A large proportion of methane (CH4) emitted into the atmosphere derives from agriculture, and specifically ruminant enteric fermentation which contributes approximately 28% of global anthropogenic CH4 emissions  .
Ruminants rely on a complex rumen microbial consortium of bacteria, protozoa, archaea, fungi, and bacteriophages  associated with fermentation of feed. Fermentation of fibrous and nitrogenous feedstuff results in the production of volatile fatty acids (VFA) and microbial protein, used by the animal for growth, metabolism, and productivity  . During fermentation, carbon dioxide (CO2) and hydrogen (H2) are utilized by methanogenic archaea in the reductive methanogenesis pathway, which reduces partial H2 pressure, but also results in CH4 being emitted into the atmosphere  . Low CH4 producing ruminants tend to be more productive and increasing productivity of animals could also lessen CH4 emissions  . As a result, many strategies are being evaluated to mitigate enteric methanogenesis, including vaccines, bacteriocins and probiotics, bacteriophage therapy, genetic selection, feeding management and feed additives, and plant secondary metabolites  .
Feed management and additives such as high-quality forages, grains, ionophores, fats, yeasts, enzymes, microbes, plant extracts and algae have the potential for CH4 abatement  . Algae products can improve ruminant health and productivity   , increase feed quality  , and inhibit methanogenesis  . However, reducing enteric methanogenisis is challenging, and to be adopted as a methodology it needs to be sustainable, practical, economically viable, and improve animal productivity  . Recently, the marine macroalgae Asparagopsis taxiformis ([Delile] Trevisan de Saint-Léon 1845; Asparagopsis) has demonstrated effective inhibition of CH4 production in vitro at a dose of 2% of substrate organic matter (OM). In addition, Asparagopsis maintains the apparent in vitro degradability of OM (IVD-OM), and increases propionate but with a concomitant decrease of acetate   . These rumen fermentation parameters are important indicators of fermentation efficiency  . Some other marine macroalgae that also reduce CH4, but to a lesser extent than Asparagopsis, have demonstrated better anaerobic fermentation in vitro compared to equal supplementation (17%) with cotton seed meal by increasing the concentration of total VFA (TVFA) when included with a low-quality grass substrate  . Therefore, it may be possible to improve rumen fermentation using combinations of macroalgae at practical dose concentrations when paired with Asparagopsis by increasing VFA production and improving the VFA profile.
It was hypothesised that macroalgae demonstrating CH4 abatement in vitro at high dose concentrations would maintain antimethanogenesis at variable potency at low dose concentrations, and that when combined with the highly potent Asparagopsis they would enhance rumen fermentation. The aim of Experiment 1 was to rank seven select macroalgae for their potential as antimethanogenic feed additives using dose concentrations practical for livestock feeding, and establish their effects on fermentation parameters. The aim of Experiment 2 was to evaluate combining the seven macroalgae with Asparagopsis to determine the effect of the combinations on methanogenesis and potential benefits to VFA production.
2. Materials and Methods
2.1. Selection and Preparation of Macroalgae and Rhodes Grass Substrates
The macroalgae species were selected to represent the major groups of marine macroalgae (red, brown, green) based on their demonstrated ability to decrease enteric CH4 andimprove fermentation  and are listed in Table 1. The biomass was sourced from either large scale culture at the Centre for Macroalgal Resources and Biotechnology (MACRO) James Cook University (JCU) in Townsville QLD (19.33˚S; 146.76˚E), Pacific Reef Fisheries in Ayr QLD (PRF; 19.58˚S, 147.40˚E), or intertidal flats near Townsville Qld at Nelly Bay (NB; 19˚16'S; 146˚85'E) with GBRMPA permit GO2/20234.1, or Rowes Bay (RB; 19˚23'S; 146˚79'E) with DPIF permit 103,256. The individual algae and sources were Caulerpa taxifolia ([M.Vahl] Agardh 1817; NB), Cladophora patentiramea ([Montagne] Kützing 1849; PRF), Cystoseria trinodis ([Forsskål] Agardh 1820; NB), Dictyota bartayresii (Lamouroux 1809; RB), Padina australis (Hauck 1887; RB), Sargassum flavicans ([Mertens] Agardh 1820; NB) and Ulva ohnoi (Hiraoka and Shimada 2004; MACRO).
Macroalgae were rinsed and centrifuged in a commercial washing machine at 1000 rpm for 5 min at ambient temperature to remove excess water. The biomass was freeze- dried (VirTis Benchtop 2K; Warminster PA, USA) at −55˚C and 120 µbar for minimum 48 h, then milled to pass a 1 mm sieve and stored at −20˚C. The basal substrate was Rhodes grass (Chloris gayana) with neutral detergent fibre (NDF) of 645 g∙kg−1 dry matter (DM) and acid detergent fibre (ADF) of 315 g∙kg−1 DM. The DM content of the various biomass was determined by drying at 105˚C to constant weight and the OM content was determined by the combustion for 8 h at 550˚C  . Crude protein and
Table 1. Compositional parameters of the macroalgae and Rhodes grass hay.
a. MJ∙kg−1 DM.b. Parameters presented as g∙kg−1 unless otherwise stated. DM, dry matter; OM, organic matter; CP, crude protein.
gross energy quantification were determined as previously described by  . Crude protein of the macroalgae was based on total nitrogen (wt %) content using a nitrogen factor of 5 for the macroalgae  and 6.25 for the Rhodes grass.
2.2. Research Animals and Preparation of Rumen Fluid Inoculum
Rumen fluid inoculum (RF) was collected from four fistulated Brahman steers (Bos indicus; LW 460 ± 20 kg) fitted with 10 cm Bar Diamond (Parma, OH, USA) rumen cannulas. The steers were maintained at the College of Public Health, Medical and Veterinary Sciences of JCU according to current guidelines  and approved by the CSIRO animal ethics committee (A5/2011). The steers were maintained on Rhodes grass hay ad libitum for 6 months before the collection of RF, which was extracted 2 h after morning feeding by sampling from four quadrants of the rumen and hand squeezing to completely fill pre-warmed 1 L stainless steel thermos flasks.
2.3. Inoculation of in Vitro Fermentations
The RF was pooled and immediately processed by filtration through a 0.5-mm sieve and combined with incubation buffer (GVB)  at a ratio of 1:4 (RF:GVB). Through- out the inoculation process the RF buffer fermentation media (RFB) was maintained at 39˚C and continuously mixed at 30% - 35% of maximum vortex speed (Major Science SWB 20 L−3; Saratoga, CA, USA) to ensure RFB homogeneity between individual fermentations. Anaerobic conditions were maintained with high purity N2 (HPN2; BOC, Wetherill Park NSW, AUS). A Dose-It pump (Integra Biosciences, Hudson NH, USA) was used to aspirate 125 mL of RFB into each incubation bottle containing the macroalgae and Rhodes grass substrates. The substrate and macroalgae were preloaded prior to RF collection into 250 mL Simax incubation bottles (Sázava, CZE). Each bottle was purged HPN2, capped, and warmed to 39˚C. The bottles were sealed with an Ankom RF1 gas production module (Macedon NY, USA), placed in mixing incubators (Ratek OM11; Boronia VIC, AUS), and maintained at 39˚C and oscillating at 85 RPM for 72 h.
2.4. Experimental Design
Experiment 1 was conducted to rank macroalgae according to their antimethanogenic potency and effects on rumen fermentation parameters. This was accomplished using in vitro fermentation batch culture (Ankom) to determine effects of macroalgae inclusion on total gas (TGP), CH4, and VFA production, and IVD-OM. A series of four incubation periods were completed and consisted of 1.0 g of Rhodes grass substrate as control and appropriate quantities of each macroalga to achieve a dose concentration of 5% of the substrate OM according to the biomass composition described in Table 1. Fermentations were also completed as controls (no macroalgae), a positive-control (P- control) of Asparagospis at 2%, and RFB blanks. The inclusion of the macroalgae at 5% was determined as an arbitrary feasible level of feeding for livestock and based on results previously described for inclusion approaching 20%  . The inclusion of Asparagopsis at 2% was set based on previously determined optimum dose for this seaweed in rumen fermentations   . The seven macroalgae and Asparagopsis were randomly assigned to the four incubation periods (n = 3), and controls and blanks were included in all periods (n = 4). Within each incubation period, there was duplication of each macroalga, and controls and blanks at each sampling time point (12, 24, 48, and 72 h).
Experiment 2 was conducted to determine if the seven macroalga when combined with Asparagopsis studied in Experiment 1 would benefit in vitro rumen fermentation. This was accomplished using the same fermentation techniques as Experiment 1. A series of five incubation periods were completed and consisted of 1.0 g of Rhodes grass substrate as control and appropriate quantities of each macroalga to achieve a dose concentration of 5% (OM basis) in combination with Asparagopsis at 2% according to the biomass composition described in Table 1. Fermentations were also completed as controls (no macroalgae), a P-control of Asparagospis at 2%, and RFB blanks. The seven combinations and Asparagospis were randomly assigned to the five periods (n = 3), and controls and blanks were included in all periods (n = 5). Within each incubation period, there was duplication of each macroalgae combination, and controls and blanks at each sampling time point (12, 24, 48, and 72 h).
2.5. Total Gas and Methane Production
The fermentation methods used in this study were similar to  but using the Ankom RF1 system and gas analysis as described by  . Pressure accumulation in the incubation bottles was measured continuously and recorded every 20 min. The maximum fermentation pressure inside the incubation bottle was set to 3 psi which, when exceeded, caused venting for 250 ms and the pressure change accounted in the cumulative pressure recording. Gas pressure was measured every 60 s and cumulative pressure was recorded at 20 min intervals. The TGP was expressed as mL of gas produced per gram of substrate OM (mL∙g−1 OM) by application of the natural gas law to the accumulation of the recorded gas pressure while accounting for individual bottle volume.
In vitro CH4 production was quantified in time series at multiple time points of 12, 24, 48 and 72 h during the fermentation. The mL of CH4 g−1 of substrate OM was estimated using concentrations in headspace at the time series points while assuming homogeneity of headspace gas. The headspace samples were collected into 10 mL Labco Exetainer vials (Lampeter, GBR) and quantified using gas chromatography (GC) according to parameters described by  . Concentrations of CH4 in headspace gas were converted to mL∙g−1 substrate OM based on TGP at the relative time series points and assuming headspace homogeneity at system venting  .
2.6. Apparent in Vitro Digestible Organic Matter and Volatile Fatty Acids
The IVD-OM and VFA production was determined as described by  and quantified to coincide with CH4 determinations at each time series point. Each fermentation was chilled to cease bacterial activity and the in vitro fluid (IVF) was then vacuum filtered through a Duran No. 1 porosity glass fritted crucible containing a 0.5 cm layer of sand filtration aid. The pH was measured on the filtrate. Crucibles and wet residue were oven-dried to constant weight at 105˚C for DM determination. Residue OM was determined as loss on ignition in a muffle furnace at 550˚C for 8 h  .
The TVFA accumulated in the IVF were quantified after termination of fermentation. Preparation of IVF prior to GC analysis consisted of addition 4 mL of IVF to 1 mL of 20% metaphosphoric acid containing 11 mM of 4-methylvaleric acid (Sigma-Al- drich; Castle Hill NSW, AUS) providing 2.2 mM internal standard. The samples were mixed and stored at −20˚C until a 1.5 mL subsample was centrifuged for 15 min at 13,500 rpm and 4˚C (Labnet Prism R; Edison NJ, USA). The supernatant was filtered through 0.2 µm PTFE syringe tip filters (Agilent; Santa Clara, CA, USA) and quantified by GC according to parameters described by  .
2.7. Statistical Analysis
Two-factor repeated-measures permutational analysis of variance (PERMANOVA) was used to test for significant differences in TGP, CH4 production, and IVD-OM over time. A one-factor PERMANOVA was used to test for significant differences in the production of VFA between treatments (fixed factor) using Primer 6 (version 6.1.13;  statistical software and PERMANOVA+ (version 1.0.3)  . For PERMANOVA, Bray-Curtis similarity matrices were produced using the untransformed raw data and dummy variables (0.0001) were used to account for zero values. The P-values were calculated from 999 (TGP) and 9,999 (CH4, IVD-OM, and VFAs) random permutations. Pair-wise a posteriori comparison was used to determine significant groupings, where applicable. For PERMANOVA, differences were considered significant if P < 0.05. The TGP data were also fitted with generalized additive models (GAM) using cubic regression spline smoothers to predict the relationship and examine differences between TGP over time. The generalized additive models were produced using the mgcv package within the R language (version 3.0.1)  . Goodness-of-fit of the individual smoothers was quantified using the hydroGOF package within the R language and was assessed from the proportion of variance in the data that was accounted for by the model (r2)  .
3.1. Experiment 1: Ranking
Based on predictions of the GAM, which had a high goodness-of-fit (r2 > 0.97), there was a significant difference (P < 0.001) in TGP in the fermentations over 72 h (Figure 1). In addition, the TGP reached its plateau approaching 48 h in all fermentations and the Asparagopsis induced a lower TGP rate compared with all other treatments (P < 0.001). There was no significant difference in cumulative TGP between the seven macroalgae with inclusion at 5% (OM basis) and the Rhodes grass control after 72 h of fermentation. The TGP for all macroalgae other than Asparagopsis ranged between 171 and 176 mL∙g−1 OM, representing an insignificant 2.4% - 4.8% reduction in TGP com-
Figure 1. The effect of inclusion of seven different macroalgae on gas production and substrate digestibility over 72 h of in vitro fermentation with rumen fluid. From top down: Total gas production (TGP); CH4 production; and apparent in vitro digestibility (IVD-OM). The Rhodes grass control substrate was equal in all fermentations, the Asparagopsis control was included at a concentration of 2% of substrate OM, and the other seven macroalgae included at 5%. No ±SE is presented for TGP because SE was smaller than the symbols.
pared to the Rhodes grass control (180 mL∙g−1 OM). However, the Asparagopsis at 2% had significantly less TGP (127 mL∙g−1 OM) representing a 26% - 29% reduction compared to all other macroalgae and controls.
The CH4 production as affected by inclusion of the seven macroalgae other than Asparagopsis was not altered significantly compared to the Rhodes grass control during the course of the 72 h incubations (Figure 1). The rate of CH4 production as defined by time series headspace sampling was variable in the same manner as for TGP. The only significant reduction in CH4 was induced by Asparagospis in the same way as previously demonstrated  and no detectable CH4 was produced. The CH4 production from the other fermentations ranged from a low of 14 mL∙g−1 OM to a high of 16 mL∙g−1 OM for U. ohnoi and the Rhodes grass control, respectively.
A typical pattern of IVD-OM increasing with time was demonstrated with or without macroalgae (Figure 1), however, the rate of digestion of the substrate varied between treatments in the first half of the fermentation period (P = 0.004). Asparagospis induced the earliest onset of substrate degradation in the same fashion as demonstrated by  for Asparagopsis at dose concentrations ≤ 5% of substrate OM. The C. patentiramea was the slowest to achieve maximum rate of IVD-OM. Approaching 48 h, IVD-OM in all the fermentations coalesced, and after 72 h had similar IVD-OM ranging from a low of 70% to a high of 72% for C. patentiramea and the Asparagopsis, respectively. In the absence of macroalgae the IVD-OM for the Rhodes grass control was 72%.
In Experiment 1 the series of in vitro fermentations of the seven macroalgae or Asparagopsis P-control did not induce significant differences between any of the treatments or Rhodes grass control for TVFA (P = 0.093) or propionate (P = 0.098). However, fermentations including the Asparagopsis were significantly lower for acetate (P = 0.002) and higher for butyrate (P = 0.004) compared to the other fermentations (Table 2). A significant increase in propionate and butyrate with 2% Asparagopsis inclusion has been previously reported  , however this feature was slightly dampened in the present study for propionate but confirmed the previously observed butyrate concentrations. Compared to the Rhodes grass control, after 72 h of fermentation the macroalgae induced marginal reductions in TVFA on a molar concentration basis from a low of 9% to a high of 23% for D. bartayresii and C. patentiramea, respectively, and Aspara- gopsis reduced TVFA by 22%. The production of acetate was marginally reduced by a low of 10% to a high of 24% by inclusion of D. bartayresii and C. patentiramea, respectively, and a significant 44% with Asparagopsis, respectively. Conversely, propionate was marginally decreased by a low of 5% to a high of 24% with inclusion of D. bartayresii and C. patentiramea, respectively, and increased by 9% with Asparagopsis. The production of butyrate was marginally decreased by a low of 11% to a high of 37% for D. bartayresii and C. patentiramea, respectively, and significantly increased by 76% with Asparagopsis.
There were not adequate differences in TGP, CH4, or VFA production to justify well defined ranking for the seven macroalgae evaluated. Clearly, none of the seven macro-
Table 2. The effect of inclusion of seven macroalgae species on accumulation of volatile fatty acids after 72 h of in vitro fermentation with rumen fluid. The Rhodes grass control substrate was equal in all fermentations, the Asparagopsis P-control was included at 2% of substrate OM, and the other seven macroalgae included at 5%.
a. Significant at P < 0.05.
algae at 5% of substrate OM compares to Asparagopsis at 2% for CH4 abatement without detriment to IVD-OM during in vitro fermentation. Therefore based solely on numerical CH4 abatement the ranking order from lowest to highest CH4 produced is as follows: 1) U. ohnoi; 2) C. taxifolia; 3) P. australis; 4) C. trinodis; 5) D. bartayresii; 6) S. flavicans; 7) C. patentiramea.
3.2. Experiment 2: Combinations
Similar to the results of Experiment 1, based on predictions of the GAM, which had a high goodness-of-fit (r2 > 0.98), there was a significant difference (P < 0.001) in TGP in the fermentations over 72 h (Figure 2). In addition, the TGP reached its plateau approaching 48 h in all fermentations, however, in contrast with Experiment 1, the combination treatments were equivalent to the Asparagopsis P-control at 2% of substrate OM in the reduction of TGP, but all were significantly different from the Rhodes grass control (P < 0.001) after 72 h of fermentation. The TGP for the combinations ranged between 124 and 136 mL∙g−1 OM representing a significant 28% - 34% reduction in TGP compared to the Rhodes grass control (188 mL∙g−1 OM). The Asparagopsis induced a TGP of 134 mL∙g−1 OM and therefore a 28% reduction, similar to the combinations demonstrating an overwhelming effect of Asparagopsis when combined with other macroalgae.
The CH4 production as affected by inclusion of the seven macroalgae combined with Asparagopsis was not different compared to the Asparagopsis alone during the course of the 72 h incubations (Figure 2). In the absence of macroalgae the fermentation initiated production of CH4 immediately after inoculation with RFB, however, the inclusion of the macroalgae combinations completely inhibited CH4 until 48 h when trace
Figure 2. The effect of inclusion of seven different macroalgae combined with Asparagopsis on gas production and substrate digestibility over 72 h of in vitro fermentation with rumen fluid. From top down: Total gas production (TGP); CH4 production; and apparent in vitro digestibility (IVD-OM). The Rhodes grass control substrate was equal in all fermentations, the Asparagopsis control was included at a concentration of 2% of substrate OM, and the seven macroalgae combinations resulted in an inclusion of Asparagopsis at 2% and each of the seven macroalgae at 5%. No ±SE is presented for TGP because SE was smaller than the symbols.
amounts were measured for the S. flavicans combination (0.2 mL∙g−1 OM) representing 98% inhibition. After 48 h of fermentation S. flavicans and D. bartayresii combined with Asparagopsis accumulated small amounts of CH4 such that at 72 h of fermentation these two combinations had reduced CH4 by 95% (0.8 mL∙g−1 OM) compared to the Rhodes grass control (16.0 mL∙g−1 OM). The Asparagopsis alone reduced CH4 by 99% (0.2 mL∙g−1 OM) after 48 h and by 96% (0.6 mL∙g−1 OM) at 72 h.
In the same way as Experiment 1 the typical pattern of IVD-OM increasing with time was demonstrated with or without macroalgae combinations (Figure 2). However, in Experiment 2 IVD-OM induced by the macroalgae combined with Asparagopsis was not as variable throughout the 72 h in vitro fermentations. Other than the Rhodes grass control, all fermentations contained 2% Asparagopsis which negated effects of the other macroalgae in the combinations. All the fermentations coalesced from onset of fermentation and after 72 h of fermentation the IVD-OM ranged from a low of 68% to a high of 72% for the D. bartayresii combined with Asparagopsis and Rhodes grass control, respectively.
In Experiment 2, in vitro fermentations with inclusion of the seven macroalgae combinations or Asparagopsis induced significant reductions in TVFA (P = 0.002) and acetate (P < 0.001), and significant increases in propionate (P < 0.001) and butyrate (P < 0.001) compared with the Rhodes grass control (Table 3). However, there was no significant difference between the Asparagopsis alone and the combinations, therefore improvements in VFA profiles due to combining Asparagopsis with any of the seven candidate macroalgae was not demonstrated. Compared with the Rhodes grass control, the macroalgae combinations induced reduction in TVFA on a molar concentration
Table 3. The effect of inclusion of seven macroalgae species combined with Asparagopsis on accumulation of volatile fatty acids after 72 h of in vitro fermentation with rumen fluid. The Rhodes grass control substrate was equal in all fermentations, the Asparagopsis P-control was included at 2% of substrate OM, and the seven macroalgae combinations resulted in inclusions of Asparagopsis at 2% and each of the seven macroalgae at 5%.
a. Significant at P < 0.05. ‡Each of the seven macroalgae represents half of a combination pair with.
basis from a low of 7% to a high of 12% and 14% by inclusion of the P. australis and C. taxifolia combined with Asparagopsis and Asparagopsis alone, respectively. The production of acetate was reduced by a low of 24% to a high of 29% and 31% by inclusion of S. flavicans and C. taxifolia combined with Asparagopsis, and Asparagopsis alone, respectively. Conversely, propionate was increased by a low of 21% to a high of 26% and 21% by inclusion of the P. australis and C. taxifolia combined with Asparagopsis, and Asparagopsis alone, respectively. The production of butyrate was increased by a low of 75% to a high of 91% and 72% for the U. ohnoi and C. taxifolia combined with Asparagopsis, and Asparagopsis alone, respectively.
This report represents the only current study of these seven macroalgae species (Table 1) at low inclusion in vitro. The purpose of this study was to rank them for beneficial effects in vitro prior to selection of one candidate for evaluation in vivo. However, there was no clear ranking order based on CH4 mitigation and improvements in in vitro rumen fermentation. In combining the seven macroalgae with Asparagopsis there was no significant difference between the combinations and in all cases the Asparagopsis overwhelmed the effects on fermentation which eclipsed potential benefits of combining these macroalgae.
These macroalgae have been evaluated in rumen fermentations in vitro at high dose approaching 20% of substrate OM and the effects on TGP was variable and mostly significant reductions compared to controls were reported   . In those studies the reductions in TGP were concomitant with reductions in CH4 and in some cases detrimental effect on IVD-OM. Fermentations with the individual (pure) macroalgae as inclusions with Rhodes grass in Experiment 1 of the present study also demonstrated variable TGP. However, lack of significant reduction can be attributed to the much lower inclusion concentration of 5% in the fermentations. It was hypothesized that the effect demonstrated at high dose would remain at 5%, however only Asparagopsis at 2% maintained its potency at low dose which was a direct result of methanogenesis inhibition. The halogenated bioactive compounds inherent in Asparagopsis spp. have potent antimethanogenic properties  that are not inherent in the other macroalgae evaluated in the present study. Their mode of action at high dose may be attributed to organic acids, tannins, phlorotannins, polyphenoloics, aminoglycans and other compounds that have antibacterial or inhibitory effect on rumen microbial metabolism, but have minimal effect at a low dose.
The reduction in CH4 induced by these macroalgae has been reproduced at high inclusion concentrations in vitro   , unfortunately in the same way as TGP, at 5% inclusion, the effect was minimal. However, the Asparagopsis at 2% was consistent with previous studies specific to that macroalga demonstrating nearly complete elimination of CH4 production in vitro   . Without significant differences in TGP and CH4 the ranking order was not clear and only numerical differences could be applied in the ranking. Thus, currently the Asparagopsis spp. are the only macroalgae demonstrating CH4 abatement ability exceeding 70% reduction at low dose and typically abatement > 99% is demonstrated.
Early in the fermentations the IVD-OM was variable with some of the macroalgae inducing a lag in the onset of fermentation. Previous research has shown a lag in TGP with addition at high dose  and this feature appears to be present at low dose, however both TGP and IVD-OM coalesced approaching 48 h of fermentation, and except for TGP induced by Asparagopsis, all fermentations were equal after 72 h. The Asparagopsis did not have different effect compared to the other macroalgae on IVD-OM demonstrating the importance of dose concentrations of macroalgae on rumen fermentation in vitro and presumably in ruminant animals. This indicates the importance of low dietary concentrations to maintain rumen efficiency and the seven macroalgae used in Experiment 1 demonstrated little benefit on IVD-OM or CH4 emissions at low dose. An important feature to note is that none of the macroalgae had a negative effect on IVD-OM at the dose concentration studied.
The effect of inclusion of antimethanogenic compounds on production of VFA during in vitro fermentations has been variable in most studies, however, a trend concomitant with significant CH4 reduction is in favour of increased propionate  . This has also been reported in previous studies using macroalgae which sometimes demonstrate a decrease in acetate in favour increased propionate and is more prominent with increasing dose. Macroalgae species that have a moderate or weak antimethanogenic capacity may not induce changes in the VFA profile   . In the present study it was apparent that the seven macroalgae are weak antimethanogenic agents in vitro and as such they have minimal effects on VFA production. The TVFA and acetate results at 5% inclusion indicate a variable and small but not significant reduction with no apparent change in propionate or butyrate. These same macroalgae induced an increase in propionate with decreasing acetate at high dose, however, at high dose some species decreased TVFA as a result of detriment to IVD-OM  . It is important to maintain or improve IVD-OM to maximize fermentation efficiency which reiterates the requirement for appropriate dose concentrations of any dietary inclusion. Notably, Experiment 1 demonstrated that six of the seven macroalgae (excluding C. patentiramea) induced production of marginally more TVFA and significantly more acetate than Asparagopsis and so demonstrated their potential to enhance fermentation efficiency when included in combination with Asparagopsis.
It was hypothesized that combining macroalgae with Asparagopsis in vitro would improve fermentation because these were identified in Experiment 1 as weakly antimethanogenic and resulted in some increase in VFA compared with Asparagopsis alone. However, at 5% in combination with Asparagopsis at 2% there was no evidence demonstrating improvements at a level to provide incentive for a follow-up in vivo study.
There was some variable but small decreases in TGP for some of the combinations compared with Asparagopsis alone, however all were significantly reduced compared to fermentations without macroalgae. The universal decrease in TGP was attributed to the Asparagopsis proportion of the combinations and a direct result of the near elimination of CH4 production. However, a minimal concentration of CH4 was detected in the final measurement (72 h) suggesting a gradual loss in antimethanogenic ability over time at the dose concentration used in this study. This phenomenon was described with Asparagopsis at doses ≤2% of substrate OM  . In the present study this occurred with S. flavicans and D. bartayresii combined with Asparagopsis, however, this does not indicate an effect specific to these combinations as a minimal level of CH4 was detected after 72 h with the Asparagopsis alone. Total depletion of CH4 is not expected in vivo where rumen fermentation is much more robust than in batch cultures. Also, feed residence time in the rumen is typically less than 72 h  .
The loss of energy as CH4 has potential to be reclaimed as productivity, however this can’t be demonstrated in vitro and in vivo studies are necessary to determine the extent of productivity gains. When demonstrated in vivo productivity gains would dramatically increase the value of Asparagopsis and macroalgae combinations for livestock production systems. Adoption of any CH4 mitigation strategy requires more than environmental benefits. The value of carbon abatement may eventually provide revenue incentive for producers to adopt macroalgae feed additives based on CH4 abatement. However, improvements in productivity enhance the environmental value of macroalgae.
The IVD-OM was not negatively affected, however, the hypothesis of improvements to fermentation was not demonstrated by the combinations compared to Asparagopsis. All the fermentations, with and without macroalgae were stable and IVD-OM was not different. Improvements in IVD-OM would provide a conduit for improved utilization of feed and offset the cost of supplementation with macroalgae. Further investigation into macroalgae on improved feed energy utilization, productivity, and feed quality is necessary, particularly relative to periods of poor grass quality for grazing livestock  .
In light of the VFA results of previous work  and Experiment 1 it was hypothesized that when combined with Asparagopsis the other macroalgae would have improved VFA production compared to the Asparagopis alone. This effect unfortunately was not demonstrated and the combinations adopted similar profiles as Asparagopsis alone and the variability between treatments observed in Experiment 1 was muted in Experiment 2, thus again demonstrating the dominant effect of Asparagopsis in vitro. It is typical with Asparagopsis that decrease in CH4 is concomitant with decrease in acetate and increase in propionate. It is common for antimethanogenic feed additives to have this effect in vitro  and in vivo   and is believed to be due to reductive propionate production being more favourable than acetogenesis in the presence of excess hydrogen  .
Although the present study did not support the use of macroalgae combinations to decrease CH4 production in vitro the utility of combinations of macroalgae to enhance ruminant animal productivity and reduce CH4 emissions is worthy of further exploration. Supplementation of high protein macroalgae such as the freshwater green Oedogonium sp. is feasible up to 25% of intake  which could increase the proportion of rumen bypass protein thus benefiting productivity  . Alternative sources of protein can also reduce CH4 emissions intensity by improving productivity of grass fed beef during those periods of decreasing diet quality. Supplementation with macroalgae can therefore directly reduce methanogenesis and reduce emission intensity by improving the product to emissions ratio.
There was not enough difference induced in rumen fermentation efficiency or CH4 production in vitro to support a conclusive ranking order between the seven individual macroalgae at the 5% inclusion concentration of this study. A numerical difference indicates U. ohnoi and C. patentiramea were the most and least antimethanogenic, respectively. When macroalgae were combined with Asparagopsis, a known potent antimethanogenic agent in vitro, there was not an adequate effect to justify proceeding to in vivo evaluation or recommendation for use of the combinations in livestock feed. However, high protein macroalgae supplemented at higher dietary concentrations may provide greater benefit when combined with Asparagopsis by contributing to reduced CH4 emissions through further improved productivity at times of low feed quality thus reducing emissions intensity per product output.
The gas and VFA analysis was accomplished via chromatography expertise of Shane Askew (Advance Analytical Centre, JCU) and collection and identification of macroalgae species assisted by Bjoern Gosch. Coral Coast Barramundi Fisheries Ltd. and Pacific Reef Fisheries Ltd. are acknowledged for supply of cultivated macroalgae. Melissa Matthews is acknowledged for reference management and construction of data tables.
 Beauchemin, K.A., Kreuzer, M., O’Mara, F. and McAllister, T.A. (2008) Nutritional Management for Enteric Methane Abatement: A Review. Australian Journal of Experimental Agriculture, 48, 21-27.
 Morgavi, D.P., Forano, E., Martin, C. and Newbold, C.J. (2010) Microbial Ecosystem and Methanogenesis in Ruminants. Animal, 4, 1024-1036.
 Patra, A.K. (2012) Enteric Methane Mitigation Technologies for Ruminant Livestock: A Synthesis of Current Research and Future Directions. Environmental Monitoring and Assessment, 184, 1929-1952.
 Beauchemin, K.A., McAllister, T.A. and McGinn, S.M. (2009) Dietary Mitigation of Enteric Methane from Cattle.CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources, 4, 1-18.
 O’Sullivan, L., Murphy, B., McLoughlin, P., Duggan, P., Lawlor, P.G., Hughes, H. and Gardiner, G.E. (2010) Prebiotics from Marine Macroalgae for Human and Animal Health Applications. Marine Drugs, 8, 2038-2064.
 Evans, F.D. and Critchley, A.T. (2014) Seaweeds for Animal Production Use. Journal of Applied Phycology, 26, 891-899.
 Rjiba Ktita, S., Chermiti, A. and Mahouachi, M. (2010) The Use of Seaweeds (Ruppia maritima and Chaetomorpha linum) for Lamb Fattening during Drought Periods. Small Ruminant Research, 91, 116-119.
 Wang, Y., Xu, Z., Bach, S.J. and McAllister, T.A. (2008) Effects of Phlorotannins from Ascophyllum nodosum (Brown Seaweed) on in Vitro Ruminal Digestion of Mixed Forage or Barley Grain. Animal Feed Science and Technology, 145, 375-395.
 Kinley, R.D., de Nys, R., Vucko, M.J., Machado, L. and Tomkins, N.W. (2016) The Red Macroalgae Asparagopsis taxiformis Is a Potent Natural Antimethanogenic That Reduces Methane Production during in Vitro Fermentation with Rumen Fluid. Animal Production Science, 56, 282-289.
 Machado, L., Magnusson, M., Paul, N.A., Kinley, R.D., de Nys, R. and Tomkins, N.W. (2016) Dose-Response Effects of Asparagopsis taxiformis and Oedogonium sp. on in Vitro Fermentation and Methane Production. Journal of Applied Phycology, 28, 1443-1452.
 Machado, L., Magnusson, M., Paul, N.A., de Nys, R. and Tomkins, N.W. (2014) Effects of Marine and Freshwater Macroalgae on in Vitro Total Gas and Methane Production. Plos One, 9, e85289.
 Angell, A.R., Mata, L., de Nys, R. and Paul, N.A. (2016) The Protein Content of Seaweeds: A Universal Nitrogen-to-Protein Conversion Factor of Five. Journal of Applied Phycology, 28, 511-524.
 Goering, H. and Van Soest, P. (1970) Forage Fiber Analyses (Apparatus, Reagents, Procedures, and Some Applications). Agriculture Handbook No. 379, U.S. Agricultural Research Service, Washington DC.
 Pellikaan, W.F., Hendriks, W.H., Uwimana, G., Bongers, L.J.G.M., Becker, P.M. and Cone, J.W. (2011) A Novel Method to Determine Simultaneously Methane Production during in Vitro Gas Production Using Fully Automated Equipment. Animal Feed Science and Technology, 168, 196-205.
 Tavendale, M.H., Meagher, L.P., Pacheco, D., Walker, N., Attwood, G.T. and Sivakumaran, S. (2005) Methane Production from in Vitro Rumen Incubations with Lotus pedunculatus and Medicago sativa, and Effects of Extractable Condensed Tannin Fractions on Methanogenesis. Animal Feed Science and Technology, 123-124, 403-419.
 R Core Team (2013) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. Vienna.
 López, S., Dhanoa, M.S., Dijkstra, J., Bannink, A., Kebreab, E. and France, J. (2007) Some Methodological and Analytical Considerations Regarding Application of the Gas Production Technique. Animal Feed Science and Technology, 135, 139-156.
 Dubois, B., Tomkins, N.W., Kinley, R.D., Bai, M., Seymour, S., Paul, N.A. and de Nys, R. (2013) Effect of Tropical Algae as Additives on Rumen in Vitro Gas Production and Fermentation Characteristics. American Journal of Plant Sciences, 4, 34-43.
 Machado, L., Magnusson, M., Paul, N.A., Kinley, R.D., de Nys, R. and Tomkins, N.W. (2016) Identification of Bioactives from the Red Seaweed Asparagopsis taxiformis That Promote Antimethanogenic Activity. Journal of Applied Phycology, 28, 3117-3126.
 Mitsumori, M., Shinkai, T., Takenaka, A., Enishi, O., Higuchi, K., Kobayashi, Y., Nonaka, I., Asanuma, N., Denman, S.E. and McSweeney, C.S. (2012) Responses in Digestion, Rumen Fermentation and Microbial Populations to Inhibition of Methane by a Halogenated Methane Analogue. British Journal of Nutrition, 108, 482-491.
 Kinley, R.D. and Fredeen, A.H. (2015) In Vitro Evaluation of Feeding North Atlantic Stormtoss Seaweeds on Ruminal Digestion. Journal of Applied Phycology, 27, 2387-2393.
 Machado, L., Kinley, R.D., Magnusson, M., de Nys, R. and Tomkins, N.W. (2015) The Potential of Macroalgae for Beef Production Systems in Northern Australia. Journal of Applied Phycology, 27, 2001-2005.
 Tomkins, N.W., Colegate, S. and Hunter, R. (2009) A Bromochloromethane Formulation Reduces Enteric Methanogenesis in Cattle Fed Grain-Based Diets. Animal Production Science, 49, 1053-1058.
 Li, X., Norman, H.C., Kinley, R.D., Laurence, M., Wilmot, M., Bender, H., de Nys, R. and Tomkins, N.W. (2016) Asparagopsis taxiformis Decreases Enteric Methane Production from Sheep. Animal Production Science, AN15883.
 Poppi, D.P. and McLennan, S.R. (1995) Protein and Energy Utilization by Ruminants at Pasture. Journal of Animal Science, 73, 278-290.