A preliminary study on the effects of red Bonnemaisonia hamifera seaweed on methane emissions from dairy cows

Graphical Abstract

Summary: The red seaweed Bonnemaisonia hamifera has shown CH₄-mitigating properties in several in vitro studies. This pilot study aimed to investigate the potential of the seaweed as an antimethanogenic feed additive when fed to dairy cows at a maximum inclusion rate of 1% on a dry matter (DM) basis. Increasing the level of B. hamifera resulted in linear decreases in daily CH₄ production, yield, and intensity by 13% to 16%. Moreover, linear increases were observed in daily H₂ emissions, yield, and intensity. Hence, larger and longer-term studies should be undertaken to fully investigate the potential of this seaweed because the concentration of the potentially harmful compound, bromoform, is remarkably lower than in the known antimethanogenic red seaweed Asparagopsis spp.

Highlights

• •Methane yield was reduced by over 15% at 1% DM inclusion rate of B. hamifera.
• •Hydrogen yield was increased by 325% at 1% DM inclusion rate of B. hamifera.
• •The exact nature of the antimethanogenic compounds in B. hamifera is unknown.

Abstract

The red seaweed Bonnemaisonia hamifera has shown potential as a CH₄-mitigating feed additive in vitro, and hence, this pilot study aimed to investigate the potential of the seaweed when fed to dairy cows. The study was conducted as a 4 × 4 Latin square using 4 Danish Holstein cows with 4 periods of 7 d where the cows were placed in respiration chambers for the last 3 d. Milk yield and feed intake were recorded daily. The cows were fed a TMR without B. hamifera (control) or the same diet with the addition of, on a DM basis, 0.33% (low), 0.66% (medium), and 1% (high) B. hamifera. Increasing inclusion levels of B. hamifera linearly decreased CH₄ emissions with up to a 13.3% reduction in daily CH₄ production (g/d), 15.7% reduction in CH₄ yield (g/kg DMI), and 16.4% reduction in CH₄ intensity (g/kg ECM). In contrast, linear increases were observed in daily H₂ production (g/d), H₂ yield (g/kg DMI), and H₂ intensity (g/kg ECM). The exact nature of the antimethanogenic compounds in B. hamifera is unknown; however, the concentration of the potentially harmful compound, bromoform, was remarkably lower in B. hamifera compared with reported concentrations in the known antimethanogenic red seaweed Asparagopsis taxiformis. In conclusion, B. hamifera showed CH₄-mitigating properties in this preliminary study, but further in vivo studies are needed to fully evaluate the potential of the seaweed as an antimethanogenic feed additive.

The CH₄-mitigating properties of the red seaweed Asparagopsis spp. have been ascribed to their high concentrations of bromoform and other halomethanes (Machado et al., 2016), which have been known for decades to be efficient inhibitors of methanogenesis in ruminants (Lanigan, 1972). Hence, several recent in vitro and in vivo studies have demonstrated that the CH₄-reducing effects are dependent on the dose of Asparagopsis spp., and thus the amount of bromoform (Kinley et al., 2016; Roque et al., 2019; Mihaila et al., 2022; Alvarez-Hess et al., 2024). However, in vivo studies with dairy cows have also reported reductions in feed intake and milk production (Roque et al., 2019; Stefenoni et al., 2021), abnormalities or damage to the rumen wall (Muizelaar et al., 2021), and transfers of bromoform metabolites into milk (Stefenoni et al., 2021; Krizsan et al., 2023) when fed Asparagopsis spp. Moreover, concerns regarding the contribution of Asparagopsis spp. cultivation to the depletion of the ozone layer due to the emission of bromoform have also been raised (Jia et al., 2022). Interestingly, Mihaila et al. (2022) reported that another red seaweed, Bonnemaisonia hamifera, reduced in vitro CH₄ production by almost 96% at an inclusion level of 6% of OM, and in contrast to Asparagopsis armata, bromoform was not detectable in B. hamifera (Mihaila et al., 2022). The exact nature of the antimethanogenic bioactive compounds in B. hamifera is unknown, but Guinguina et al. (2023) speculated that 1,3,3-tetrabromo-2-heptanone and 1-iodo-3,3-dibromo-2-heptanone could be among the compounds as they are known to possess antibacterial and antimicrobial properties (Siuda et al., 1975; Nylund et al., 2008; Enge et al., 2012). To our knowledge, B. hamifera has never been evaluated in vivo as an antimethanogenic feed additive in ruminants, and hence, the objective of the current pilot study was to investigate the effects of increasing in practice applicable doses (0%–1% on a DM basis) of the red seaweed B. hamifera on the enteric CH₄ emissions from dairy cows. It was hypothesized that increasing levels of B. hamifera would result in decreasing enteric CH₄ emissions from dairy cows.

The experiment was conducted at Aarhus University, AU Viborg–Research Centre Foulum, Denmark, under a license from the Danish Animal Experiments Inspectorate. The experiment was planned under consideration of the ARRIVE Guidelines (Percie du Sert et al., 2020).

Four lactating Danish Holstein cows (2 first parity and 2 fifth parity) were included in a 4 × 4 Latin square design study. During the 4 periods, the cows received (1) basal control (CON; diet without any seaweed), (2) basal diet with 0.33% B. hamifera on a DM basis (LO), (3) basal diet with 0.66% B. hamifera on a DM basis (MED), or (4) basal diet with 1% B. hamifera on a DM basis (HI; Table 1). Each of the 4 experimental periods lasted 1 wk with 4 d of adaptation to the diet followed by 3 d of gas exchange measurements using respiration chambers. All cows were fed the CON diet in a pre-period (3 d) before the first period. At the initiation of the experiment, milk yield was 25.7 ± 12.0 kg/d (average ± SD), DIM was 209 ± 14.0 d, BCS was 3.0 ± 0.20 on a scale from 1 to 5, and BW was 685 ± 79.6 kg. The cows were housed in individual pens (400 × 450 cm) with slatted floor and a cubicle bed with a mattress and sawdust during adaptation periods. The cows had free access to water throughout the experiment.

Table 1.

Dietary and chemical composition (means ± SD; % of DM unless otherwise stated) of the diet in the pre-period and a control diet without any seaweed and the same diet diluted with 3 different levels of the red seaweed Bonnemaisonia hamifera

Item	Treatment1
	PRE	CON	LO	MED	HI
Dietary composition
Spring barley	7.52	7.52	7.50	7.47	7.45
Rapeseed meals, 4% fat	17.5	17.5	17.5	17.4	17.4
Rapeseed cakes, 10.5% fat	4.18	4.18	4.16	4.15	4.14
Sugar beet pulp, rolled	7.10	7.10	7.08	7.06	7.03
Sugar beet molasses	4.18	4.18	4.16	4.15	4.14
Maize silage, silo 11–22	29.2	29.2	29.1	29.1	29.0
First cut grass/clover silage	14.6	14.6	14.6	14.5	14.5
Second regrowth grass/clover silage	14.2	14.2	14.2	14.1	14.1
Limestone	0.084	0.084	0.083	0.083	0.083
Sodium bicarbonate	0.627	0.627	0.625	0.623	0.620
Sodium chloride	0.125	0.125	0.125	0.125	0.124
Mineral premix2	0.418	0.418	0.416	0.415	0.414
Vitamins	0.146	0.146	0.146	0.145	0.145
Bonnemaisonia hamifera	0.00	0.00	0.33	0.66	1.00
Chemical composition
DM (% of fresh feed)	43.2	44.5 ± 0.49	44.5 ± 0.52	44.7 ± 0.46	45.1 ± 0.53
Ash	7.23	7.35 ± 0.14	7.37 ± 0.05	7.43 ± 0.07	7.40 ± 0.05
CP	16.1	15.8 ± 0.59	16.0 ± 0.51	16.4 ± 0.33	15.9 ± 0.24
Crude fat	3.90	3.40 ± 0.18	3.48 ± 0.26	3.40 ± 0.26	3.43 ± 0.17
aNDFom3	32.4	32.4 ± 0.73	32.7 ± 0.25	32.5 ± 0.04	32.2 ± 0.62
Starch	14.8	14.3 ± 0.71	15.3 ± 1.04	15.0 ± 0.79	15.6 ± 1.26
NEL204 (MJ/kg of DM)	6.46	6.46	—	—	—

¹PRE = diet in pre-period; CON = control diet; LO = 0.33% B. hamifera; MED = 0.66% B. hamifera; HI = 1% B. hamifera on a DM basis.

²Vilofoss Komix Type 3, declared macromineral composition (g/kg DM): Ca = 147, Mg = 141, Na = 116, S = 1. Added vitamins and microminerals (per kg DM): vitamin A = 600,000.10 IU, vitamin D₃ = 190,000.10 IU, vitamin E = 4,000 IU, Mn = 4,000 mg, Cu = 1,500 mg, Zn = 4,500 mg, Co = 25 mg, Se = 50 mg in combination with Vilofoss Suplex ADE, analyzed or declared macromineral composition (g/kg DM): Ca = 139, Mg = 91, Na = 95. Added vitamins and microminerals (per kg DM): vitamin A = 900,000 IU, vitamin D₃ = 200,000 IU, vitamin E = 2,000 IU, Se = 50 mg.

³aNDFom = NDF determined on an OM basis.

⁴Net energy for lactation at 20 kg DMI/d, calculated according to NorFor (Volden, 2011).

Total mixed rations were prepared once a day and fed to the cows on an ad libitum basis with approximately 40% of the daily ration at 0615 h and 60% at 1600 h. The composition of TMR is shown in Table 1. Rations were formulated according to the Nordic Feed Evaluation System (Norfor; Volden, 2011) with an expected milk yield of 10,500 kg ECM per year. The roughage:concentrate ratio (on a DM basis) was 58:42 for all diets. Net energy for lactation in CON was calculated according to NorFor (Volden, 2011). Contents of crude fat, protein, starch, ash, and NDF on an OM basis (aNDFom) were determined as described by Thorsteinsson et al. (2023).

Bonnemaisonia hamifera was continuously cultivated in a land-based system by Maripure ApS (Aalborg, Denmark) throughout the experiment. After harvest, the biomass was freeze-dried and ground to a particle size of 2 to 4 mm and delivered in 4 different batches. Bromoform was analyzed in seaweed biomass using SIM mode on a GC-MS with a detection level of 1 µg of bromoform/g of DM as described by Nørskov et al. (2021). Across biomass batches, contents of ash were 182.4 ± 10.5 g/kg DM and bromoform 0.101 ± 0.027 mg/g DM.

Allocated feed and refusals were weighed daily throughout the experiment to determine feed intake, whereas DM in feed and refusals was measured from d 5 to 7 in each period by drying at 60°C for 48 h (Åkerlind et al., 2011). The DMI was calculated as the amount of DM in refusals subtracted from DM offered. The cows were milked twice daily at 0515 and 1615 h. Milk yield was recorded by continuous flowmeters at every milking throughout the experiment, while milk was analyzed for fat, protein, lactose monohydrate, and urea on d 5 to 7 in each period using mid-infrared reflection (MilkoScan 7 RM, Eurofins Steins Laboratorium A/S, Vejen, Denmark). Milk yield expressed in ECM (3.140 MJ/kg) was calculated as ECM (kg) = milk yield × [(38.3 × fat + 24.2 × protein + 15.71 × lactose + 20.7)/3,140], with ECM and milk yield in kilograms; and fat, protein, and lactose monohydrate in grams per kilogram.

Four individual transparent respiration chambers based on open-circuit indirect calorimetry were used for measurement of gas exchange on d 5 to 7 in each period. The chambers were modified versions of Hellwing et al. (2012), and were operated as described by Thorsteinsson et al. (2023). The cows were assigned to the same specific respiration chamber throughout the experiment. Before, during, and after the experiment, recovery tests (n = 17 for CO₂ and n = 18 for CH₄) were performed by infusing a known amount of pure CO₂ and CH₄ into the chambers and comparing it with the amount of gas measured by the system. Across chambers, average recovery values ± SD were 98.6 ± 1.66% for CO₂ and 99.8 ± 1.84% for CH₄. Recovery tests were used to correct the measured gas concentrations. The average of CH₄ and CO₂ recoveries was used to correct O₂ and H₂. Gas exchange was measured as flows at standard temperature and pressure (STP; 0°C/273.15 K and 101.325 kPa) and subsequently converted into grams per day as described by Thorsteinsson et al. (2023). The respiratory coefficient was calculated as the ratio between CO₂ produced and O₂ consumed (L/L), and gas yield and intensity were calculated based on the DMI and ECM yield, respectively, during each chamber period.

All statistical analyses were conducted in R v.4.3.2 (R Core Team, 2024). For all parameters displayed in tables, the observations were averaged within cows and periods, resulting in a dataset with 16 observations; however, due to an unexpected lower growth rate of B. hamifera in the final stage of the experiment and hence lack of biomass delivered in the fourth batch, the cow initially allocated to the MED diet in period 4 was fed CON instead. The effect of the seaweed inclusion on the various animal responses was analyzed with the following linear mixed model fitted:

Y_dpc = μ + α_d + γ_p + A_c + Ε_dpc,

where Y_dpc is the dependent response variable, μ is the overall mean, α is the fixed effect of diet (d = CON, LO, MED, or HI), γ is the fixed effect of period (p = 1 to 4), A is the random effect of cow (c = 1 to 4), and Ε_dpc is the random residual error assumed to be independent with constant variance and normally distributed.

To obtain diurnal patterns of CH₄ and H₂, hourly emissions of the gases were averaged over the 3-d measurement period within cow and period, resulting in 24 observations per cow per period. The data were analyzed with the following model:

Y_dhpc = μ + α_d + τ_h + α_t × τ_h + γ_p + A_c + Ε_dhpc,

where Y_dhpc is the dependent response variable, μ is the overall mean, α is fixed effect of treatment (d = CON, LO, MED, or HI), τ is the fixed effect of hour (h = 0 to 23), α_t × τ_h is the interaction, γ is the fixed effect of period (p = 1 to 4), A is the random effect of cow (c = 1 to 4), and Ε_dhpc is the random residual error assumed to be independent with constant variance and normally distributed. Data were analyzed as repeated measurements using a first-order autoregressive covariance structure with heterogeneous variance (AR1).

All data were evaluated for normality and homogeneity of the variance during the statistical analysis. Data are presented in tables as estimated marginal means and SEM. Differences between estimated marginal means were evaluated using Tukey's method for comparison. Statistical significance was declared when P ≤ 0.05 and statistical tendencies were declared when 0.05 < P ≤ 0.10.

To the best of our knowledge, this is the first in vivo study evaluating the enteric CH₄-mitigating potential of the red seaweed species B. hamifera. We are not aware of the existence of any large-scale commercial cultivation or harvest of B. hamifera anywhere in the world, and due to limited production capacity in a quite newly established Danish land-based facility, we were limited to a duration of 7 d of the individual experimental periods in the present pilot study. The CH₄-mitigating potential of feed additives is often overestimated in vitro versus obtained reductions in vivo (Honan et al., 2022). Therefore, despite the short duration of experimental periods, this first in vivo experiment provides a far better foundation for the estimation of the antimethanogenic potential of B. hamifera than in vitro trials, which would have been the alternative with the seaweed supply limitations. Nonetheless, it is important to stress that only the short-term effects have been evaluated and will be discussed in the following sections, and that larger and longer-term in vivo trials should be undertaken.

Increasing inclusion levels of B. hamifera linearly decreased CH₄ emissions with up to 13.3% reduction in daily CH₄ production (g/d), 15.7% reduction in CH₄ yield (g/kg DMI), and 16.4% reduction in CH₄ intensity (g/kg ECM; Table 2). A similar dose-dependent response of B. hamifera on CH₄ production has been reported in vitro by Mihaila et al. (2022) and Guinguina et al. (2023). From Figure 1, it can be observed that the lower daily CH₄ production, yield, and intensity observed on the HI diet compared with CON was caused by a generally lower production throughout the day. In alignment with studies investigating Asparagopsis spp. as antimethanogenic feed additives (Roque et al., 2019; Stefenoni et al., 2021; Krizsan et al., 2023), reductions in CH₄ emissions also resulted in increased H₂ emissions in the current study. Hence, HI resulted in a 331% increase in daily H₂ emission (g/d), 325% increase in H₂ yield (g/kg DMI), and a 324% increase in H₂ intensity (g/kg ECM; (Table 2).

Table 2.

Dry matter intake and gas exchange of dairy cows fed increasing amounts of the red seaweed Bonnemaisonia hamifera

Item	Treatment1				SEM2	P-value
	CON	LO	MED	HI		Treatment	Linear
DMI (kg/d)	23.1	23.5	23.4	23.7	1.72	0.63	0.30
Milk (kg)	28.2	29.1	29.6	29.1	4.72		0.22
ECM (kg/d)	27.1	28.1	28.6	27.8	4.23		0.22
Gas exchange (g/d)
CO2	15,371	15,283	15,263	15,387	915	0.90	0.96
O2	10,274	10,270	10,267	10,326	569	0.96	0.72
CH4	384a	362ab	348ab	333b	34.7	0.04	<0.01
H2	1.79b	2.29b	4.24ab	7.71a	1.51	0.03	<0.01
Respiration coefficient3	1.09	1.08	1.08	1.08	0.0152	0.55	0.34
Gas yield (g/kg DMI)
CO2	667	652	656	652	12.9	0.32	0.19
O2	447	439	442	438	14.0	0.94	0.77
CH4	16.6a	15.5ab	14.8ab	14.0b	0.930	0.01	<0.01
H2	0.0789b	0.0983ab	0.190ab	0.335a	0.0611	0.04	<0.01
Gas intensity (g/kg ECM)
CO2	587	568	561	572	51.0	0.58	0.39
O2	393	382	377	385	35.2	0.74	0.49
CH4	14.6a	13.4ab	12.7ab	12.2b	1.38	0.02	<0.01
H2	0.0715b	0.0873ab	0.163ab	0.303a	0.0652	0.04	<0.01

^a,bValues within the same line with different superscripts differ (P < 0.05).

¹CON = control diet; LO = 0.33% B. hamifera; MED = 0.66% B. hamifera; HI = 1% B. hamifera on a DM basis.

²Largest SEM is reported.

³Calculated as ratio between CO₂:O₂ (L/L).

Figure 1.

(a) Estimated hourly CH₄ emission from dairy cows fed increasing amounts of Bonnemaisonia hamifera (mean ± SEM). (b) Estimated hourly H₂ emission from dairy cows fed increasing amounts of B. hamifera. CON = control diet; LO = 0.33% B. hamifera; MED = 0.66% B. hamifera; HI = 1% B. hamifera on a DM basis. Each time point represents the average CH₄ or H₂ production from half an hour before to half an hour after that time (i.e., time point 0630 h represents the average gas production from 0600 to 0700 h). Different letters shown below the figures for a given time point indicate significantly different values between treatments at P < 0.05.

Studies with Asparagopsis spp. have demonstrated reductions in CH₄ yield from dairy cows ranging from 43% to 54% at an inclusion level of up to 1% on an OM basis (Roque et al., 2019; Krizsan et al., 2023). In the current study, HI (1% on a DM basis) corresponds to approximately 0.9% OM inclusion rate of B. hamifera. Thus, B. hamifera appears to be a less potent seaweed compared with Asparagopsis spp. in terms of reducing enteric CH₄ emission from dairy cows. The exact nature of the antimethanogenic compounds in B. hamifera is yet to be resolved; however, the concentration of bromoform in B. hamifera in the current study was low compared with reported levels in studies evaluating Asparagopsis spp. as CH₄ inhibitor in dairy cows (Roque et al., 2019; Muizelaar et al., 2021). Hence, reported bromoform concentrations in Asparagopsis spp. across studies have been around 1.29 ± 0.04 mg/g DM (Roque et al., 2019; Muizelaar et al., 2021) in contrast to 0.101 ± 0.027 mg/g DM in B. hamifera biomass in the present study. This might explain the lower CH₄-mitigating potential of B. hamifera compared with observed reductions in studies with Asparagopsis spp., but it should be noted that the difference between reduction potential is not proportional with bromoform concentration, indicating that other antimethanogenic compounds may be present in B. hamifera. Untargeted metabolomic analysis followed by in vitro fermentation studies should be undertaken to further identify and validate the antimethanogenic compounds in B. hamifera. Bromoform is categorized as a potential human carcinogen by the US Environmental Protection Agency (2000), and thus, as discussed by Wasson et al. (2022), concerns could be raised regarding the long-term impact of feeding Asparagopsis spp. on animal health as dairy cows persist within a herd for several years. Therefore, B. hamifera could potentially be a safer candidate for a seaweed-based CH₄-mitigating feed additive in both in relation to animal and possibly also consumer health than Asparagopsis spp.

The inclusion of the red seaweed Bonnemaisonia hamifera in diets of dairy cows in this preliminary study resulted in dose-dependent reductions of daily CH₄ production, CH₄ yield, and CH₄ intensity amounting to 13% to 16% reduction at the highest inclusion (1% in dietary DM) compared with the control diet. The concentration of the potentially harmful compound, bromoform, was remarkably lower in B. hamifera compared with previously reported concentrations in the antimethanogenic red seaweed, Asparagopsis taxiformis. Thus, it is highly relevant to undertake larger and longer-term in vivo trials to be able to fully investigate the potential of B. hamifera as a CH₄ inhibitor in cattle.