Assessing the methane mitigation potential of Canadian red seaweeds using in vitro batch culture

Abstract Seven red Canadian seaweeds (Callophyllis flabellulata, Graciliariopsis verrucosa, Mastocarpus papillatus, Mazzaella splendens, Mazzaella japonica, Palmaria mollis, and Prionitis lanceolata) and a positive control (Asparagopsis taxiformis) were selected to evaluate their chemical and elemental composition and their effects on in vitro fermentation and methane (CH4) production in an alfalfa hay or barley straw diet. The in vitro batch culture was conducted as a completely randomised design with a control (alfalfa hay or barley straw) and seven increasing concentrations of seaweed. Chemical and elemental composition varied greatly across seaweed genera. Increasing supplementation of A. taxiformis linearly decreased (P < 0.001) dry matter disappearance (DMD) and gas production (GP; mL, mL/g DMD) with CH4 production eliminated (P < 0.001) at 1.0% inclusion of A. taxiformis in both diets. Inclusion of Mastocarpus papillatus, Mazzaella japonica, Mazzaella splendens, Palmaria mollis, and Prionitis lanceolata increased (P ≤ 0.05) GP (mL/g DMD) at 0.5% and 1.0% in alfalfa diets. Graciliariopsis verrucosa linearly decreased (P = 0.01) CH4 production (mL/g DMD) in the straw diet, but no doses were different compared to the control. In conclusion, the Canadian red seaweeds examined in this study did not exhibit anti-methanogenic potential when incubated with alfalfa hay or barley straw.


Introduction
Seaweed or macro-algae are macroscopic, multicellular organisms that live within a diverse range of marine environments.They are chemically and physically diverse, possessing a range of complex carbohydrates, fatty acids, minerals, and bioactive compounds that have been suggested to be readily degraded by the rumen microbiota (Maia et al. 2016;Makkar et al. 2016).An international effort is being made to profile various seaweeds to assess their potential for inclusion in ruminant diets as a dietary enteric methane (CH 4 ) mitigant.One of the benefits of producing seaweeds is that they have a higher photosynthetic efficiency than land plants and can be grown in a variety of water sources, potentially making them viable to be mass produced for livestock feed (McCauley et al. 2020).
There are three distinct groups of seaweeds commonly referred to by colour: green (Chlorophyta), brown (Ochrophyta), and red (Rhodophyta).Of these, red seaweeds are rich in a number of various bioactive compounds with diverse nutritional and biological activities (Abbott et al. 2020).Specific to red seaweeds is the presence of halogenated low molecular weight compounds, including bromoforms and haloforms (McCauley et al. 2020).These bromoformic compounds have been shown to effectively inhibit ruminal CH 4 production; however, the safety of these compounds within ruminant diets has not yet been proven.Most recent interest is in the red Asparagopsis sp. which has been shown to decrease CH 4 production by 99% in an in vitro batch culture (Machado et al. 2014;Kinley and Fredeen 2015), 80% in Merino-cross wethers (Li et al. 2018), 67% in lactating dairy cows (Roque et al. 2019b), 98% in Brahman-Angus steers (Kinley et al. 2020), and over 80% in Angus-Hereford steers (Roque et al. 2021) with small dietary inclusion rates.
Although several other macroalgae genera have been investigated in vitro (Machado et al. 2014;de la Moneda et al. 2019;Bikker et al. 2020), none have been identified as having an anti-methanogenic property similar to that of Asparagopsis sp.However, sustainable production and the overall environmental impact of farming, processing, transport, and feeding of Asparagopsis sp. is uncertain.Therefore, research is required to identify alternative seaweeds that may show promise for being sustainably produced for ruminants.This study describes the nutrient profiles of eight red seaweeds, including Asparagopsis taxiformis, Callophyllis flabellulata, Graciliariopsis verrucosa, Mastocarpus papillatus, Mazzaella splendens, Mazzaella japonica, Palmaria mollis, and Prionitis lanceolata and evaluates the effect of dose (0%, 0.5%, 1.0%, 2.0%, 4.0%, 8.0%, 100% dry matter (DM)) and substrate (alfalfa hay or barley straw) on in vitro fermentation and gas and CH 4 production.

Ethics statement
Animal handling and care procedures of the donor heifers were approved by the Lethbridge Research and Development Centre Animal Care Committee in accordance with the guidelines of the Canadian Council on Animal Care (2009).

Seaweeds
The A. taxiformis used in this study was provided by CSIRO, Queensland, Australia, and the University of California, Davis, USA.The other algal species used in this study were selected based upon their availability and natural abundance across the east and west coast of Canada, and all were harvested off the coast of British Columbia, Canada.The Canadian harvested seaweeds provided by AK Green (British Columbia, Canada)
For each run, rumen inoculum was collected from three ruminally cannulated beef heifers fed a 50% alfalfa hay and 50% barley silage diet (DM basis).Animals were adapted to the diet for three weeks before rumen content collection.Rumen contents (∼300 g) were taken from the reticulum and the ventral, caudal, and dorsal-ventral sacs within the rumen of each animal and pooled in equal proportions.Rumen contents were squeezed through two layers of PECAP nylon (pore size 355 μm; Sefar Canada Inc., Ville St. Laurent, Canada) and the filtrate was immediately transported to the laboratory in an insulated, air-tight container and kept at 39 • C. The pH of the filtrate was measured using a pH meter (Orion model 260A;Fisher Scientific,Toronto,ON,Canada).
Inoculum was prepared by mixing rumen fluid with Goering and Van Soest (1970) buffer at a 1:3 ratio (15 mL rumen fluid: 45 mL buffer).Inoculum (60 mL) was transferred into each vial under a stream of O 2 -free CO 2 .The vials were then sealed with rubber stoppers and placed on a rotary shaker (120 rpm) in an incubator maintained at 39 • C. Each substrate, dose, and seaweed treatment were replicated over a total of 4 runs (1 replicate per run) and vials containing only inoculum and filter bags were included as blank controls for a total number of 99 vials per run.

Measurements
Headspace gas pressure was measured at 3, 6, 12, 18, 24, 36 and 48 h of incubation by inserting a 23 gauge (0.6 mm) needle attached to a pressure transducer (model PX4200-015GI; Omega Engineering, Inc., Laval, QC, Canada) with a visual display unit (Data Track, Christchurch, UK).A 15 mL gas sample was subsequently taken at each time from each vial using a 20 mL graduated plastic syringe connected to a three-way stopcock, which was then injected into a preevacuated 6.8 mL Exetainer (Labco Ltd., High Wycombe, England, UK) for gas composition analysis.After gas sampling, all headspace gas was released from the vial and returned to the incubator.
After 48 h of incubation, vials were removed from the incubator, gas was sampled, and the rubber stopper was removed.A 1 mL sample of inoculum from each vial was taken and mixed with 0.2 mL metaphosphoric acid within a microtube (2 mL) and frozen (−20 • C) until analysed for VFA concentration.The bags were removed from the vials and rinsed with cold water until the excess water ran clear.Bags were dried in the oven at 55 • C for 24 h and hot-weighed to determine in vitro DMD.

Chemical analysis
The elemental analysis was conducted by a commercial laboratory (Cumberland Valley Analytical Services, Waynesboro, PA, USA).Phosphorus, Ca, Mg, K, Na, Fe, Mn, Zn, and Cu were determined using Association of Official Analytical Chemists (AOAC) method 985.1 (AOAC 2000a) with modifications where seaweeds were ashed for 1 h at 535 • C, digested in open crucibles for 20 min in 15% nitric acid on a hot-plate, diluted to 50 mL, and then analyzed using inductively coupled plasma.Samples used for Mo analysis were ashed at 480 • C for 4 h, digested in an open crucible for 20 min in 15% nitric acid on a hot-plate, diluted to 50 mL, and then analysed on axial view inductively coupled plasma.Selenium was analysed using AOAC method 996.16 (AOAC 2000b).
Unincubated seaweeds and substrates were analysed for analytical DM and ash content by drying samples at 135 • C for 2 h (AOAC 2005; method 930.15) and then combusting samples in a muffle furnace at 550 • C for 5 h, respectively (AOAC 2005; method 942.05).Samples were analysed for NDF concentration as described by Van Soest et al. (1991) with modifications for using a fibre analyzer (F57 Fiber Filter Bags, 200 Fiber Analyzer, ANKOM Technology Corp., Macedon, NY, USA)  Flawil, Switzerland).Incubated substrates were freeze-dried for 72 h for the determination of DM followed by the NDF procedure as described above.
The concentration (%) of CH 4 in headspace gas was analysed by gas chromatography (Varian 4900; Agilent Technologies Canada Inc., Mississauga, ON, Canada) equipped with a 10 m PPU column and thermal conductivity detector with helium gas used as a carrier gas.The concentration of VFA was analysed using gas-liquid chromatography (model 6890; Agilent, Wilmington, DE, USA) with a polar capillary column (30 m × 0.32 mm × 1 μm; ZB-FFAP; Phenomenex Inc., Torrance, CA, USA), a flame ionization detector, and helium as carrier gas.

Calculations and statistical analysis
Disappearances of DM and NDF were calculated as the difference between the chemical composition of the substrate before and after incubation.Gas volume was calculated from gas pressure (psi) using the following quadratic equation (Romero-Pérez and Beauchemin 2018) developed under our laboratory-specific conditions: gas volume (mL) = 4.7047 × (gas pressure) + 0.0512 × (gas pressure) 2 .
Methane production was calculated from gas volume and CH 4 concentration.GP and CH 4 production were expressed as the cumulative volume over 48 h.
Data were analysed using the mixed model procedure of SAS (SAS 2018).Data and measurements from alfalfa hay and barley straw treatments were analysed separately.The model included seaweed, dose, and seaweed × dose as fixed effects and run as a random effect.For analysis of 100% seaweed, seaweed was included as fixed effect and run as a random effect.The univariate procedure was used to verify that data were normally distributed.Degrees of freedom were adjusted us-ing the Kenward-Roger option, and differences among treatments were tested using the PDIFF option.Differences between means were declared significant when P < 0.05, and orthogonal contrasts were used to test for linear and quadratic responses of seaweed dose.

Elemental composition
The chemical composition of the forage substrates and the eight species of seaweed are shown in Table 1.The seaweeds varied largely in DM, organic matter (OM), CP, and NDF concentrations with averages (±SD) of 27.9 ± 23.5, 66.2 ± 7. 4, 20.8 ± 4.2, and 44.2 ± 13.3, respectively.In contrast, all seaweeds had low ether extract concentration (0.3 ± 0.1).Elemental concentrations varied greatly among seaweed species (Table 2).All seaweed presented with undetectable concentrations of Hg, Pb, and TI with A. taxiformis, Mastocarpus papillatus, and Prionitis lanceolata presenting with the highest concentrations of As.Moreover, A. taxiformis also had the highest concentration of Ca, Al, Fe, I, and Ba.
Mazzaella splendens was the only Canadian seaweed to linearly decrease (P < 0.01) DMD with increasing dose; however, only at the dose of 8.0% DM was DMD less (P < 0.001) than the control.Additionally, Mazzaella splendens dosed at 0.5% to 2.0% DM increased (P < 0.001) GP (mL/g DMD) and 0.5% Mazzaella splendens increased (P < 0.001) CH 4 production (mL/g DMD) compared with the control.Palmaria mollis increased (P < 0.001) GP (mL/g DMD) at 0.5%, 1.0%, and 8.0% DM compared with the control with 0.5% Palmaria mollis having greater (P < 0.001) CH 4 production (mL/g DMD) than the control.There was a tendency for a quadratic effect of Prionitis lanceolata on GP mL/g DM (P = 0.06) and GP mL/g DMD (P = 0.08) where both variables were most dramatically increased from the control at 0.5% and 1%.
There was a seaweed × dose effect (P < 0.001) on total VFA production (mmol/L) and the molar percentages of acetate, propionate, and butyrate as well as acetate:propionate (A:P) ratio (Table 4).Increasing dose of A. taxiformis linearly decreased (P < 0.001) total VFA production (mmol, mmol/g DMD).The molar percentage of acetate responded quadratically (P = 0.01) to increasing doses of A. taxiformis with smaller proportions observed at 0.5%, 1.0%, 2.0%, and 4.0% compared to the 8.0% DM dose; the control was lower than all doses.Additionally, A. taxiformis quadratically increased (P < 0.001) the molar percentage of propionate from the control at 0.5%-4% DM inclusion but not at 8.0% DM inclusion.The per-centage of butyrate linearly increased (P = 0.03) with increasing doses of A. taxiformis.The A:P responded quadratically (P < 0.01) to increasing doses of A. taxiformis where all but the 8.0% DM dose was significantly less (P < 0.001) than the control.

Barley straw substrate
There was a quadratic response (P < 0.01) of DMD to A. taxiformis, where DMD was greater at the 0.5% dose and lower at doses >2% compared to the control (Table 5).The DMD responded quadratically (P = 0.03) to Graciliariopsis verrucosa, where DMD was greater than the control at all doses except for 8.0%.The DMD was linearly decreased (P = 0.03) by Mastocarpus papillatus and Mazzaella japonica and linearly increased (P < 0.001) for Palmaria mollis at increasing doses of seaweed.GP responded quadratically (P < 0.01) to A. taxiformis, where the greatest reduction in GP occurred at 4.0% and 8.0% seaweed inclusion.Increasing Graciliariopsis verrucosa inclusion resulted in a quadratic (P ≤ 0.05) response of GP (irrespective of unit), with GP being greatest at 2.0% DM.Methane production irrespective of unit of expression was quadratically (P < 0.001) decreased by A. taxiformis, with CH 4 production eliminated at doses greater than 0.5% DM.Additionally, CH 4 production (mL/g DM) responded quadratically (P ≤ 0.05) to Graciliariopsis verrucosa, where CH 4 was greatest at 2% and decreased from the control at 8.0%.
Total VFA production (mmol, mmol/g DMD) responded quadratically (P = 0.05) to increasing doses of A. taxiformis, where 4% and 8% decreased total VFA (Table 6).Total VFA on a mmol/g DMD basis was linearly decreased by increasing the dose of Graciliariopsis verrucosa.Proportions of acetate and propionate responded quadratically (P < 0.01) to increasing doses of A. taxiformis, where acetate was lowest and propionate was greatest at 1% and 2%, respectively.All doses of A. taxiformis increased (P < 0.001) butyrate production and decreased A:P; however, A:P responded quadratically (P < 0.001) to A. taxiformis, where the A:P was lowest at 0.5%, 1.0%, 2.0%, and 4.0%.Increasing doses of Graciliariopsis verrucosa, Palmaria mollis, and Prionitis lanceolata linearly increased (P < 0.01) the percentage of butyrate.

Seaweeds
Palmaria mollis had the greatest (P < 0.001) DMD and NDFD (Table 7).Mastocarpus papillatus had the lowest (P < 0.001) DMD, while A. taxiformis had the lowest (P < 0.001) NDFD.Asparagopsis taxiformis incubated alone had the lowest (P < 0.001) GP (mL, mL/g DM, mL/g DMD) and did not produce any CH 4 or VFA.Prionitis lanceolata and Mazzaella japonica were associated with the highest (P < 0.001) production of gas per gram DMD and CH 4 per gram DMD.Aside from A. taxiformis, Mazzaella splendens and Palmaria mollis had the lowest CH 4 production (mL/g DMD).Palmaria mollis had the highest (P < 0.001) total VFA production, followed by Mazzaella splendens.

Discussion
The nutrient composition of the seaweeds analysed in this study demonstrates the high variability observed across seaweed species despite their similarity in seaweed type (red).There was a large variability in OM (52.6%-74.6%),CP (16.0%-28.7%),and NDF concentrations (27.3%-66.9%)among the examined seaweeds, with a low fat content consistent across all seaweeds (0.31% DM ± 0.09).This variation across seaweed genera is consistent with literature where environmental fac-tors like water temperature, salinity, harvest stage, and season of harvest can result in large differences in chemical composition (Makkar et al. 2016;Bikker et al. 2020).
Seaweeds are known for their ability to accumulate large quantities of heavy metals, with the concentrations of these metals limiting their use in animal diets (Øverland et al. 2019).Maximum allowable levels for aluminum (Al), arsenic (As), cadmium (Cd), and lead (Pb) are 1000, 8, 0.4, and 8 mg/kg, respectively, as defined by the Canadian Food Inspection Agency for inclusion in livestock feeds.Included at less than 8.0% of the diet DM, these seaweeds pose limited risk of exceeding dietary inclusion for ruminants.Alternatively, with some studies proposing a greater inclusion rate (>10% DM) of seaweed into ruminant diets (Maia et al. 2016), these elements may restrict their use at larger quantities.
In the present study, there were small differences observed for the impact of seaweed on alfalfa hay vs. barley straw, with the suppression of CH 4 production by A. taxiformis consistent across both forages.Previous studies have indicated that the response to seaweed supplementation depends on the diet composition.For example, supplementation of Oedogonium at 0.2% OM decreased CH 4 at different rates (15%-40%) when incubated in three different forages, including Rhodes grass, Rhodes grass hay, and Finders grass (Dubois et al. 2013;Machado et al. 2014Machado et al. , 2015)).Similarly, Ulva sp. and Gigartina sp.only decreased CH 4 when incubated with corn silage and not meadow hay (Maia et al. 2016).To our knowledge, the seaweeds used in the present study have not been assessed elsewhere, but it is possible that they may show more efficacy if incubated with substrates that are more fermentable than those used in the present study.
Although Prionitis lanceolata numerically increased GP and CH 4 production at all doses in the alfalfa diet, this was not due to an increase in DMD or NDFD, and total VFA was not affected.Interestingly, Palmaria mollis had the greatest DMD and NDFD compared to all other seaweeds but only increased DMD and NDFD when incubated with the lower quality barley straw substrate (DMD = 57.0%;NDFD = 49.8%)rather than the higher quality alfalfa hay substrate (DMD = 70.7%;NDFD = 46.2%).Other Palmaria species, including Palmaria palmata, have been previously shown to have the highest value for in situ disappearance (Tayyab et al. 2016)  This study only examined red seaweeds as they have been associated with a decrease in enteric CH 4 production due to the presence of halogenated low molecular compounds, including bromoforms and chloroforms (Machado et al. 2014).However, the concentration of these compounds has been shown to affect their antimethanogenic potential with a large variation observed even within the same species (Machado et al. 2015;Li et al. 2018;Roque et al. 2019b;Abbott et al. 2020;Kinley et al. 2020).Due to the limited amount of material, we were not able to quantify the concentrations of these compounds within the seaweed used.
Table 5.In vitro dry matter and neutral detergent fibre disappearances and cumulative gas and methane production after 48 h incubation of barley straw dosed with different seaweeds at increasing inclusion levels.In agreement with previous studies (Machado et al. 2014(Machado et al. , 2015;;Roque et al. 2019a), A. taxiformis effectively eliminated in vitro CH 4 production when included at >1.0% diet DM when included to both barley straw and alfalfa hay sub-strates.The red seaweed, A. taxiformis, has now been established as an effective CH 4 inhibitor as shown in this study and elsewhere (Machado et al. 2014;Kinley and Fredeen 2015).The efficacy of this seaweed has also been shown in vivo resulting in a 80% reduction in enteric CH 4 in Merinocross wethers fed 3% A. taxiformis on an OM basis (Li et al. 2018).Similarly, up to 60% reduction in CH 4 emissions adjusted for milk production was reported upon feeding lactating dairy cows Asparagopsis armata at 1.0% OM for a 21 day period (Roque et al. 2019b).Similarly, A. taxiformis was fed at up to 0.2% of feed OM and resulted in up to a 98% decrease in enteric CH 4 production over a 90 day feeding period in growing beef cattle (Kinley et al. 2020).It is the bromoform and chloroform contents of the Asparagopsis species that are attributed to the decrease in CH 4 production (Abbott et al. 2020;Kinley et al. 2020).In their purified forms, both of these compounds have shown to be effective at inhibiting enteric CH 4 production across a short period of time; however, even at low concentrations, they are associated with toxicity in animals and humans (Chipperfield et al. 2020).
Bromoform is a volatile ozone-depleting molecule, and it is estimated that up to 70% of naturally occurring bromoforms in the environment are produced by algae (McCauley et al. 2020).These substances can be photochemically oxidized or react with hydroxyl radicals, contributing to the depletion in ozone (McCauley et al. 2020), hence the concern with their purified use.Alternatively, seaweeds produce and store rather than release bromoforms, making them an effective method of administering these compounds without their release into the environment (McCauley et al. 2020 ).However, the metabolism of seaweeds and their secondary compounds within the rumen remains to be comprehensively examined.
Several other studies have reported that red seaweeds resulted in a decrease in GP.Maia et al. (2016) examined in vitro fermentation of two red (Gigartina sp. and Graciliariopsis vermiculophylla), one green (Ulva sp.), and two brown (Laminaria ochroleuca and Saccharina latissima) seaweeds in either a meadow hay or corn silage substrate and found that all re-duced GP; however, this did not result in a decrease in total VFA production and fermentation efficiency and seaweed was included at 25.0% of diet DM.Other red seaweeds, including Halymenia, Hypnea, and Laurencia, reduced in vitro CH 4 production by 26.5%, 42.5%, and 39.8% when included in a Flinder's hay substrate at 16.7% OM basis compared to decorticated cottonseed meal (Machado et al. 2014).It remains unclear whether the reduction in enteric CH 4 is linked only to the presence of these chloroform/bromoform compounds or whether the presence of other secondary compounds like phlorotannins may also contribute to the alteration in rumen CH 4 production.Phlorotannins are a unique class of polyphenolic compounds that have been associated with antioxidant (Morais et al. 2020) and antimicrobial effects; however, these phlorotannins are more prolific in brown seaweeds (Abbott et al. 2020) and not found in high concentrations in red seaweeds.Alternatively, red seaweeds are rich in galactans that have a diverse range of biological activities, including antimicrobial, antioxidant, pre-biotic, and antiviral properties (Abbott et al. 2020).A comprehensive analysis on the concentrations of both phlorotannins and galactans within the examined seaweeds may support the lack of effect on fermentation characteristics by the seaweeds examined within this experiment.

Conclusions
In conclusion, the seaweeds examined in this study did not have an inhibitory effect on in vitro ruminal CH 4 production.This study demonstrates the variability in chemical composition and in vitro ruminal fermentation of various red seaweeds.Although the examined seaweeds were ineffective at reducing CH 4 production, at applicable inclusion rates (1%-2%) other fermentation characteristics were not negatively af-fected.Elevated heavy metal concentration may limit inclusion rate of some red seaweeds in ruminant diets.
compared to other seaweeds, including Mastocarpus, as demonstrated in this study.Several other studies confirm the lack of effect of Palmaria sp.(Molina-Alcaide et al. 2017; de la Moneda et al. 2019; Ramin et al. 2019) on in vitro CH 4 production; however, the Palmaria species observed in the present study (Palmaria mollis) has not been examined elsewhere.

Table 1 .
Nutrient composition of forages and red seaweeds used as substrates in the in vitro incubations.

Table 2 .
Elemental analysis of seaweeds used in the in vitro incubations.

Table 3 .
In vitro dry matter and neutral detergent fibre disappearances and cumulative gas and methane production after 48 h incubation of alfalfa hay dosed with different seaweeds at increasing inclusion levels.
within a variable with different letters differ significantly from the control at P ≤ 0.05 for T × D. Differences among other treatments are not indicated to simplify the presentation of results 1 Dose is on a % DM basis. 2 SEM presented for T × D. 3 T × D, treatment × dose interaction P value.

Table 4 .
In vitro volatile fatty acid production after 48 h incubation of alfalfa hay dosed with different seaweeds at increasing inclusion levels.
DMD, dry matter disappearance; NDF, neutral detergent fibre disappearance; GP, gas production over 48 h of in vitro incubation; DM, dry matter.a-e, means within a variable with different letters differ significantly from the control at P ≤ 0.05 for T × D. Differences among other treatments are not indicated to simplify the presentation of results.
1Dose is on a % DM basis. 2 SEM presented for T × D. 3 T × D, treatment × dose interaction P value.

Table 6 .
In vitro volatile fatty acid production after 48 h incubation of barley straw dosed with different seaweeds at increasing inclusion levels.
3T × D, treatment × dose interaction P value

Table 7 .
In vitro dry matter and neutral detergent fibre disappearances, cumulative gas, methane, and total volatile fatty acid production of seaweeds incubated alone for 48 h.DMD, dry matter disappearance; NDFD, neutral detergent fibre disappearance; GP, gas production over 48 h of in vitro incubation; DM, dry matter; VFA, volatile fatty acids.a-g, values within a column with different letters differ significantly at P ≤ 0.05. Note: