting the growth of methanogenic microorganisms and mitigating enteric methane production without directly targeting methanogenesis pathways. Honan et al. [5] report that 3-nitroxypropanoland halogenated compound-containing organisms such as macroalgae act as rumen inhibitors, whilst lipids, nitrates, plant secondary compounds including essential oils can be defined as rumen modifiers. Some of these compounds are directly incompatible with water and others, despite retaining solubility and stability in solution, may require longer periods to deliver a methane-reducing effect than can be replicated in a short-term in vitro fermentation.
Despite those limitations, the analysis described herein was able to provide evidence for a suitable candidate for addition to the uPRO supplement for drinking water delivery. Beeocitrix+ and saponin were removed after FTIR analysis due to the precipitation observed in the former, and limited evidence for key peak retention in the latter. Nitrate at a dose rate of 4 g NO3/L was dismissed due to its negative impact (−32.7%) on digestibility during in vitro fermentation. Martins et al. [36] also reported a decrease in DM digestibility associated with nitrate in their meta-analysis.
Whilst the dose rate of 2 g NO3/L resulted in a just over 53% reduction in methane production compared to the control, the risk of nitrate toxicity is a limiting factor for its deployment in drinking water. Tomkins et al. [37] note that spreading nitrate intake over two intake events reduces the risk of toxicity. It is likely that spreading nitrate intake out through multiple drinking events during the day would reduce this risk further still, but the ad libitum nature of water-based supplementation limits the ability to control dosage to safe levels, especially if there are concentration changes due to evaporative loss of water in the trough. While existing dosage control technologies could mitigate this risk, introducing such variables during animal trials may be undesirable during the initial evaluation stage.
There are conflicting reports regarding the efficacy of tartaric acid as a methane mitigator when analysed in vitro, with studies reporting both significant [38] and non-significant [39] reductions. The results in this study agree with the latter, leading to the exclusion of tartaric acid from consideration. The fermentation of SilvaFeed in aqueous solution resulted in a slight, albeit non-significant, reduction in methane production. This contrasts with previous in vitro studies where significant methane reductions were observed [40,41] using similar quebracho tannins, and there is the potential that the methane-mitigating properties of this compound may be impacted in some way when combined with uPRO. However, Beauchemin et al. [13] note that methane reductions associated with tannins are also highly dependent on diet as well the capacity for rumen microbes to degrade it, which may also explain the observed result and encourages further investigation.
Existing research on the use of choline chloride as a methane mitigant is limited. The observed increase in methane production in the choline chloride fermentation compared to the control (22.3 mL/g DM) is noteworthy. This finding aligns with another in vitro study where initial 48-h methane production increased before decreasing by 50% after 15 days [42]. These results suggest that while choline chloride may be feasible for water-based supplementation in concert with uPRO, further investigation is required to fully understand its potential efficacy and long-term effects.
Finally, small reductions in total gas and methane production (19.9 and 1.5 mL/g DM respectively) coupled with a slight increase in digested DM (3.3%) were observed in the Agolin fermentations. Although only the total gas production saw a significant reduction (p = 0.009), these results were expected due to documented observations that, as an essential oil-based rumen modifier, Agolin may require up to four weeks to be fully effective in reducing methane emissions [43]. Several articles, including a meta-analysis, have established in vivo that by promoting a more efficient digestion of feed and altering the microbial composition, Agolin mitigates methane formation without compromising animal performance [43,44,45]. There are other in vivo studies which challenge those findings, however, where methane production or yield was not impacted in dairy cattle provided with Agolin [46,47]. Agolin also benefits from existing approvals for animal use and reliable commercial supply. This readiness for deployment and proven track record makes it an ideal selection to be coupled with the uPRO supplement. The mixed results in the current literature underscore the need for additional in vivo analysis, however, and our research group has progressed the combination of uPRO and Agolin to in vivo experimentation not reported here.
The findings of this study establish the potential for methane-reducing compounds to be used in conjunction with nutrient supplementation currently being delivered in animal drinking water. They also highlight the limitations of in vitro assays for assessing such compounds, but nonetheless provide a methodology which can serve as a starting point on which to base future research. To that end, the current work represents a first step of ongoing research which includes long term stability/solubility testing and screening potential new compounds as they appear on the market.
In the case of the selected compound for use in the uPRO supplement line, our results align with previous research indicating that the inclusion of Agolin in ruminant diets is likely to lead to a significant decrease in methane production. This reduction can be attributed to the mode of action of Agolin, which primarily involves modulation of the rumen microbiota and fermentation processes. While previous studies have provided valuable insights into its mode of action, there is still a need for a comprehensive understanding of the biochemical pathways involved, particularly when delivered via drinking water, a highly novel strategy. Future research should focus on elucidating the specific microbial populations affected by Agolin and the metabolic pathways through which methane production is inhibited. Moreover, attention should be paid to exploring the synergistic effects this compound might have with other ingredients in water supplementation and the basal diet. The use of water medication to deliver methane-reducing compounds carries significant implications for sustainable livestock production, particularly in extensive grazing systems where there has been no establishment of an effective approach using additives in the solid form.

4. Materials and Methods

4.1. uPRO ORANGE® Supplement

The additive uPRO ORANGE® (DIT AgTech, Wilsonton, QLD, Australia) is a supplement comprised of urea, urea phosphate, and ammonium sulfate to supplement the basal diet with extra N, P and S. It is a clear yellow/orange coloured liquid with a viscosity comparable to water.

4.2. Selected Compounds

The compounds selected for in vitro testing with uPRO included four types of nitrate (calcium, magnesium, sodium, and potassium nitrate), choline chloride, Quillaja saponin, tartaric acid (Sigma-Aldrich, Bayswater, Australia), a blend of essential oils (Agolin Ruminant L®, Feedworks, Romsey, Australia), soluble Quebracho tannins (SilvaFeed®, San Michele Mondovì, Italy), and green propolis (Beeocitrix+®, Beeotec, Santa Rita do Sapucaí, Brazil).

4.3. Feed Substrate

Rhodes grass (Chloris gayana Kunth) is a tropical C4 grass recognised for its adaptability and resilience in various climates. Originating from Africa, it has gained global popularity for its exceptional drought tolerance and ability to thrive in poor soil conditions. With its rapid growth and capacity to regenerate after grazing, Rhodes grass stands as a reliable choice for sustainable pasture management, particularly in arid and semi-arid regions such as the rangelands of Australia. Table 7 outlines the essential nutritional composition of the Rhodes grass employed in this study.

4.4. Solubility and Stability Testing Using FTIR-ATR

4.4.1. FTIR Spectroscopy and Measurement of Neat MRC Spectra

Two separate FTIR instruments were used to expedite analysis. The MRCs were only being compared with and without uPRO and against the treatment conditions individually and did not need to be compared directly to other compounds in the study, so there was no need to run all analysis on the same instrument. The compounds Beeocitrix+, Agolin, SilvaFeed, and tartaric acid were examined using a Bruker Alpha FTIR spectrophotometer equipped with a platinum diamond attenuated total reflectance (ATR) single reflection module. Meanwhile, magnesium nitrate, sodium nitrate, potassium nitrate, calcium nitrate, choline chloride, and saponin were analysed using a PerkinElmer Spectrum 100 FTIR spectrophotometer fitted with a Diamond/ZnSe universal ATR.
For neat product analysis, aqueous solutions (40 µL) of MRCs were applied to the ATR crystal and, where possible, allowed to dry before measuring the spectra. Concentrations of the MRCs were chosen to ensure that a signal could be detected on the FTIR instrument (Table 8). Magnesium nitrate, sodium nitrate, potassium nitrate, calcium nitrate, and tartaric acid, due to extended drying times and potential crystallization, had their spectra recorded after 20 min or once initial crystallization was observed. Air served as the reference background. The Bruker Alpha FTIR used OPUS software (v7.5), recording spectra between 4000 and 400 cm−1 as the average of 24 scans at a resolution of 4 cm−1. The PerkinElmer FTIR utilized Spectrum IR software (v10.6.2.1159), recording spectra between 4000 and 650 cm−1 as the average of 8 scans at a resolution of 4 cm−1. The FTIR spectra from raw compounds were used to identify key peaks from approximately 3500 to 1100 cm−1.

4.4.2. FTIR Sample Preparation for Combinations of uPRO and MRC Baselines and Treatments

Water used to prepare the solutions was premixed with uPRO at a concentration of 1.7 mL/L, prior to adding the MRC at the same concentration as used in the neat spectral analysis. Exactly 5 mL of each solution was pipetted into six 10 mL test tubes and sealed. Two tubes were incubated at 45 °C (±1 °C) in an oven, two at ambient room temperature (approximately 25 °C), and two in a refrigerator at 4 °C (±1 °C). After 24 h, one tube from each temperature group was sampled and analysed using FTIR spectroscopy in the same manner described in Section 4.4.1. The remaining tubes were left for 48 h before analysis. This process was replicated where the pH of water used to make the initial uPRO solution was altered (pH 5.5 and 8.5), and tubes were incubated at room temperature for 24 and 48 h before analysis.

4.5. pH Values of Prepared Solutions

To identify potential issues stemming from alterations in the pH of solutions containing additives and the uPRO supplement, pH measurements (Eutech Instruments PC 2700, Thermo Fisher Scientific, Adelaide, Australia) were recorded.

4.6. Nitrogen Content

The nitrogen content of the feed substrate was determined using a LECO TruMac Series Carbon and Nitrogen Analyser (Sydney, Australia) and converted to protein content using a conversion factor of 6.25.

4.7. Neutral Detergent Fibre (NDF)

The neutral detergent fibre of the feed substrate was measured using an Ankom (Macedon, NY, USA) model 200 fibre analyser as per the manufacturer’s instructions.

4.8. In Vitro Apparent Digestibility, Total Gas, and Total Methane Assessment

4.8.1. Feed Substrate Preparation

The digestibility analysis utilised a medium-quality Rhodes grass (Chloris gayana) as the substrate. Forage samples were oven dried (Memmert, Schwabach, Germany) until reaching a constant mass, then ground in a blender and sieved to a size of 2mm.

4.8.2. Rumen Collection

Rumen fluid was collected from eight grass-fed steers post-slaughter at a certified abattoir (JBS Australia, Rockhampton, Australia), following established protocols [48]. Standard operating procedures were strictly followed for animal slaughter and waste product collection, ensuring compliance with AUS-MEAT standards. Animals were provided access to water and were withdrawn from feed approximately twenty-four hours before slaughter.

4.8.3. In Vitro Fermentation Experimental Design

Two separate in vitro fermentation runs were conducted, incorporating blank, control, and treatment groups in duplicate, and were based on the method described by Kinley et al. [49] with some minor modifications. Each fermentation vessel received approximately 1 g of Rhodes grass substrate and 100 mL of Goering and van Soest buffer maintained at 39 °C. The vessels were placed in a shaking water bath (Julabo SW22, Julabo GmbH, Seelbach, Germany) set to 39 °C to mimic rumen conditions. Additionally, 20 mL of fluid was added to each vessel as follows: blanks and controls received 20 mL of water, while treatments were administered with 20 mL of test compounds diluted with an aqueous uPRO solution to recommended dose rates (Table 9). Due to limitations in the number of replicates that could be completed, only two nitrates were tested representing two different dose rates.
An additional 2 mL of reducing solution was added to the vessels, followed by 25 mL of rumen fluid. The fermentation vessels were then purged with CO2, capped with an Ankom RF gas production module (Macedon, NY, USA), purged with CO2 once more, and gently stirred. The shaking water bath was set to 85 RPM, and the fermentations proceeded for 72 h. Stirring occurred twice daily to redistribute any substrate accumulation on the vessel walls.

4.8.4. In Vitro Fermentation Total Gas Production

The Ankom RF gas production system monitored the total gas generated during the 72-h period. The system was configured with the following parameters: maximum pressure—3 psi, live interval—60 s, recording interval—20 min, valve open time—250 ms. Cumulative pressure, following the principles of natural gas law, was utilized to calculate the volume of gas produced, expressed as mL/g of substrate DM.

4.8.5. In Vitro Fermentation Methane Production

The vented gas from the Ankom RF gas production system modules was collected in FlexFoil® PLUS gas sample bags (SKC, Dorset, UK), connected to the Ankom modules via Teflon tubing and an Ankom vent valve adapter (Macedon, NY, USA). Methane levels were analysed using gas chromatography on an Agilent Technologies 6890N GC system (Santa Clara, CA, USA), equipped with a Supelco custom 80/100 HAYESEP Q 3ft x 1/8 in stainless steel column and a flame ionization detector (FID). Operating temperatures were set as follows: column at 65 °C, injector at 100 °C, and FID at 100 °C. Nitrogen served as the carrier gas at a flow rate of 10mL/min, with an injection volume of 250 µL. Samples were extracted from the foil gas sample bags using a gas-tight syringe before direct injection into the GC instrument.

4.8.6. In Vitro Apparent Digestibility

At the conclusion of each run, vessels were cooled on ice to minimise fermentation. The in vitro fluid was subsequently vacuum-filtered through size 1 Duran glass fritted crucibles, which were pre-filled with about 1 cm of fine sand. Following filtration, the crucibles were oven-dried at 105 °C until a constant mass was attained, and digestibility was assessed gravimetrically.

4.9. Statistical Analysis

Data analysis was conducted with IBM SPSS (v26). For FTIR analysis, two-sided one sample t-tests were used. For DMD, gas production, methane production, and total gas to methane ratio, independent t-tests were used. Significance was determined at a threshold of p < 0.05. Visualization and analysis of FTIR spectra was performed using Spectragryph (v1.2.16) (Friedrich Menges; Oberstdorf, Germany).

5. Conclusions

This study evaluated the suitability of several commercially available methane-reducing compounds for deployment in animal drinking water along with uPRO, a non-protein nitrogen, phosphate, and sulfate supplement. The compounds examined in this study, apart from Beeocitrix+ and saponin, appeared to retain their structure and dissolve readily when combined with an aqueous uPRO solution at the tested concentrations using FTIR spectroscopy as a rapid analysis tool. The results also suggest that the compounds retain this stability and solubility across several different conditions which water in troughs would experience. This indicates their suitability for use in cattle drinking water in combination with a nutrient supplement. An Agolin and uPRO solution, in which Agolin was shown to retain its solubility and stability with significant increases (p < 0.001, < 0.001, and p = 0.02, 0.02) in the absorbances of key peaks when analysed using FTIR, emerged as the most suitable compound. After in vitro fermentation, a modest increase in DM digestibility (3.3%), a significant reduction in total gas produced (19.9 mL/g DM, p = 0.009), and a reduction in methane production (1.5 mL/g DM; p = 0.375), demonstrated promise for deploying Agolin in drinking water. Greater methane reductions using Agolin would be expected in long term studies in vivo, due to the time required for it to modify the rumen microbiota, and it has been selected for further validation in an animal trial.

6. Patents

Australian Innovation Patent number 2021105299—A method of reducing methane production in a ruminant animal—11/08/2021.

Author Contributions

Conceptualization, D.F.A.C. and R.J.B.; methodology, D.F.A.C., M.N. and R.J.B.; validation, D.F.A.C.; formal analysis, R.J.B., D.F.A.C. and J.B.J.; investigation, R.J.B., J.B.J. and D.F.A.C.; resources, D.F.A.C. and G.F.d.S.C.; data curation, R.J.B.; writing—original draft preparation, D.F.A.C. and R.J.B.; writing—review and editing, D.F.A.C., E.P.R., W.L.d.S., M.N., G.F.d.S.C. and R.J.B.; visualization, D.F.A.C. and R.J.B.; supervision, D.F.A.C. and M.N.; project administration, M.G.T., S.P.Q. and D.F.A.C.; funding acquisition, M.G.T., S.P.Q. and D.F.A.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Meat and Livestock Australia (MLA-MDC-P.PSH.1378) and the State Government of Queensland (AQIRF169-2021RD4). One of the authors (R.J.B) was the recipient of an MLA Donor Company Research Stipend Scholarship.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data can be shared upon reasonable request via email to the corresponding author.

Acknowledgments

We acknowledge DIT AgTech for providing the uPRO ORANGE® supplement for testing and all collaborators (Beeocitrix+, Feedworks, Silva Team) that provided their commercial products for testing. Additionally, the authors express their gratitude to JBS Australia for providing rumen fluid, Tania Collins for laboratory assistance, and Andrew Bryant for conducting protein analysis.

Conflicts of Interest

The authors have read the journal’s guideline and have the following competing interests: the co-author (E.P.R) is an employee of DIT AgTech. The other authors have no competing interests.

Appendix A

Appendix A contains the FTIR spectra from analysis conducted in the experiment.
Methane 03 00025 g0a1
Figure A1. Fourier transform infrared-attenuated total reflectance spectra of undried neat non-protein nitrogen, phosphate and sulfate supplement (uPRO) on Bruker Alpha and Perkin Elmer FTIR spectrophotometers.
Figure A1. Fourier transform infrared-attenuated total reflectance spectra of undried neat non-protein nitrogen, phosphate and sulfate supplement (uPRO) on Bruker Alpha and Perkin Elmer FTIR spectrophotometers.
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Figure A2. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) Agolin but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) Agolin prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) Agolin prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Figure A2. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) Agolin but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) Agolin prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) Agolin prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
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Figure A3. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) Beeocitrix+ but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) Beeocitrix+ prepared in aqueous solution containing uPRO (1.7 mL/L).
Figure A3. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) Beeocitrix+ but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) Beeocitrix+ prepared in aqueous solution containing uPRO (1.7 mL/L).
Methane 03 00025 g0a3
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Figure A4. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) calcium nitrate but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) calcium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) calcium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Figure A4. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) calcium nitrate but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) calcium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) calcium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
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Figure A5. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) magnesium nitrate but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) magnesium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) magnesium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Figure A5. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) magnesium nitrate but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) magnesium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) magnesium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Methane 03 00025 g0a5
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Figure A6. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) potassium nitrate but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) potassium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) potassium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Figure A6. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) potassium nitrate but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) potassium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) potassium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Methane 03 00025 g0a6
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Figure A7. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) sodium nitrate but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) sodium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) sodium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Figure A7. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) sodium nitrate but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) sodium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) sodium nitrate prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
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Methane 03 00025 g0a8
Figure A8. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) choline chloride but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) choline chloride prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) choline chloride prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Figure A8. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) choline chloride but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) choline chloride prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) choline chloride prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Methane 03 00025 g0a8
Methane 03 00025 g0a9
Figure A9. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing saponin but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), saponin prepared in aqueous solution containing uPRO (1.7 mL/L), and saponin prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Figure A9. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing saponin but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), saponin prepared in aqueous solution containing uPRO (1.7 mL/L), and saponin prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Methane 03 00025 g0a9
Methane 03 00025 g0a10
Figure A10. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) Silvafeed but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) Silvafeed prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) Silvafeed prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Figure A10. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) Silvafeed but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) Silvafeed prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) Silvafeed prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Methane 03 00025 g0a10
Methane 03 00025 g0a11
Figure A11. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) tartaric acid but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) tartaric acid prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) tartaric acid prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Figure A11. Fourier transform infrared-attenuated total reflectance spectra for dried aqueous solutions containing (a) tartaric acid but no non-protein nitrogen, phosphate and sulfate supplement (uPRO), (b) tartaric acid prepared in aqueous solution containing uPRO (1.7 mL/L), and (c) tartaric acid prepared in aqueous solution containing uPRO (1.7 mL/L) subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions starting at 5.5 and 8.5 pH and held at 25 °C).
Methane 03 00025 g0a11

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Table 1. Comparison of absorbance values of methane-reducing compounds in aqueous solutions, aqueous solutions containing non-protein nitrogen, phosphate and sulfate supplement (uPRO), and treatments for stability/solubility analysis. Two-sided one sample t-tests were used for statistical analysis, with a threshold of significance of p < 0.05.
Table 1. Comparison of absorbance values of methane-reducing compounds in aqueous solutions, aqueous solutions containing non-protein nitrogen, phosphate and sulfate supplement (uPRO), and treatments for stability/solubility analysis. Two-sided one sample t-tests were used for statistical analysis, with a threshold of significance of p < 0.05.
CompoundKey Peak Wavenumber (cm−1)Key Peak Wavenumber AbsorbancesSEMp-Value
Neat 1 Baseline 2Treatments 3
Agolin29250.120.120.130.01<0.001
28600.130.110.120.020.02
17300.060.050.060.020.02
11000.250.240.280.08<0.001
Calcium nitrate13340.440.450.490.010.003
Magnesium nitrate13290.510.550.560.120.046
Potassium nitrate13460.270.250.300.01<0.001
Sodium nitrate13440.420.350.470.02<0.001
Choline chloride14790.140.120.130.001<0.001
10840.130.100.110.002<0.001
SilvaFeed16120.140.230.240.010.044
15190.090.100.100.0040.684
14500.110.150.150.0040.885
Tartaric acid17140.560.550.540.010.358
11240.560.530.570.010.272
10780.600.590.580.010.184
1 The absorbance values for a dried aqueous solution containing the methane-reducing compound (MRC) but no uPRO. 2 The absorbance values for a dried aqueous solution containing uPRO (1.7 mL/L) and the MRC. 3 The average absorbance values for a dried aqueous solution containing uPRO (1.7 mL/L) and the MRC subjected to treatments (solutions held for 24 and 48 h periods at 4, 25, and 45 °C, and 24 and 48 h periods with water used to prepare solutions started at 5.5 and 8.5 pH and held at 25 °C).
Table 2. pH values of prepared solutions of methane-reducing compounds and non-protein nitrogen, phosphate and sulfate supplement (uPRO).
Table 2. pH values of prepared solutions of methane-reducing compounds and non-protein nitrogen, phosphate and sulfate supplement (uPRO).
CompoundFinal pH
(Neutral Water)		Final pH
(Neutral Water + uPRO)		Final pH
(Water pH 5.5 + uPRO)		Final pH
(Water pH 8.5 + uPRO)
Agolin3.442.682.972.93
Beeocitrix+8.935.675.735.84
Silvafeed4.632.962.932.99
Saponin4.412.752.772.83
Magnesium nitrate5.012.242.142.21
Sodium nitrate4.852.412.392.41
Potassium nitrate4.982.642.642.65
Calcium nitrate5.252.262.252.28
Tartaric acid2.091.691.761.78
Choline chloride6.442.452.462.45
Table 3. In vitro apparent dry matter digestibility (IVDMD) (mean ± SD) after additive treatment, ranked in order of greatest positive variance to the control fermentation. Threshold of significance p < 0.05.
Table 3. In vitro apparent dry matter digestibility (IVDMD) (mean ± SD) after additive treatment, ranked in order of greatest positive variance to the control fermentation. Threshold of significance p < 0.05.
AdditiveIVDMD (%)Variance to Control (%)p-Value
Choline chloride69.07 ± 5.43.80.4305
Agolin68.52 ± 2.43.3 0.2338
Tartaric acid67.80 ± 0.42.550.1230
Sodium nitrate64.69 ± 0.70.49 0.4559
SilvaFeed64.92 ± 1.1−0.320.8168
Calcium nitrate31.49 ± 0.4−32.70.0015
Table 4. Total gas production (mean ± SD) after additive treatment, ranked in order of greatest positive variance to the control fermentation. Threshold of significance p < 0.05.
Table 4. Total gas production (mean ± SD) after additive treatment, ranked in order of greatest positive variance to the control fermentation. Threshold of significance p < 0.05.
AdditiveTotal Gas Production (mL/g DM)Variance to Control (mL/g DM)p-Value
Choline chloride185.73 ± 7.192.90.0029
SilvaFeed80.30 ± 5.9−0.320.0953
Tartaric acid87.11 ± 2.8−5.70.1085
Agolin72.99 ± 2.5−19.9 0.0086
Sodium nitrate68.09 ± 5.8−30.5 0.0291
Calcium nitrate0.00 ± 2.0−104.00.0012
Table 5. Total methane production (mean ± SD) after additive treatment, ranked in order of greatest negative variance to the control fermentation. Threshold of significance p < 0.05.
Table 5. Total methane production (mean ± SD) after additive treatment, ranked in order of greatest negative variance to the control fermentation. Threshold of significance p < 0.05.
AdditiveMethane Production (mL/g DM)Variance to Control (mL/g DM)p-Value
Calcium nitrate0.00 ± 0.0−10.00.0646
Sodium nitrate5.42 ± 1.3−4.60.2461
Agolin8.17 ± 0.1−1.50.3752
SilvaFeed9.24 ± 1.7−0.40.8379
Tartaric acid9.24 ± 1.70.20.8905
Choline chloride31.93 ± 2.022.30.0075
Table 6. Ratio of methane to total gas produced (mean ± SD) after additive treatment, ranked in order of greatest negative variance to the control fermentation. Threshold of significance p < 0.05.
Table 6. Ratio of methane to total gas produced (mean ± SD) after additive treatment, ranked in order of greatest negative variance to the control fermentation. Threshold of significance p < 0.05.
AdditiveMethane: Total Gas Variance to Control p-Value
Calcium nitrate0.00 ± 0.0−10.30.0790
Sodium nitrate7.91 ± 1.2−2.3 0.5408
Agolin11.2 ± 0.20.8 0.6120
Tartaric acid11.33 ± 0.70.90.5800
SilvaFeed11.46 ± 1.31.10.5809
Choline chloride17.18 ± 0.46.80.0391
Table 7. Nutritional composition of Rhodes grass substrate (± standard deviation) used in fermentations.
Table 7. Nutritional composition of Rhodes grass substrate (± standard deviation) used in fermentations.
VariableRhodes Grass
Dry matter (g/kg)951 ± 0.002
Organic matter (g/kg DM)931 ± 0.0008
Crude protein (g/kg DM)53 ± 0.17
Neutral detergent fibre (% as fed)69.9 ± 0.035
Table 8. Concentrations of methane-reducing compounds used in Fourier transform infrared-attenuated total reflectance spectral analysis.
Table 8. Concentrations of methane-reducing compounds used in Fourier transform infrared-attenuated total reflectance spectral analysis.
AdditiveConcentration
Nitrates100 g/L
Agolin10 mL/L
SilvaFeed10 mL/L
Choline chloride100 g/L
Tartaric acid100 g/L
Table 9. Concentrations of compounds used in the in vitro fermentations.
Table 9. Concentrations of compounds used in the in vitro fermentations.
AdditiveConcentration 1
Sodium nitrate2 g NO3/L
Calcium nitrate4 g NO3/L
Agolin30 mg/L
SilvaFeed1.7 mL/L
Choline chloride27.92 g/L
Tartaric acid15 mg/L
1 In non-protein nitrogen, phosphate and sulfate supplement (uPRO) mixed at 1.7 mL/L.
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Batley, R.J.; Romanzini, E.P.; Johnson, J.B.; de Souza, W.L.; Naiker, M.; Trotter, M.G.; Quigley, S.P.; de Souza Congio, G.F.; Costa, D.F.A. Rapid Screening of Methane-Reducing Compounds for Deployment via Water with a Commercial Livestock Supplement Using In Vitro and FTIR-ATR Analyses. Methane 2024, 3, 437-455. https://doi.org/10.3390/methane3030025

AMA Style

Batley RJ, Romanzini EP, Johnson JB, de Souza WL, Naiker M, Trotter MG, Quigley SP, de Souza Congio GF, Costa DFA. Rapid Screening of Methane-Reducing Compounds for Deployment via Water with a Commercial Livestock Supplement Using In Vitro and FTIR-ATR Analyses. Methane. 2024; 3(3):437-455. https://doi.org/10.3390/methane3030025

Chicago/Turabian Style

Batley, Ryan J., Elieder Prates Romanzini, Joel B. Johnson, William Luiz de Souza, Mani Naiker, Mark G. Trotter, Simon P. Quigley, Guilhermo Francklin de Souza Congio, and Diogo Fleury Azevedo Costa. 2024. "Rapid Screening of Methane-Reducing Compounds for Deployment via Water with a Commercial Livestock Supplement Using In Vitro and FTIR-ATR Analyses" Methane 3, no. 3: 437-455. https://doi.org/10.3390/methane3030025

APA Style

Batley, R. J., Romanzini, E. P., Johnson, J. B., de Souza, W. L., Naiker, M., Trotter, M. G., Quigley, S. P., de Souza Congio, G. F., & Costa, D. F. A. (2024). Rapid Screening of Methane-Reducing Compounds for Deployment via Water with a Commercial Livestock Supplement Using In Vitro and FTIR-ATR Analyses. Methane, 3(3), 437-455. https://doi.org/10.3390/methane3030025

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