Changing the Proportions of Grass and Grain in Feed Substrate Impacts the Efficacy of Asparagopsis taxiformis to Inhibit Methane Production in Vitro


Benefits of the red seaweed Asparagopsis taxiformis as an ingredient to manage methane (CH4) emissions from the red meat and dairy industries continue to evolve. Asparagopsis has been demonstrated to eliminate enteric CH4 emissions in vitro and reduce it greater than 80% in animals. Variability in animal studies is suspected to be associated with variable inclusion and proportions of grass and grain in the diet. This in vitro study aimed to elucidate effects of gradient grass to grain proportions in the fermentation using five steps from 100% Rhodes grass (RG) to 100% barley grain (BG). Gradient inclusion of Asparagopsis was in six steps of Control with no inclusion (C), Low (L), Low-Medium (LM), Medium (M), Medium-High (MH), and High (H) levels tested in three fermentation durations (24 h, 48 h, 72 h). There was significant effect of RG/BG and inclusion of Asparagopsis such that CH4 production decreased with increasing Asparagopsis independent of RG/BG; however, there was enhanced reduction at greater proportions of BG. Thus, the level of Asparagopsis required to completely inhibit CH4 production in vitro was decreased with decreasing RG/BG. Increasing the duration of fermentation had greatest effect on CH4 at C, L, and LM levels of Asparagopsis independent of RG/BG, although magnitude of CH4 production was greater for higher proportions of BG for the C and L levels. Digestibility of in vitro substrate increased with fermentation duration and increasing BG; however, there was no change associated with inclusion levels of Asparagopsis. Increases in total volatile fatty acids (tVFA) were observed with increased fermentation duration and concomitant with increasing substrate digestion. Increasing proportions of BG induced increase in tVFA. In contrast, and independent of changes in substrate, increasing inclusion of Asparagopsis had little effect on tVFA. The acetic and propionic acid ratio (AA:PA) decreased with decreasing RG/BG and increasing Asparagopsis. This pattern was most pronounced with 100% BG and MH-H Asparagopsis levels. Compared to control, there was decrease in the AA:PA ratio with addition of even L levels of Asparagopsis and with L compared to LM inclusion levels. Increasing levels of BG and Asparagopsis resulted in significant decreases in AA:PA ratios and CH4 production. This study has confirmed that gradient levels (ratio) of grass and grain in a feed mix impact the antimethanogenic efficacy of Asparagopsis during in vitro fermentation with rumen fluid.

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Kinley, R. , Tan, S. , Turnbull, J. , Askew, S. and Roque, B. (2021) Changing the Proportions of Grass and Grain in Feed Substrate Impacts the Efficacy of Asparagopsis taxiformis to Inhibit Methane Production in Vitro. American Journal of Plant Sciences, 12, 1835-1858. doi: 10.4236/ajps.2021.1212128.

1. Introduction

The relevance of antimethanogenic innovations to livestock feeds and feeding systems is evolving under petition from the red meat and dairy industries for methods to manage greenhouse gas (GHG) emissions in effort to meet emissions targets [1]. In Australia, the red meat industry has invested in an ambitious target of reaching carbon neutrality by 2030 (CN30), a program led by Meat and Livestock Australia [2]. The dairy and sheep industries have also shown awareness and willingness to adopt innovative technologies in production systems intended to provide significant GHG emissions reductions.

Diverse innovations with variable impact on enteric methane (CH4) emissions with variable levels of applicability are being validated, most with specific targets in an industry with a wide range of feeding systems [1] [3] [4]. The challenge for achieving emissions abatement and subsequent speed to impact is intrinsically linked to the feeding system. The challenge increases with decreased influence in the daily behavior of the livestock. For example, the beef feedlot industry is fundamentally well suited to fit-for-purpose total mixed rations (TMR) containing ingredients with a myriad of purposes including CH4 management. Similarly, but in a reduced capacity, grazing livestock receiving a feed supplement is well suited to receive a formulated daily provision albeit in a pulse format with intake frequency being dependent on the system of delivery. It is common for grazing dairy cattle to receive a supplemental feed at or near milking once or twice per day [5]. Livestock access to supplemental feeds, and voluntary intake of them, varies widely in grazing systems and availability is dependent on the level of nutritional intervention inherent in the system. Extensive systems typical of northern Australia do not typically receive supplemental feeds although they may have access to lick blocks or pots intermittently. Nutritional intervention in ruminant livestock feeding systems commonly targets increasing intake of efficiently digested and energy dense feed components. In grazing systems, this is commonly as supplemental and/or an improved grassland management. More intensive systems commonly involve provision of higher grain to grass ratio [6]. Nutritional intervention provides a conduit for CH4 management indirectly through improved feed use efficiency and directly through antimethanogenic feed ingredients.

Reference [7] demonstrated in vitro that the tropical legume Desmanthus spp. had antimethanogenic potential of up 36% compared to commonly grazed tropical Rhodes grass. That study induced further legume research in vivo where provision of increasing Desmanthus in a basal diet of low-quality grass demonstrated a linear increase DMI and reduction of CH4 [8]. Considering the large numbers of cattle on grasslands in northern Australia a moderate reduction in CH4 emissions from this sector would make a significant reduction in Australia’s total agricultural GHG inventory [8]. Approximately 90% of Australia’s beef cattle production occurs on grasslands, however over 40% of Australia’s beef is produced on feedlots [9]. The feedlot sector can be rapidly impacted toward reductions in enteric CH4 emissions using antimethanogenic feed ingredients including the red seaweed Asparagopsis [10] [11] and 3-nitrooxypropanol (3-NOP) [12]. Likewise, these products are effective in mitigation of enteric CH4 for the dairy industry [13] [14]. The paradox in applicability of antimethanogenic feeds is variable efficacy in inhibition of enteric CH4 as is demonstrated with variable diets and delivery systems.

Inherent aspects of a feeding system are intrinsic to the magnitude of response to antimethanogenic feeds. This highlights the need for knowledge in how to manage the intake of and inclusion levels of these products relative to their antimethanogenic efficacy in variable feeding systems. Inconsistency in the frequency of intake of a supplement containing the antimethanogenic material is expected to be relevant in management of CH4 reduction potential. The basal diet itself is expected to be relevant because there is evidence that high concentrate diets produce a more pronounced response to Asparagopsis [11]. The latter aspect can be investigated in vitro therefore the present study focuses on the efficacy of Asparagopsis to inhibit CH4 production when the basal diet changes with respect to inclusion level and the grass and grain proportions.

Asparagopsis is a potent antimethanogenic feed ingredient for ruminant livestock. The effectiveness has been shown in vitro [15] [16] [17] [18] and in vivo with variable efficacy [10] [11] [13] [19] [20]. The mode of action is reported to derive from the synthesis of halogenated CH4 analogues such as bromoform and bromochloroacetic acid [21] [22] that directly inhibit methyl-coenzyme M reductase (MCR) [23] [24] [25] at the final step in the CH4 formation pathway. Inclusion of Asparagopsis in vitro and in vivo has resulted in some of the largest CH4 reductions from a single feed ingredient added to ruminant diets, however response varies between studies. Variability in response to Asparagopsis may be due to a collective of factors including bioactive content of the Asparagopsis product, animal breed, intake of the product relative to basal diet DMI, and basal diet composition.

This study investigates the in vitro impact of changing the grass to grain ratio which is a prominent contrast in nutritional intervention between extensive and intensive ruminant livestock production. Published research suggests that increasing the dietary grain to grass ratio increases the antimethanogenic efficacy of Asparagopsis [10] [11] which has relative implications for utility of the seaweed for TMR feeding systems and supplemented or non-supplemented grazing systems. The hypothesis is that there is no difference in the minimum effective inclusion level (MEIL) based on antimethanogenic efficacy, or impact on rumen fermentation with inclusion of Asparagopsis in a scenario of increasing grain to grass ratio. Specific objectives include: 1) quantification of in vitro total CH4 production (mL/g DMI) as affected by Asparagopsis inclusion while increasing the grain to grass ratio; 2) characterization of the of in vitro MEIL of a single Asparagopsis product required to reduce CH4 production below the limit of detection as affected by increasing the grain to grass ratio; and 3) characterization of the effect of objectives 1) and 2) on rumen fermentation based on in vitro digestibility of substrate dry matter (IVDDM) and in vitro volatile fatty acid (VFA) profiles.

2. Materials and Methods

2.1. Preparation of Feed Substrate and Macroalgae

The basal feed substrates used were Rhodes grass (RG; Chloris gayana) locally grown under irrigation and barley grain (BG; Hordeum vulgare) grown in southern Queensland. Both were air-dried and ground to 1 mm, proximate of basal substrates analysis is presented in Table 1. Dry matter (DM) was determined by dehydration of substrate at 105˚C until constant weight and organic matter (OM) was determined as loss on combustion at 550˚C for 8 hours [26]. Neutral and acid detergent fibre were determined using an Ankom (Macedon, NY, USA) model 200 fibre analyser. Crude protein content was determined using a LECO (St. Joseph, MI, USA) model CHN628 series nitrogen analyser.

The Asparagopsis taxiformis (hereafter Asparagopsis) used in this study was harvested from Humpy Island, Keppel Bay, QLD (23˚3'01''S, 150˚54'01''E) by the Centre for Macroalgal Resources and Biotechnology of James Cook University, Townsville, Queensland AUS. Asparagopsis in the gametophyte lifecycle stage was rinsed in clean seawater, placed in mesh bags, and spun for 6 min at 1000 rpm in a commercial washing machine (Fisher and Paykel, Macquarie Park, NSW, AUS) to remove excess water. The fresh biomass was frozen and stored at −20˚C then freeze dried (Forager Food Company, Red Hills, Tas, AUS) to maximise retention of volatile bromoform (CHBr3) [27] as the demonstrated bioactive ingredient [21]. The freeze dried Asparagopsis biomass was crushed to 2 - 3 mm (Hobart D340 mixer, Troy, OH, USA) to ensure a uniform product and

Table 1. Nutritional composition of the Rhodes grass and barley grain substrates and Asparagopsis biomass (g/kg DM unless stated otherwise).

stored at −15˚C. Major bioactives concentration (only CHBr3 is reported here) in the Asparagopsis was quantified by methanol extraction and subsequent analysis by gas chromatography-mass spectrometry (GCMS) using a modified version of the protocol described by [22].

2.2. Donor Animals and Preparation of Rumen Fluid Inoculum

Rumen fluid inoculum (RF) was collected from four fistulated Brahman (Bos indicus) steers of approximately 430 kg live weight (LW) and fitted with 10 cm Bar Diamond (Parma, OH, USA) rumen cannulas. The steers were maintained at the Commonwealth Scientific and Industrial Research Organization (CSIRO) Lansdown Research Station near Townsville, Qld, AUS (19˚39'27.000''S, 146˚50'04.60''E) according to current guidelines [28] and approved by CSIRO’s Qld animal ethics committee on Ethical Clearance Certificate 2018-37. The steers grazed on mixed grasses dominated by RG and were supplemented with irrigated RG ad libitum perpetually. Rumen fluid (RF) was extracted through the cannulas at 7:00 am by sampling from four quadrants of the rumen and hand-squeezing into pre-warmed 1-L stainless steel thermal flasks. The RF was pooled and immediately processed by filtration through a 0.5 mm sieve and combined with an artificial saliva buffer described by [29] buffer (GVB) at a ratio of 1:4 (RF: GVB). Maintenance of 39˚C and mixing of the RF buffer fermentation media (RFB) (Major Science SWB 20 L-3; Saratoga, CA, USA) was continuous to ensure homogeneity. Each fermentation bottle was prepared prior to RF collection with prescribed treatments of RG, BG, and Asparagopsis then purged with N2 and capped to maintain anaerobic conditions. Purging was continuous during inoculation and 125 mL of RFB was aspirated into the fermentation bottles using a Dose-It pump (Integra Biosciences, Hudson, NH, USA). The fermentation bottles were then sealed with Ankom RF1 gas production modules (Macedon, NY, USA) and placed in one of six Ratek OM11 incubators (Boronia, Victoria, Australia) maintained at 39˚C and oscillating at 85 RPM.

2.3. In Vitro Set up and Experimental Design

The study was a triplicated 5 × 6 × 3 factorial design of five grasses: grain ratios, six inclusion levels of Asparagopsis, and three fermentation durations. The design was developed to compare impact on the efficacy of Asparagopsis to reduce CH4 emissions as affected by gradient inclusion levels of Asparagopsis on gradient dietary forage and grain ration formulations using RG and BG, respectively. The factorial consisted of a total of 90 experimental treatment groups. Nutritional composition of RG, BG, and Asparagopsis is reported in Table 1. Five formulation levels of feed substrate [content total 1.0 g OM (≈1.2 g DM)] expressed as a ratio of RG to BG inclusion in the range of; 1) 100% RG [1:0]; 2) 75% RG - 25% BG [3:1]; 3) 50% RG - 50% BG [1:1]; 4) 25% RG - 75% BG [1:3]; and (v) 100% BG [0:1], respectively. Asparagopsis inclusion levels included six increasing Asparagopsis inclusion levels standardized as mg CHBr3 in 1.0 g OM (≈1.2 g DM) of the feed substrate content (Table 1) in the range of: 1) 0.00 [control]; 2) 0.05 [low, L]; 3) 0.08 [low-medium, LM]; 4) 0.11 [medium, M]; 5) 0.14 [medium-high, MH]; and 6) 0.16 [high, H], respectively. Over the course of five in vitro incubation periods all the individual treatment fermentations were incubated (within period) in triplicate to allow for sacrifice of one fermentation bottle at each of three fermentation durations of: 1) 24 h, 2) 48 h, and 3) 72 h, respectively. All treatment groups were monitored in triplicate over the course of the five randomized incubation periods where treatment combinations were different in each period and substrate and Asparagopsis treatment groups did not recur in the same incubator to eliminate any chance of incubator or RF batch effect. The data from all sampling periods was combined to provide time series curves representing the effect of RG/BG combinations and gradient Asparagopsis inclusion at three fermentation durations over 72.

2.4. Sampling and Analysis

2.4.1. Total Gas and Methane Production

Total gas production (TGP) and CH4 production were determined using Ankom RF gas production technology (Macedon, NY, USA) with protocols and parameters as described by [15]. Briefly, the Ankom RF modules were set to maximum pressure of 3 psi which when exceeded would vent for 250 milliseconds. Live interval (LI) was set at 60 seconds monitoring of gas production each measurement was corrected for ambient pressure change via ambient Ankom RF monitors. The recording interval (RI) was set to 20 minutes thus LI cumulative pressure change was recorded at each RI as 20 min contributions to the cumulative pressure change over the duration of the fermentation (24, 48, or 72 h). Total cumulative pressure change was converted to TGP using the natural gas law and corrected for absolute volume of individual fermentation bottles.

The TGP was applied in the determination of total CH4 (mL) expressed as mL/g substrate digested. In vitro CH4 production was determined by analysis of headspace gas relative to TGP while assuming constant homogeneity of bottle headspace. Time series CH4 production curves were prepared by collection of samples at the three designated timepoint samplings (24, 48, and 72-h). At termination of each incubation period headspace gas samples from individual fermentation bottles were collected through the Ankom RF module vent tube into 10-mL Labco Exetainer vacuum vials (Lampeter, Great Britain). Gas samples were analysed by gas chromatography (GC) on a Shimadzu GC-2014 (Kyoto, Japan) equipped with a Restek (Bellefonte, PA, USA) ShinCarbon ST 100/120 column (2 m × 1 mm × micropacked) with a flame ionisation detector (FID). Column temperature was set to 150˚C, injector at 240˚C, and FID at 380˚C. Ultra high purity N2 was the carrier gas at 25 mL/min and total injection volume was 250 μL.

2.4.2. In Vitro Apparent Digestibility of Substrate DM and OM

In vitro digestibility of substrate DM (IVDDM) and OM (IVDOM) was quantified to coincide with CH4 determinations during 24, 48, and 72-hour fermentation periods as previously described [15]. Immediately following collection of headspace gas samples, the fermentation bottles were chilled to terminate fermentation activity then in vitro fluid (IVF) was collected and vacuum filtered through a weighed Duran No. 1 porosity glass fritted crucible containing a 0.5 cm layer of sand filtration aid. The crucible and fermentation residue were oven-dried to constant weight at 105˚C in determination of the remaining substrate DM and subsequent IVDDM determination. The OM in the fermentation residue DM subsequent IVDOM determination was determined as loss on ignition in a muffle furnace at 550˚C for 8 h (Carbolite AAF 11/18; Derbyshire, Great Britain).

2.4.3. Volatile Fatty Acid Production

Volatile fatty production (VFA) for each of the 28, 48, and 72-hour fermentation periods was measured in the IVF as previously described [15]. The sample preparation of IVF for VFA analysis was at a ratio of 4:1 of IVF to 20% metaphosphoric acid spiked to 11 mM with 4-methylvaleric acid (Sigma-Aldrich; Castle Hill, NSW, Australia) as internal standard to achieve a sample concentration of 2.2 mM internal standard. Samples were prepped in 1.5 mL microcentrifuge vials and stored at −20˚C. Sample vials were thawed at 4˚C and centrifuged for 20 min at 13,500 g and 4˚C (Labnet Prism R; Edison, NJ, USA). A 0.5 mL subsamples of clear supernatant was extracted using glass Pasteur pipettes and analysed by GC using a Shimadzu GC-2010 (Kyoto, JPN) equipped with a Restek Stabilwax (30 m × 0.25 mm × 0.25 mm) fused silica column and FID. Initial column temperature was 90˚C and ramped up at 3˚C/min until 155˚C temperature was achieved and was held for 8.3 min. Injector temperature was held at 220˚C and FID at 250˚C. Ultra high purity N2 was used as the carrier gas at 1.5 mL/min and the injection was 1.0 mL.

2.4.4. Statistical Analyses

The effect of gradient RG/BG in the feed substrate (1:0, 3:1, 1:1, 1:3, 0:1) and gradient Asparagopsis inclusion (0, L, LM, M, MH, H) at three fermentation durations (24 h, 48 h, 72 h) was analysed for CH4 and VFA production, IVDDM, and IVDOM in a 5 × 6 × 3 factorial experiment as a univariate repeated-measures ANOVA using the General Linear Model procedure of SPSS Statistics 27 (IBM Corp, Armonk, NY). Differences among means were tested by One Way ANOVA with LSD Post Hoc Multiple Comparisons procedure of SPSS for significant differences between the substrate treatments, Asparagopsis treatments. Differences in treatments were tested by pairwise comparisons (LSD test). Effects were declared significant at P < 0.05 and P = 0.05 - 0.10 were considered as a trend.

3. Results

3.1. Methane Production

Inhibition of CH4 production during simulated rumen fermentations in vitro as affected by gradient inclusion levels of Asparagopsis and RG/BG in the substrate is displayed as reduction response over 24 (a), 48 (b), and 72-hour (c) incubation periods (Figure 1). The CH4 production decreased with increasing inclusion level of Asparagopsis independent of RG/BG ratio (P < 0.001) and the efficacy of reduction was further enhanced by greater proportions of BG. Thus, the level of Asparagopsis required to completely inhibit CH4 production was decreased concomitant with decreasing RG/BG ratio as presented in Figure 1. Therefore, it was demonstrated that increasing the dietary grain content relative to grass increases the antimethanogenic response to Asparagopsis during in vitro rumen

Figure 1. Effect of inclusion levels of Asparagopsis (C, L, LM, M, MH, H) on methane production (mL/g DMD) at different Rhodes grass to barley grain ratios (1:0, 3:1, 1:1, 1:3, 0:1) after 24 h (a), 48 h (b) and 72 h (c) in vitro fermentation. Data points are treatment means and error bars represent standard errors.

fermentation. Compared to the Control, the L inclusion level of Asparagopsis in 1:0 RG/BG (100% RG) resulted in significant CH4 reduction of 70% (P < 0.001) in the first 24 h of fermentation. Antimethanogenic efficacy in 100% RG was reduced over time but remained significant with CH4 inhibition of 43% (P < 0.001) and 41% (P < 0.001) after 48 h and 72 h of fermentation, respectively. Increasing the level of Asparagopsis increased the efficacy and consistency of CH4 inhibition and the LM inclusion level of Asparagopsis in 100% RG induced 99% CH4 reduction for up 48 h and reducing in efficacy to 84% reduction approaching 72 hours. The M and MH, levels of Asparagopsis inclusion in 100% RG eliminated CH4 production for up to 48 h then marginally reduced in efficacy to 93% and 96% reduction approaching 72 hours, respectively. In contrast when the substrate was 0:1 RG/BG (100% BG), the response to Asparagopsis at the L inclusion level was marginal at 0%, 20%, and 12% for the 24 h, 48 h, and 72 h fermentation durations respectively. However, with each gradient step in Asparagopsis level the response magnified such that the LM level induced 99%, 100%, and 45% reductions over 24 h, 48 h, and 72 h, respectively. The M level represented the inclusion level where elimination of CH4 was virtually sustained over time. The H level of inclusion eliminated CH4 production in all the fermentations independent of RG/BG.

The contrasting antimethanogenic response to increasing Asparagopsis inclusion when using fermentation substrates of 100% RG and 100% BG was further elucidated by examination of the gradient effect with reduction in the proportion of RG and increase in BG. The RG/BG ratio was a influencing factor in efficacy of reduction of CH4 with increasing BG by inducing more sensitivity to Asparagopsis (Figure 1). The response to RG/BG at 3:1 (25% BG) of diet substrate was similar 1:0 (100% RG) and sustained elimination of CH4 was only achieved at the H level of Asparagopsis inclusion. The 1:1 RG/BG ratio (50% RG, 50% BG) demonstrated similar CH4 reductions as the 1:3 (75% BG) with 21%, 24%, and 15% for the L inclusion level and 97%, 98%, and 58% for the LM level over 24 h, 48 h, and 72 h, respectively. With RG/BG at 1:1 and 1:3 a sustained elimination of CH4 was achieved at the M Asparagopsis inclusion level signaling a shift to improved antimethanogenic response.

3.2. Dry Matter Digestibility

In vitro digestion of dry matter (IVDDM) is shown in Table 2 for fermentations over 24 h, 48 h, and 72 h incubation periods. Significant increases in IVDDM were demonstrated at all fermentation durations with decreasing RG and increasing of BG proportions in the diet substrate over time (P < 0.001). This is a demonstration of the lower inherent RG-IVDDM of 63% 72%, and 74% compared to BG-IVDDM of 83%, 90%, and 91% at 24 h, 48 h, and 72 h, respectively. This is typical of rumen in vitro batch culture fermentations such as Ankom where the IVDDM and IVDOM rates of digestion peaks before 36 h and slows down such that it is approaching completion by 48 h independent of the diet

Table 2. Effect of inclusion levels of Asparagopsis (C, L, LM, M, MH, H) on dry matter digestibility at different Rhodes grass (RG) to barley grain (BG) ratios (1:0, 3:1, 1:1, 1:3, 0:1) and incubation periods (24 h, 48 h, 72 h). Means with different letters within a column differ significantly at P < 0.05.

[15]. However, independent of the IVDDM changes induced by gradient RG/BG, there was no differences in IVDDM induced by increasing inclusion of Asparagopsis after 24 h, 48 h, or 72 h with P-values of P = 0.626, P = 0.084, and P = 0.145, respectively.

3.3. Volatile Fatty Acid Production

Total volatile fatty acids (tVFA) including the major subspecies acetic acid (AA), propionic acid (PA), and butyric acid (BA) are captured in the in vitro fluid in the fermentation bottles during in vitro batch culture fermentation and accumulate for the specified duration of the experimentation. For simplicity, Table3 presents the impact of RG/BG and Asparagopsis on VFA production at the maximum accumulation point in this study which is response over 72 h of fermentation. However, VFA production during fermentations of 24 h and 48 h are provided as supplement materials (TableS1 and TableS2, respectively).

In addition to the expected increases in tVFA (mM) that typically occur as result of increased time and subsequent increasing IVDDM, for all levels of Asparagopsis including control, the gradient change in dietary composition had significant effect. Increasing proportions of BG in the diet substrate induced increase in tVFA during 72 h of in vitro fermentation (P < 0.001). The 100% RG diet substrate produced the least tVFA, then a plateau was observed for mixed rations, and the highest tVFA production occurred for the 100% BG substrate. In contrast, and independent of changes in substrate, increasing levels of Asparagopsis had little effect on production of tVFA. Supplementary TableS1, TableS2 and Table3 show that there was no difference in tVFA within the fermentation durations as confirmed with the minimum P-values for all the combined RG/BG ratios of P = 0.352, P = 0.247, and P = 0.246 for the 24 h, 48 h, and 72 h fermentations, respectively.

Table 3 shows the major subspecies VFA’s as relative proportions (%) of tVFA produced during 72 h of fermentation. Acetic acid is consistently produced in the highest proportion independent of the diet compositions (P < 0.001) and levels of Asparagopsis inclusion (P < 0.001). However, although AA remains proportionately the highest of tVFA, increasing levels of BG and Asparagopsis both decrease AA independently (P < 0.001) such that at the highest of

Table 3. Effect of inclusion levels of Asparagopsis (C, L, LM, M, MH, H) on volatile fatty acid profile at different Rhodes grass (RG) to barley grain (BG) ratios (1:0, 3:1, 1:1, 1:3, 0:1) after 72 h in vitro fermentation. Means with different letters within a column differ significantly at P < 0.05.

levels of both (0:1 and MH-H) the PA were marginally higher than AA. The effect of Asparagopsis on AA was more pronounced with the higher inherent AA when RG/BG was high (1:0 and 3:1) demonstrated by larger drops in AA proportion. This effect is evident but less pronounced with increasing BG and the pattern is more stable as the AA proportion in the Control decreases. As the proportion of BG increases in the substrate, Asparagopsis seems to have a larger effect on reduction of AA between L and LM inclusion rates. In contrast to AA, the PA proportion increased in a stepwise manner following both decreasing RG/BG gradient levels (P < 0.001) and increasing Asparagopsis inclusion (P < 0.001). The largest increases in PA were demonstrated to occur with the combination of 100% BG (0:1) and highest level of Asparagopsis (H). The production of BA was similar compared to PA and increased with decreasing RG/BG (P < 0.001) and increased with increasing Asparagopsis inclusion (P < 0.001).

Figure 2 illustrates that the ratio of AA:PA decreases with decreasing RG/BG (P < 0.001) and increasing Asparagopsis inclusion (P < 0.001). The pattern was most pronounced with 100% BG and MH-H Asparagopsis inclusion levels. Compared to control there was significant drops in the AA:PA ratio with addition of even L levels of Asparagopsis (P < 0.001 - 0.05) and with L compared to LM inclusion levels (P < 0.001 - 0.05). Comparatively, the between level decrease in AA:PA was diminished with inclusion levels M-H but the decreasing AA:PA pattern remained.

4. Discussion

4.1. Methane Production

This study demonstrated a large degree of contrast in CH4 production using in vitro fermentation compared to in vivo animal studies. A notable observation of the current study is the increase in CH4 emissions when BG is added in gradient levels to the fermentation substrate. This result is contrary to results found when measuring CH4 emissions from animals [30] [31], however does align with other in vitro studies. For example, three independent in vitro studies found elevated CH4 production in high starch feeds when compared to high fiber feeds [32] [33] [34]. All three studies concur that this is likely due to the increase in rapidly fermentable carbohydrates compared to the more structural carbohydrates

Figure 2. Effect of inclusion levels of Asparagopsis (C, L, LM, M, MH, H) on acetic acid: propionic acid ratio (AA:PA) at different Rhodes grass (RG) to barley grain (BG) ratios (1:0, 3:1, 1:1, 1:3, 0:1) after 72 h in vitro fermentation. Data points are treatment means and error bars represent standard errors. Effects of inclusion levels were significant at P < 0.001 independent of RG/BG.

found in high fiber feeds which typically take longer for microbial populations to ferment. Another explanation for the increase in CH4 with highly digestible feeds is due to the symbiotic nature of rumen ciliates, which are prolific fermenters of readily fermentable carbohydrates, and methanogens through hydrogen transfer [35] and the fact that in vitro cultures cannot simulate variable rumen retention rates for feeds of variable digestibility. Reference [34] also reported that with rapid fermentation of starchy feeds there is typically a concomitant drop in rumen pH levels which are protected against pH drop in a closed system during buffered in vitro batch culture. A significant drop in ruminal pH can inhibit fibrolytic bacteria in vivo thus reducing digestion and subsequently carbon dioxide (CO2) and hydrogen (H2) available for methanogenesis. However, this scenario is unlikely in the strongly buffered in vitro batch cultures lending to higher CH4 production per kg DM from higher levels of BG in vitro compared to in vivo.

Even with increases in CH4 emissions with high grain diets in vitro the MEIL of Asparagopsis required for sustained CH4 mitigation is consistent with previous work done in vivo. Reference [11] tested two inclusion levels of Asparagopsis compared to a control group using growing Angus-Hereford steers. In three diet formulation phases of starter-transition-finishing diet of 63 d, 21 d, and 63 d, respectively. In their study with the diet phases of high, mid and low forage inclusion the researchers found that as forage (alfalfa and wheat hay + distillers grain) content decreased, and grain (rolled corn) increased, the efficacy of antimethanogenesis increased concurrently. This is consistent with the current study and Asparagopsis was demonstrated to have increased antimethanogenic efficacy with higher BG as RG/BG was decreased. This was also demonstrated with other CH4 reducing feed additives, and 3-NOP was reported as having a linear relationship between MCR and dietary NDF concentrations in the rumen, thus an increase in efficacy of Asparagopsis to inhibit MCR enzymes when NDF concentrations are low may be observed [12].

The implications of variable antimethanogenic response to Asparagopsis and how it would manifest in practice is of interest to the livestock industry. The feeding systems, and hence diet formulations, can vary widely and include but limited to, livestock type, breed, format of feeding, and feed formulation, which are features impacting the CH4 emissions profile for those systems. Feedlot beef and sheep systems, as the name suggests, receive formulated rations designed to drive high productivity through increasing feed conversion efficiency and the diet is typically high in energy dense component, namely grain. This provides for an opportune system predisposed to high levels of relative CH4 mitigation with a low MEIL of Asparagopsis. This in vitro study confirms the indication derived from collective, but less diet directed, in vivo studies [10] [11], thus more grain reflects less Asparagopsis required for high level CH4 mitigation. Feedlot systems are inherently the “low hanging fruit” for ease of delivery of Asparagopsis and efficacy of antimethanogenic response. However, grazing systems make up much larger contingents to cattle and sheep in Australia and for GHG emissions inventory management it is important to mitigate CH4 emissions in grazing systems. Evolution of Asparagopsis delivery into supplemented feeding systems would be the logical subsequent step. Supplemented systems are largely grass diets of variable digestibility and knowledge derived from this study will assist in planning the delivery system and loading of Asparagopsis in supplements. Dairy systems generally receive good quality forage and commonly offer a supplement at, or near, time of milking once or twice per day. Knowledge of a slightly higher MEIL that may be partially impacted by forage quality and grain supplementation will drive further investigation into supplement formulation and palatability management. Currently there is no technology for consistent delivery of feed supplements in large scale beef and sheep grazing systems in Australia. These extensive systems have low level of animal and producer interaction thus delivery and confirmation of intake is problematic. Further confounding efficacy of antimethanogenesis is seasonal changes in forage availability and quality. To have important and sustained GHG emissions reduction realized in extensive systems requires significant evolution in knowledge and technology which would be profitable to the sector. This study has confirmed that all feeding systems can realize large CH4 emissions reduction but the strategies to achieving it varies just as widely as the livestock industry.

4.2. Digestibility

Significant increases in IVDDM were observed with increasing levels of BG compared to RG in the fermentation substrate composition. As described in Table 1 this is the result of proportionally higher levels of readily digestible carbohydrates found in BG compared to RG which is a more fibrous substrate [34]. Beneficially, from the perspective of using Asparagopsis in ruminant livestock feed, the seaweed did not induce significant changes in digestibility at any of the Asparagopsis inclusion rates. This is consistent with other in vitro studies that reported little effect on IVDOM when Asparagopsis was included at levels up to 10% OM [16] [17]. Reference [15] further examined the effect of in vitro inclusion levels of Asparagopsis with a gradient series of inclusion from 0% to 10% OM. The study demonstrated there was no impact on IVDOM at inclusions level up to 5%, however approaching levels of 10% there was reduction in IVDOM. The highest level of inclusion (H) in the present study was equivalent to 1.2% OM, comparatively in vivo studies feeding Asparagopsis have demonstrated antimethanogenic efficacy at MEIL as low as 0.2% OM [10] and 0.5% [11]. Our findings are consistent with other studies and contribute to the growing body of evidence demonstrating that digestibility is not impacted by the inclusion of Asparagopsis and that it is independent of the grass and grain composition. Clearly, there is an extensive margin of consistency for digestibility when feeding Asparagopsis at the MEIL.

4.3. Volatile Fatty Acid Production

Microbial fermentation of feed in the rumen accounts for approximately 50% - 70% of the animal’s available dietary energy and is largely in the form of VFAs [36]. The VFA profile is dominated by AA, PA, and BA which are the most abundant VFA’s produced in the rumen [37]. This study is consistent with previous in vitro research that has reported little to no changes in total VFA production induced by Asparagopsis inclusion [15] [18]. The same observation was also reported in Asparagopsis in vivo studies [10] [19]. Changes in tVFA production were induced by substrate RG/BG changes, with the lowest tVFA observed in high RG diets with gradual increases as dietary BG increased. As with CH4 production in vitro the increased tVFA may be associated to inherently greater digestibility of BG diets compared to RG diets as well as strongly buffered pH within the fermentation bottles. Due to increased production of VFAs in the rumen it is typical for a drop in pH due to the acidic nature of VFAs. This causes negative feedback for microbes because VFA absorption rate is slower than the rate of production in the rumen [38] [39]. The relative abundance of VFA species in the profile is largely reliant on the types of feed substrates being fermented. Dominant with grass based diets, AA is mainly liberated by bacterial and fungal populations responsible for the degradation of fibrous and structural carbohydrates. In contrast PA and BA are typically produced by bacterial and ciliate protozoal populations responsible for the degradation of starchy, more soluble carbohydrates [40] [41].

Concomitant with VFA during feed substrate degradation CO2 and H2 are also produced and the animal is not able to utilize these directly and may be detrimental when accumulating in the rumen [38] [39]. Methanogens can take advantage of these excess end products in their own metabolism which results in feed energy waste as CH4 which is an inefficiency of rumen digestion. Increased H2 pressure in the rumen is of interest when CH4 production is dramatically reduced as is possible with Asparagopsis inclusion in the diet. Pathways that utilize H2, and offer H2 sinks, including VFA production but more specifically the longer chain species of PA (C3H6O2) and BA (C4H8O2) compared to AA (C2H4O2) [42]. This study demonstrated that increasing Asparagopsis inclusion has a positive impact on increasing the proportion of PA and BA in the tVFA profile indicating that these pathways are being upregulated to utilize excess H2 present because of CH4 inhibition. Furthermore, AA/PA ratios decreased with the increase of Asparagopsis inclusion rates which has been demonstrated both in vitro [15] [18] and in vivo [10] [19] and may suggest increased energy availability to the animal because PA is the main glucose precursor in ruminants [43].

5. Conclusion

This study has confirmed that gradient levels (ratio) of grass and grain in a feed mix impact the antimethanogenic efficacy of Asparagopsis during in vitro fermentation with rumen fluid. It was demonstrated that feed formulations with higher levels of grass respond to Asparagopsis less effectively than formulations with higher levels of grain. Therefore, the MEIL is higher for diets high in grass compared to diets high in grain. Subsequently, less Asparagopsis is required to reduce CH4 to below detection with in vitro substrates typical of feedlot diets compared to grazing systems. This study provides evidence and implications for the livestock industry in that feedlot style diets are particularly well suited to Asparagopsis for dramatic reduction in GHG emissions. However, the evidence suggests grazing systems will respond well to Asparagopsis but technology for stabilization and delivery aimed at consistent intake of the appropriate MEIL is deficient. Changes in IVDDM were due to fermentation duration and change in grass and grain components in the substrate, such that increasing the fermentation duration and BG component both resulted in greater IVDDM. Asparagopsis inclusion did not induce changes in IVDDM independent of RG/BG, and IVDDM was approaching completion after 48 h of fermentation. Likewise, tVFA production was increased by increasing BG and fermentation time, concomitant with increase in IVDDM. Notably, Asparagopsis did not induce changes in tVFA. The VFA species were impacted by RG/BG such that AA and PA proportions of tVFA were inversely related such that high RG produced the most AA, while high BG produced the most PA, and BA followed the same pattern as PA but at lower proportions of tVFA. Increasing Asparagopsis inclusion decreased the tVFA proportion of AA, and increased the proportion of PA, and BA which was concomitant with decrease in CH4 production. Therefore, AA:PA decreased with increase in BG and Asparagopsis, and with decrease in CH4 production.


Table S1. Effect of inclusion levels of Asparagopsis (C, L, LM, M, MH, H) on volatile fatty acid profile at different Rhodes grass (RG) to barley grain (BG) ratios (1:0, 3:1, 1:1, 1:3, 0:1) after 24 h in vitro fermentation. Means with different letters within a column differ significantly at P < 0.05.

Table S2. Effect of inclusion levels of Asparagopsis (C, L, LM, M, MH, H) on volatile fatty acid profile at different Rhodes grass (RG) to barley grain (BG) ratios (1:0, 3:1, 1:1, 1:3, 0:1) after 48 h in vitro fermentation. Means with different letters within a column differ significantly at P < 0.05.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.


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