# The anti-methanogenic efficacy of Asparagopsis armata: Could it be attributable solely to its bromoform content? Eslam Ahmed a,b,* , Takehiro Nishida a a,b,* a Eslam Ahmed, Takehiro Nishida a*Department of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro 080-8555, Japan* b*Department of Animal Behavior and Management, Faculty of Veterinary Medicine, South Valley University, Qena 83523, Egypt* ## ARTICLE ## INFO *Keywords:* Feed additive Halogenated compounds *In vitro* fermentation Methane mitigation Red seaweed Ruminants ## ABSTRACT The seaweed *Asparagopsis armata* has been shown to be a successful intervention to mitigate methane (CH4) emissions from ruminants due to the content of halogenated components, including bromoform. Some groups have been exploring extracting or synthesizing bromoform to be used as a feed additive rather than using whole seaweed biomass. This study was conducted to understand the differences between whole *A. armata* biomass and bromoform in their ability to reduce CH4 production when included in ruminant diets. A series of *in vitro* experiments were conducted, with a control group consisting of a 50 % hay:50 % concentrate. *A. armata* biomass supplementation levels of 0.25 and 0.50 %, containing 0.0037 and 0.0077 mg bromoform on a dry matter (DM) basis, respectively, were evaluated. Bromoform was evaluated at 0.0018, 0.0036, and 0.0073 mg. The two highest levels of bromoform were comparable to the bromoform content in the seaweed biomass. Adding *A. armata* biomass led to significant reductions in CH4 yield (mL/g) of 62.5 and 95.6 %, respectively, without compromising *in vitro* fermentation characteristics. The addition of bromoform resulted in a decrease in the CH4 yield (mL/g) by 7.6, 23.0, and 59.6 %, respectively. In conclusion, while bromoform led to a dose-dependent reduction in CH4 production, *A. armata* has a greater effect at equivalent doses of bromoform, suggesting that the other components in the biomass, which were responsible for up to 40 % of the reduction, play a significant role in the anti-methanogenic efficiency of *A. armata*. ## 1. Introduction It is estimated that about 30 % of anthropogenic CH4 emissions are produced by ruminants (Jackson et al., 2020). According to the International Panel on Climate Change (IPCC, 2014), CH4 has up to 80 times more warming potential than carbon dioxide (CO2) over a 20-year period; therefore, research on reducing greenhouse gas emissions, particularly CH4, is increasing. Since 2021, more than 150 countries worldwide have committed to a global pledge to reduce CH4 emissions by 30 % by 2030 as a measure to combat climate change (Malley et al., 2023). Given that the largest source of anthropogenic CH4 is agriculture, more specifically enteric CH4, there has been heightened interest in exploring methods to safely reduce CH4 emissions from cattle (Smith et al., 2021). Seaweeds are gaining increasing public interest and media attention as a potential strategy to reduce enteric CH4 emissions from ruminants. Among these seaweeds, the genus *Asparagopsis* with species *A. taxiformis* and *A. armata* stand out for their potent efficacy in inhibiting methanogenesis from sheep and cattle by over 80 % with a supplementing level of 0.5–2 % on a DM basis of the basal diet (Bhowmick and Hayes, 2023). This effect has been attributed to the volatile halogenated compounds in *Asparagopsis,* particularly bromoform. ## 2. Materials and methods ### 2.1. Basal diet and tested materials The basal substrate for the *in vitro* incubations was composed of Kleingrass (*Panicum coloratum*) hay and a commercial concentrate mixture ground at 1 mm. The AA biomass at the gametophyte stage was harvested from Rakiura Big Glory Bay, New Zealand. The material was visually inspected for contaminants, which were manually removed. The seaweed biomass was washed and dewatered before blast freezing at -18 ◦C, then delivered via refrigerated transport. The biomass was freeze-dried and stored at -18 ◦C until being used for the *in vitro* trials. A sample of the ground AA biomass was analyzed for BF content at Zukosha Co., Ltd., Obihiro, Japan. ### 2.2. Rumen fluid collection, experimental design, and in vitro incubation Rumen fluid was collected from two ruminally-fistulated, non-lactating Holstein cows approximately 8 years old with an average weight of 894 kg. Cows were fed at the maintenance level on a diet of orchard grass (*Dactylis glomerata*) hay. About 1.5 L of rumen fluid was collected and immediately transferred to the laboratory. An *in vitro* batch culture with six experimental treatments was conducted. The control group was composed of a basal diet consisting of a 50 % Kleingrass hay and 50 % concentrate mixture. ### 2.3. Chemical analyses The chemical compositions of the basal diet and the AA biomass were conducted according to AOAC standard procedures. The DM content was determined by drying the samples in an air-forced oven. The ether extract (EE) was determined according to method 920.39. The chemical compositions of the tested materials are presented in Table 1. ### 2.4. Data analysis Data were analyzed using SAS statistical software version 9.4. The model regarded the treatments as a fixed effect and statistical significance was determined for differences at a *P*-value of less than 0.05. ## 3. Results and discussion Recently, seaweed, and specifically *Asparagopsis* spp., has emerged as a promising approach for mitigating enteric emissions from ruminants due to its structure that allows the accumulation of halogenated volatile organic compounds. The major component in the AA biomass is BF. The current study demonstrated that the tested AA biomass was effective in decreasing CH4 yield at supplementing levels of 0.25 and 0.50 % by 62.5 % and 95.6 %, respectively. While the tested BF levels led to a reduction of CH4 yield by 23.0 and 59.6 %, respectively. This significant reduction could be attributed to the role of active compounds in the biomass that may act synergistically to increase the effectiveness in reducing the formation of CH4. ## 4. Conclusions To our knowledge, this *in vitro* study is the first to provide insights on the difference in CH4-reduction potential when whole AA biomass and pure BF were used as feed additives for ruminants. Adding whole AA seaweed biomass at 0.25 and 0.50 % decreased CH4 emissions by 62.5 and 95.6 %, respectively, without any adverse impacts on *in vitro* fermentation characteristics. Therefore, the use of the whole biomass with natural bioactive compounds to achieve effective CH4 reduction is preferred over increasing the dosage of BF.