Research on enteric methane mitigation in Chile: Ten years of scientific insights

Abstract

Research on enteric methane abatement in Chile began approximately ten years ago and has steadily accelerated. A key challenge in developing strategies to decrease enteric methane emissions in Chile and other countries where ruminant production is largely pasture-based is that globally, most mitigation strategies have been developed and evaluated with confined ruminants fed total mixed rations, requiring adaptation and validation in pasture-based systems. In this review, we discuss in vitro and in vivo applied research of anti-methanogenic strategies conducted in Chile. The reviewed studies included the evaluation of grazing management, different genotypes and species of forages, seasonal forage crops, by-products, oilseeds, and algae supplementation, as well as the use of a chemical inhibitor of methanogenesis, 3-nitrooxypropanol, and the combination of more than one anti-methanogenic strategy. Global meta-analyses and reviews show that intensification of ruminant production consistently decreases methane output per unit of meat or milk and is often economically attractive to producers and acceptable to governments and consumers alike; however, it is generally associated with increased emissions of other greenhouse gases and can result in greater output of carbon dioxide equivalents. Results obtained in Chile with production intensification varied depending on the intensification strategy. We consider that at present, none of the anti-methanogenic strategies investigated thus far in Chile can be recommended for adoption in pasture-based systems. Important aspects to be addressed are the lack of published life cycle assessments, in vivo research in ruminants other than dairy cows, and a considerable proportion of research results available only as conference summaries. Efforts are ongoing to generate solutions to effectively and persistently decrease the emissions of greenhouse gases from Chilean ruminant livestock; however, more research is required to design, adapt, and evaluate enteric methane mitigation strategies applicable to pasture-based systems, which require greater funding from the public and private sectors.

Introduction

Increasing emissions of anthropogenic greenhouse gases (mainly carbon dioxide (CO₂), methane (CH₄), and nitrous oxide (N₂O)) have caused the average planetary temperature to rise by 1.1 °C in the 2011 - 2021 decade with respect to the 1850 - 1900 period, triggering extreme climatic events that negatively affect both natural systems and human populations worldwide (IPCC, 2023). There are currently ongoing efforts to limit the anthropogenic emissions of the main greenhouse gases to cap global warming to 1.5 °C with no or limited overshoot with respect to preindustrial temperatures to avoid the worst consequences of climate change in terms of frequency and severity of extreme climatic events, loss of ecosystems, food insecurity, negative impacts on human health, and social and economic disruptions (Hoegh-Guldberg et al., 2018; IPCC, 2023). Compared to CO₂, CH₄ traps 28 times more heat in a 100-year period and has a much shorter lifetime in the atmosphere (Szopa et al., 2021). Therefore, mitigating CH₄ emissions offers an opportunity to ameliorate global warming in the short term (IPCC, 2021; United Nations Environment Programme and Climate and Clean Air Coalition, 2021). However, the rate of increase in atmospheric CH₄ concentration has been rapidly accelerating since 2006, with a record in 2021 (Shindell et al., 2024). In a concerted effort, 158 countries, including Chile, have agreed to decrease global CH₄ emissions by at least 30% by 2030, relative to 2020 levels (Global Methane Pledge, 2024).

Approximately one-third of anthropogenic CH₄ emissions originate from the rumen of domestic ruminants as a consequence of microbial digestion and fermentation of ingested feed, that is, enteric CH₄, which is released to the atmosphere mainly through eructation and respiration (Szopa et al., 2021; United Nations Environment Programme and Climate and Clean Air Coalition, 2021). Whilst on the one hand it is highly important to decrease CH₄ emissions from ruminants, the global demand for livestock products, including ruminants meat and milk, is projected to continue to increase by 2050, particularly in developing economies (FAO, 2023). The challenge of at the same time increasing ruminant production to satisfy human demands and decreasing enteric CH₄ emissions has prompted a global research effort to find solutions that can decrease CH₄ production in the rumen without negatively affecting ruminant production (Ungerfeld, 2022).

As the challenge of mitigating enteric CH₄ emissions from ruminant production has been gaining global attention (Beauchemin et al., 2020), Chile has been no exception. In vitro and in vivo studies to evaluate applied solutions to mitigate enteric CH₄ in Chilean ruminant production systems began at Instituto de Investigaciones Agropecuarias (INIA), Centro Regional de Investigación Remehue, Universidad Austral de Chile, and Universidad de Concepción in the early 2010s. A key challenge to develop strategies to control enteric CH₄ emissions in Chile and other countries where most ruminant production is pasture-based is that most enteric CH₄ mitigation strategies, although not all, have been designed and developed for confined ruminants fed total mixed rations (Vargas et al., 2022). It has been estimated that 27.5% of global emissions of enteric CH₄ are generated by ruminants in pasture-based systems, 70.3% come from mixed systems, and only 2.19% come from feedlots (FAO, 2022). However, up to 2020, only 7% of scientific papers on enteric CH₄ mitigation corresponded to studies in pasture-based systems, compared to 39% with confined ruminants (with the balance corresponding to in vitro studies, literature reviews, meta-analyses, and others) (Vargas et al., 2022).

In this review, we present and discuss in vitro and in vivo applied research conducted in Chile for the evaluation and development of anti-methanogenic strategies that can potentially be adopted by Chilean producers. We identify and discuss knowledge gaps and the applied research that we think needs to be conducted to develop practical solutions to mitigate enteric CH₄ in Chile. Basic research on rumen biochemistry and microbiology related to the fundamental aspects of CH₄ production in the rumen and its inhibition has also been conducted in Chile (for example, Ungerfeld (2013); Ungerfeld (2015); Ungerfeld et al. (2024)). Because this fundamental research is not specific to Chilean production systems, which are the focus of this review, we do not discuss this work herein.

As in any area of science, applied research on enteric CH₄ mitigation must meet rigorous scientific standards to provide society with credible knowledge. This ensures that governments, industry, the public, and other decision-makers can engage in informed discussions about the application of enteric CH₄ mitigation options to ruminant production. Claims of enteric CH₄ mitigation should only be seriously considered and discussed if published in peer-reviewed scientific journals to ensure scientific quality and credibility. Therefore, this review is primarily based on scientific papers on applied research on enteric CH₄ mitigation conducted in Chile. We also present and discuss conference papers as non-peer reviewed, preliminary information with the purpose of enlarging the national body of research results, which otherwise might be considered somewhat constrained to merit a review. However, we are cautious about non-peer-reviewed information, noting its incompleteness, and emphasize the importance of publishing research results in peer-reviewed journals. In this review, we do not discuss technical bulletins or claims appearing in commercial brochures, webpages, or outreach presentations.

Research on enteric methane mitigation in chile: general aspects

Research on the abatement of CH₄ emissions from ruminants has been increasing at an accelerating pace over the last two decades owing to growing concerns about the role of CH₄ emissions in global warming (Beauchemin et al., 2020). Several strategies for decreasing enteric CH₄ emissions are currently being investigated worldwide. These include supplementation with concentrates and other feedstuffs, improving forage quality, supplementation with oils and oilseeds, breeding for feed efficiency, use of legumes, supplementation with phytochemicals, supplementation with plants and algae containing secondary compounds, ionophores, inhibitors of CH₄ production, genetic selection of ruminants producing less CH₄, and vaccination against rumen methanogens (Beauchemin et al., 2022).

In Chile, research on enteric CH₄ mitigation strategies began at INIA Remehue in 2012 by setting up the sulfur hexafluoride (SF₆) tracer gas technique to determine CH₄ emissions in grazing dairy cows (Figure 1). The canisters and holsters used in this technique were later shown to cause minimal alterations in dairy cow behavior (Pereira et al., 2021). Research papers evaluating CH₄ emissions by scientists in Chile span from 2014 to 2025, with 21 studies conducted in vitro and 14 in vivo (Figure 2). There is a clear trend towards increasing research output, rising from one or two studies per year published in peer-reviewed journals or conference abstracts between 2014 and 2021 to three or more in 2022 and 2024, with the exception of 2023, where only one in vivo study was reported.

Figure 1. Representation of the SF₆ gas tracer technique for measuring methane emissions from dairy cows. 1) Cows receive orally a pre-calibrated SF₆ permeation tube with a known release rate; 2) Exhaled gas is captured though a sampling unit consisting of an evacuated V-shaped PVC canister positioned on the cows’ neck connected to a sampling line with a restricted flow continually sampling eructed and exhaled gases from near to the cow’s muzzle; 3) Evacuated PVC canisters allow collection of a sample of gases emitted in a 24-h period and are replaced daily for a minimum of 5 d; 4) Canisters are pressurized to above atmospheric pressure with nitrogen, left to rest for at least 1 h, and then four subsamples of gas collected in canisters are obtained in chromatography vials; 5) Samples are analyzed in duplicate by gas chromatography; 6) Methane emissions are calculated as the product of the SF₆ permeation tube release rate and the ratio of CH₄:SF₆ concentration in samples, adjusted for background gas concentrations.

Figure 2. Total number of publications on applied in vivo and in vitro studies on enteric methane mitigation in Chile, including peer-reviewed scientific papers and conference proceedings.

The drop in 2023 reflects the fact that, as all research is externally funded, the number of studies conducted and papers published can vary from year to year depending on the number of research projects funded and experiments being executed, independently of overall longer-term tendencies. Six institutions have been involved in this area of research; applied in vitro studies have been conducted primarily at Universidad de Concepción (57.2%), followed by INIA Remehue (19.0%) and Carillanca (14.3%), and Universidad Austral de Chile (9.52%). In contrast, in vivo research has been predominantly conducted by INIA Remehue (71.4%), with additional contributions from Universidad Austral de Chile (14.3%), Pontificia Universidad Católica (7.1%), and Universidad de Los Lagos (7.1%). Notably, all in vivo studies in which CH₄ production was measured were conducted exclusively in dairy cattle, highlighting a gap in research with other domestic ruminants. From a regional standpoint, 92.9% of the in vivo research was conducted in the center-south of the country under a humid and cool climate, and only 7.1% was conducted in the central region with a Mediterranean climate. This would not create a bias for research conducted to date with dairy cows, as most dairy cows and milk production are concentrated in the Los Ríos and Los Lagos regions in the center-south region of the country (ODEPA, 2024).

Of the in vivo studies, nine were conducted under confined conditions, four under pasture-based conditions, and in one case, information was not available. In 13 of 14 in vivo studies, CH₄ emissions were measured using the SF₆ technique, with the remaining study using a GreenFeed unit (C-Lock Inc.). The establishment and widespread use of the SF₆ technique responds to its affordability and suitability for measuring CH₄ emissions from grazing ruminants (Hammond et al., 2016; Hristov et al., 2025). Most importantly, only 57.1% of the studies were published in peer-reviewed scientific journals, with the remaining 42.9% available only as conference proceedings, most of them available at the national level only, and published in Spanish (86.7%). Various strategies to mitigate enteric CH₄ emissions have been investigated in Chile, including production intensification through dietary modifications, type of forage, supplementation of oilseeds, by-products and their extracts, supplementation of algae, and chemical inhibitors of CH₄ production. In the following sections, we present and discuss the research results grouped by type of anti-methanogenic strategy. In order to clearly distinguish preliminary findings available as conference summaries from peer-reviewed papers published in scientific journals, only peer-reviewed papers are listed in Tables 1 and 2.

Intensification through dietary modifications

Intensifying ruminant production through improvements in nutrition, management, genetics, and health decreases the output of CH₄ per kilogram of ruminant meat and milk, that is, CH₄ intensity (Beauchemin et al., 2020). This is largely because of the dilution of the maintenance requirements (Capper, 2011). However, intensification usually increases the total CH₄ emissions per animal (Arndt et al., 2022; Beauchemin et al., 2022). If the decrease in CH₄ emissions per kilogram of meat or milk of ruminant products is accompanied by a larger increase in the total output of meat or milk, the total enteric CH₄ emissions will increase (Leahy et al., 2020). Chang et al. (2021) showed that, despite a decrease in global CH₄ intensity, global absolute emissions of enteric CH₄ increased between 2000 and 2018, with regional increases occurring in South Asia, Latin America and the Caribbean, Sub-Saharan Africa, North Africa, and the Near East. Furthermore, intensification most often increases upstream emissions of CO₂ and N₂O from feed production and downstream emissions of manure CH₄ and N₂O, as more manure is produced (Beauchemin et al., 2022). Therefore, the overall effects of anti-methanogenic interventions on the entire production system must be evaluated by modelling life-cycle assessments (LCA) (Beauchemin et al., 2020; Beauchemin et al., 2022).

An examination of case studies on beef, lamb, and milk production in nine countries and regions revealed that production intensification consistently decreased the output of CO₂ equivalents (CO₂e) per kilogram of ruminant meat or milk, but changes in the total output of CO₂e were variable (Ungerfeld et al., 2022). In agreement with global trends, it is estimated that CO₂e emission intensity in Chile has consistently decreased for beef, lamb, and milk production between 2004 and 2021 along with increasing production intensification (Figure 3); FAO (2024). Due to a steady decrease in total cattle numbers coupled to the decrease in CO₂e emissions intensity that has been taking place, total CO₂e emissions from beef and dairy cattle decreased steadily in Chile between 2000 and 2021, stabilizing in 2022 (Figure 4) data from Basoa et al.(2024). The decrease in the total cattle numbers is, in turn, attributed to declining profits (Basoa et al., 2024).

Figure 3. Evolution of the emissions of CO₂e per kilogram of beef, lamb, and milk (CO₂e intensity) in Chile between 2004 and 2021. Data are from FAO (2024).

Figure 4. Evolution of total emissions of CO₂e from cattle (beef and dairy) in Chile between 1990 and 2022. Data are from Basoa et al.(2024) .

In the first in vivo study on enteric CH₄ mitigation conducted in Chile, Muñoz et al.(2015) compared supplementing dairy cows in early lactation grazing perennial ryegrass in spring with 1 or 5 kg/d of a commercial concentrate. They found that intensifying production by boosting the amount of concentrate offered to dairy cows increased milk production, although CH₄ yield (CH₄ emitted per kilogram of dry matter intake, DMI) and CH₄ intensity remained unchanged, and total emissions of CH₄ increased (Table 1). A meta-analysis by Arndt et al.(2022) reported no effects of supplementing concentrates to grazing ruminants on total CH₄ emissions or CH₄ yield, and a tendency towards decreased CH₄ intensity. It is difficult to compare studies evaluating the supplementation of concentrates to grazing dairy cows, as animal responses, especially grass intake, are influenced by the amount and type of concentrate supplemented, and the availability and quality of grass (Pulido et al., 2009). Possibly, high availability of good quality spring grass in the study by Muñoz et al.(2015) resulted in a predominantly substitutive effect of the supplemented concentrate on grass, as evidenced by the lack of effects on total DMI, limiting milk production, and CH₄ intensity. A follow-up study evaluated the response of late-lactation cows fed grass and conserved forage to extra concentrate supplementation (Muñoz et al., 2018). Supplementing with 8 vs. 4 kg/d of concentrate increased total DMI but had no effect on milk yield in late lactation, increased total daily CH₄ emissions by cow, decreased CH₄ yield, and did not affect CH₄ intensity (Table 1). It was concluded that because late-lactation cows respond little in milk production to increased concentrate supplementation, providing extra concentrate is ineffective at decreasing CH₄ intensity in this dairy cow category.

Improving the nutritional quality of grazed grass increases pasture intake and ruminant meat and milk production, thus decreasing CH₄ intensity (Arndt et al., 2022). This is because a greater rate of digestion and lower rumen retention time increases DMI (Allen, 1996) and animal productivity, diluting maintenance requirements (Capper et al., 2009). A study by Muñoz et al.(2016) compared CH₄ production in dairy cows managed under an optimal pre-grazing pasture mass of 2,200 kg/ha with a supra-optimal pre-grazing pasture mass of 5,500 kg DM/ha. Lower pre-grazing pasture mass resulted in higher grass quality, as assessed by lower neutral detergent fiber and higher crude protein (CP) concentration and in vitro digestibility. The higher quality pasture resulted in greater DMI and milk yield and lower CH₄ yield and intensity; however, despite the large difference in pre-grazing pasture mass, the optimal grazing management with lower pasture mass did not mitigate the total daily emissions of CH₄ per cow (Table 1). Likely, similar total CH₄ production occurred because the lower CH₄ yield was compensated by a greater intake of higher quality grass, allowed by lower NDF content and lower rumen filling capacity (Allen, 1996), resulting in greater fermentation and more CH₄. This result agrees with the meta-analysis by Arndt et al.(2022), who found that increasing grass quality decreased CH₄ yield and intensity, but resulted in a numerical increase in absolute CH₄ emissions. Importantly, the higher quality pasture had more nitrogen (N) at a level that exceeded dairy cows’ requirements, as reflected by increased milk urea N (Muñoz et al., 2016). Greater N content in the pasture, along with greater grass digestibility, can result in greater rates of ammonium release in the rumen and absorption through the rumen wall, and greater elimination of urea in urine (Wallace et al., 1997; Bach et al., 2005). Once in the soil, urea is rapidly hydrolyzed to ammonia. Ammonia oxidation can contaminate water courses as nitrate, and also produces N₂O, a very potent greenhouse gas when released to the atmosphere (Hristov et al., 2013; Arndt et al., 2022).

Table 1. Summary of in vivo research of enteric methane mitigation with dairy cows conducted in Chile published in peer-reviewed papers.

Reference	Type of antimethanogenic strategy	Treatments	DMI§	Methane			Milk production	Comments
				Absolute	Yield	Intensity
Muñoz et al. (2015)	Intensification through increasing concentrate supplementation	1 vs. 5 kg/d concentrate	0†	↑	0	0	↑	High grass availability and quality
Muñoz et al. (2016)	Intensification through improving grass quality	High vs. low pre-grazing pasture mass	↑	0	↓	↓	↑	Higher quality pasture had excess N content
Muñoz et al. (2018)	Intensification through increasing concentrate supplementation	4 vs. 8 kg/d concentrate	↑‡	↑	↓	0	0	Late lactation cows did not respond to extra concentrate
Muñoz et al. (2019)	Oilseeds supplementation	Inert fat vs. unprocessed cottonseed, rapeseed, or linseed in a TMR§	0 or ↓⁋	0 or ↓⁋	0⁋	0⁋	0	Oilseeds affected N excretion and milk fatty acid profile
Enriquez-Hidalgo et al. (2020)	Dietary reformulation	Replacing alfalfa hay and corn silage with fresh ryegrass and berseem clover in a TMR	↓	0	0	0	↓	Lower crude protein in the TMR resulted in less N voided in urine
Muñoz et al. (2021)	Oilseeds supplementation	Long-term effects of unprocessed cottonseed, rapeseed, or linseed in grazing dairy cows	0 or ↓	0	0 or ↓	0	0 or ↓	Cottonseed moderately but not persistently decreased methane
Garcia et al. (2022)	Methanogenesis inhibitor	Supplementation with 3-NOP§ or its carrier with or without urea inclusion	↓	↓	↓	↓	↓	An unintended dietary change affected especially DMI and milk production
Muñoz et al. (2024)	Methanogenesis inhibitor	Supplementation with 3-NOP§ or its carrier with or without cottonseeds	↓	0 or ↓	0	0	0	Time gap between 3-NOP supplementation and grass ingestion caused mild methanogenesis inhibition

†↑ = increase, 0 = no change, ↓ = decrease compared to control treatments; ‡numerical increase, as individual intake was not determined in that study; ⁋In comparison with the control treatment; §Abbreviations: TMR = total mixed ration; 3-NOP = 3-nitrooxypropanol (Bovaer®).

In another study, Muñoz et al.(2022a) evaluated under a 2 × 2 factorial arrangement two pre-grazing pasture masses, combined with 6 kg/d of a corn- or wheat-based concentrate, on enteric CH₄ emissions from dairy cows. They reported that the greatest total CH₄ emissions and CH₄ intensity occurred with the wheat and high pre-grazing pasture mass combination, so that pasture management effectively decreased CH₄ with wheat but not with corn. Previous work on non-grazing dairy cows showed that wheat produced less total CH₄ than corn (Moate et al., 2017), although this could not be confirmed by Muñoz et al.(2022a). At this point, it is difficult to explain the interaction between pre-grazing pasture mass and the type of concentrate. An analysis of rumen variables and other physiological aspects may be required to understand how this interaction occurred.

Pasture growth, quality, and nutrient composition, and hence CH₄ emissions from grazing ruminants, are influenced by season (Delagarde et al., 2000; Elgersma et al., 2005). Thus, it may be strategic to prioritize the adoption of anti-methanogenic strategies during seasons in which the greatest CH₄ production occurs. Thus, another study evaluated the effects on CH₄ emissions of feeding dairy cows exclusively with a cut and carried ryegrass pasture harvested daily across early and advanced autumn and early and advanced spring (Muñoz et al., 2022b). There was seasonal variation between periods within autumn and spring on CH₄ emissions. Absolute CH₄ emissions in autumn were higher early in the season, associated with higher forage quality and greater intake, but there were no differences between early and advanced spring (Table 1). Methane intensity in spring was lower early in the season and was associated with higher forage quality and milk production, but CH₄ intensity did not differ between early and advanced autumn (Muñoz et al., 2022b). This study confirmed that pasture quality, particularly its digestibility and nutrient concentration, significantly affects dairy cow productivity and CH₄ emissions.

As a conclusion about intensifying dairy production as an anti-methanogenic strategy, overall and partially contrary to existing global analyses (Gerber et al., 2011; Arndt et al., 2022), increasing concentrate supplementation and improving pasture quality has not consistently resulted in decreased CH₄ intensity in all the studies conducted in Chile. In agreement with previous research (Arndt et al., 2022; Beauchemin et al., 2022), total CH₄ emissions per dairy cow were unaffected or increased when dairy production was intensified by increasing concentrate supplementation or pasture quality was improved.

Type of forage

Given the advantages that ruminants have in terms of transforming human-inedible feedstuff such as grazed forage into milk and meat, it has been proposed that an important CH₄ mitigation option in pasture-based systems should be forage manipulation itself (Pacheco & Keim, 2018). Perennial ryegrass is an important forage crop in Chile and other temperate regions worldwide. Keim et al.(2014) evaluated the in vitro fermentation of four Chilean pastures differing amply in botanical composition and proportion of perennial ryegrass, which varied between 16 and 754 g/kg DM, in three regrowth periods. No differences in CH₄ production and CH₄ proportion of total gas produced were observed.

Ryegrass with high sugar content has been proposed as a strategy to decrease CH₄ emissions from grazing ruminants (Beauchemin et al., 2022). It has been reported that grazing frequency, N fertilization and ryegrass cultivar can modify the sugars to CP ratio (Bryant et al., 2012; Loaiza et al., 2017), which influences CH₄ production (Vera-Aguilera et al., 2022). Rivero et al.(2020) combined a reduced grazing frequency with a low N fertilization rate and a high-sugar tetraploid ryegrass cultivar to achieve a high sugar ryegrass, increasing the sugars to CP ratio by 52 and 89% in the spring and autumn, respectively, in comparison with the conventional management and cultivar. Incubating the different forages so obtained in in vitro batch cultures, they found that the high-sugar treatment harvested in autumn and spring produced 10 and 5% less CH₄ in total gas, respectively. In another in vitro study, Vera-Aguilera et al.(2022) reported a broad range of in vitro CH₄ production among 13 different ryegrass genotypes at vegetative stage, with the least methanogenic genotype producing 52% less CH₄ per gram of organic matter disappeared than the most methanogenic genotype. In their study, the range in sugar content was relatively narrow (17.3 - 21.5% DM), and, although CH₄ production was negatively associated with sugar concentration, the association was weak. Instead, CH₄ production per gram of organic matter disappeared was negatively associated with organic matter disappearance (Vera-Aguilera et al., 2023a).

In the following year, Vera-Aguilera et al.(2023b) compared the two most and two least methanogenic ryegrass genotypes reported by Vera-Aguileraet al. (2022) harvested from the same plots of the previous year at three phenological stages: vegetative, elongation, and reproductive. No interaction was found between the phenological stage and genotype. As ryegrasses matured, they produced more CH₄, in agreement with the observed increase in neutral detergent fiber and decrease in sugar content (Janssen, 2010). Unlike the ryegrasses harvested in the previous year (Vera-Aguilera et al., 2022), ryegrass genotypes did not differ in CH₄ production per gram of organic matter disappeared, although they numerically ranked as they had in the previous year. Vera-Aguilera et al.(2022) harvested ryegrass plants from the same plots and used the same collection and processing methods in 2021 and 2022; hence, the lack of significant differences in the methanogenic capacity of ryegrass genotypes may have been related to differences in climate conditions and plant maturation between both years. More research across different years and locations is needed to better interpret the results and to identify and generalize the main factors causing variation among ryegrass genotypes in their methanogenic capacity.

Legumes are digested faster in the rumen than grasses, and hence have a more rapid rumen passage rate (Beauchemin et al., 2022), which can result in a rumen fermentation profile lower in CH₄ (Janssen, 2010). Higher passage rate is also associated with increased feed intake, which lowers CH₄ intensity, although the effects of legume inclusion on CH₄ production are highly influenced by the phenological stage of the forages being compared and the type of legume (Beauchemin et al., 2022). In this regard, results regarding whether non-tannin-containing temperate legumes can effectively ameliorate CH₄ production from ruminants are inconsistent (Vargas et al., 2022). Muñoz et al.(2022c) compared ryegrass and alfalfa hay constituting 40% of the dry matter (DM) of a total mixed ration fed to dairy cows. Although there was a tendency towards greater absolute CH₄ emissions in cows fed alfalfa hay associated with greater feed intake, there were no differences in CH₄ yield or intensity. Feeding alfalfa hay resulted in greater N excretion in urine (Muñoz et al., 2022c), which, as discussed above, can result in augmented N₂O emissions from soils.

Enriquez-Hidalgo et al.(2020) replaced 40% of alfalfa hay and corn silage DM in a total mixed ration fed to dairy cows with a mixture of predominantly fresh ryegrass and lower proportions of berseem clover. There were no changes in total CH₄ emissions, CH₄ yield, or CH₄ intensity, although DMI and milk yield decreased when fresh ryegrass and berseem clover replaced alfalfa hay and corn silage in the total mixed ration (Table 1).

Chilean dairy producers are increasingly searching for drought-resistant crops to supplement cow diets during dry summers. Studies in New Zealand have shown that sheep fed forage rape produced less CH₄ than those fed perennial ryegrass (Sun et al., 2016). These authors suggested that this decrease could be attributed to a higher concentration of readily fermentable carbohydrates, lower rumen pH, changes in the rumen microbiome, and faster digesta passage rate. Chicory has also been proposed as a potential CH₄ mitigating forage due to its higher concentration of condensed tannins (Niderkorn et al., 2019). The inclusion of forage rape and chicory in dairy cow diets has been evaluated as an option in Chile to supplement forage during the summer, in cows receiving grass silage and a concentrate basal diet (Keim et al., 2024). In that study, forage rape, chicory, or an irrigated ryegrass pasture was offered to dairy cows at an inclusion level of 25% of the diet DM. Dry matter intake and N excretion were lower in cows fed chicory, whereas milk and protein yields were higher in cows fed forage rape. Forage rape and chicory showed potential benefits compared to irrigated ryegrass pasture in terms of milk production and nitrogen efficiency, although they did not decrease CH₄ emissions (Keim et al., 2024). In a follow-up study, the effects of supplemental chicory or forage rape (45% of the diet DM) on CH₄ emissions were compared in dairy cows fed a basal grass silage and concentrate diet. A reduction of 8% in total CH₄, 6% in CH₄ intensity, and 32% in milk urea concentration was observed in cows supplemented with chicory compared to the forage rape-supplemented group (León et al., 2024). Therefore, for summer feeding, chicory appears as an alternative to forage rape to maintain milk production while mitigating CH₄ production and N excretion.

Supplementation of oilseeds

The inclusion of oils and oilseeds in ruminant diets induces a moderate decrease in CH₄ production and can increase ruminant productivity owing to the supply of extra energy (Beauchemin et al., 2022). Research on in vitro semi-continuous cultures conducted in a Rusitec system evaluated the inclusion of ground linseed at 15% DM in combination with glycerol at 5 or 10% DM (Table 2) (Gutierrez-Gomez et al., 2020). The authors hypothesized that linseed, as a source of lipids, and glycerol, a reported propionogenic biofuel by-product, could act additively to decrease CH₄ production. Linseed inclusion resulted in a 30% decrease in total CH₄ production and CH₄ per gram of digested DM. However, glycerol failed to further decrease CH₄ production, likely because its conversion to propionate is stoichiometrically neutral in terms of electron balance (Avila-Stagno et al., 2013). Linseed inclusion in the substrate increased ammonium concentration by 2-fold, possibly due to increased digestible CP content. However, this effect was partially offset by glycerol, which may have contributed to the availability of energy for microbial protein synthesis. The authors suggested that care should be taken when supplementing oilseeds to mitigate CH₄ because N excretion can increase (Gutierrez-Gómezet al., 2020). A subsequent semi-continuous study with Rusitec confirmed the anti-methanogenic effect of linseed, which, however, was offset by glycerol (Vera et al., 2025b) (Table 2). In this study, where substrates were balanced in N, the addition of linseed did not increase ammonium concentration, whereas glycerol numerically decreased it.

Table 2. Effects of various antimethanogenic strategies on in vitro methane production. Research conducted in Chile published in peer-reviewed papers.

Reference	Type of antimethanogenic strategy	Treatments	In vitro system	Total methane	Methane per gram of digested DM, OM, or total gas	Comments
Keim et al. (2014)	Pasture composition	Pasture composition in three different periods	Batch	0†	0	There were differences in OM and NDF digestibility and total VFA
Vera et al. (2018)	Tannin-containing by-product	Pine bark at various doses	Batch	0	0	Decrease in ammonium
Rivero et al. (2020)	Defoliation management, N fertilization, and ryegrass genotype	High sugar ryegrass combined with lower N fertilization and longer regrowth period vs. control	Batch	0	↓	High sugar ryegrass had higher total gas production and total VFA
Gutierrez-Gomez et al. (2020)	Oilseeds and glycerol	Linseed and glycerol	Semi-continuous	↓	↓	Glycerol did not affect methane. Linseed increased and glycerol decreased, ammonium
Vera et al. (2021)	Tannin-containing by-product	Pine bark extract	Batch	0	0	Decrease in ammonium
Suescun-Ospina et al. (2022a)	Tannin-containing by-product	Grape marc with substrates varying in forage to concentrate ratio	Batch	↓	↓	Decrease in DM digestibility, total VFA and ammonium
Vera et al. (2022)	Tannin-containing by-product	Pine bark and quebracho extracts	Batch	↓	0	Decreases in DM digestibility, gas production, total VFA, and ammonium
Suescun–Ospina et al. (2023)	Tannin-containing by-product	Different drying methods, temperatures and duration	Batch	Differences between treatments	Differences between treatments
Vera et al. (2025a)	Tannin-containing by-product	Pine bark extract with high forage or high concentrate substrate	Semi-continuous	0	↓ (high forage) 0 (high concentrate)	Total VFA and ammonium decreased
Vera et al. (2025b)	Linseed and glycerol	Linseed with 0, 5 or 10% DM glycerol	Semi-continuous	↓	↓	Ammonium was unaffected or numerically decreased by glycerol

†↑ = increase, 0 = no change, ↓ = decrease compared to control treatments

In a double Latin square design study, Muñoz et al.(2019) compared CH₄ production, N balance, and milk fatty acid profile of early lactation dairy cows offered cottonseed, rapeseed, linseed, or an inert fat control, mixed in their total mixed rations. Cottonseed moderately decreased absolute CH₄ production and yield with respect to rapeseed and linseed, and CH₄ intensity compared to rapeseed (Table 1). However, although the diets were isonitrogenous, the effects of cottonseed supplementation on CH₄ emissions were offset by an increase in N excretion and by a greater ratio of N excreted in urine plus feces per unit of N ingested. This unfavorable result in N balance can potentially result in increased N₂O emissions from soils, partially or completely compensating the decrease in CH₄. In addition, oilseed supplementation increased the milk content of beneficial fatty acids, such as vaccenic acid and mono- and polyunsaturated fatty acids; rumenic acid in milk was increased by rapeseed, and α-linolenic acid was increased by linseed. This study showed that the effects of oilseeds and other anti-methanogenic strategies on CH₄ production should be evaluated alongside other effects on the environment and consumers’ health (Muñoz et al., 2019).

In a follow-up long-term longitudinal study, Muñoz et al.(2021) evaluated the supplementation of the same unprocessed oilseed cottonseed, rapeseed, and linseed compared to a control treatment supplemented with concentrate that did not contain oilseeds. By investigating the anti-methanogenic effects of oilseeds offered to grazing dairy cows for a longer experimental period (27 weeks), Muñoz et al.(2021) extended the few existing mid- to long-term studies evaluating oilseed supplementation as an anti-methanogenic strategy (Jordan et al., 2006; Grainger et al., 2010). Grazing dairy cows were offered experimental supplements during spring and summer, and oilseed supplementation was discontinued in autumn, but CH₄ and other measurements continued to investigate the possibility of residual effects. Consistent with the results of Muñoz et al.(2019), cottonseed effectively decreased CH₄ production moderately in spring; however, these effects were no longer sustained after 19 weeks of oilseed supplementation (Table 1). No carryover effects were observed in the autumn after oilseed supplementation ceased.

Reduced CH₄ mitigation occurs when oilseeds are offered unprocessed compared to breaking the seed hull through grinding, crushing, or rolling (Beauchemin et al., 2022). Although Muñoz et al.(2019) anticipated that offering whole oilseeds would have a lesser effect on CH₄ decrease than offering processed oilseeds, the authors aimed to evaluate unprocessed oilseed supplementation as a potential methane-mitigation strategy that could be adopted by most dairy producers in Chile, who lack specialized mills to process oily feedstuffs. Overall, it seems that moderate decreases in CH₄ emissions in the first few months of supplementation can be obtained with unprocessed oilseeds, although at the cost of a slight increase in the urine N output. As cottonseed supplementation did not improve milk production compared with a commercial concentrate containing no oilseeds (Muñoz et al., 2021), the adoption of this anti-methanogenic strategy may largely depend on its cost-effectiveness.

By-products and their extracts

Plant secondary metabolites, including saponins, polyphenols, and essential oils, can alter rumen microbiota and fermentation, and ruminant productivity and health (Calsamiglia et al., 2007; Tedeschi et al., 2021). Chile has a great diversity of ecosystems with different climates, topographies, and soil types, which harbor native plant species that could be candidates as a source of secondary compounds with the potential to modify rumen fermentation. Polyphenols, a vast family of plant secondary metabolites, are widely distributed in the plant kingdom, and can be divided into phenolic acids and flavonoids (Tedeschi et al., 2021). Flavonoids include flavanols, flavones, anthocyanidins and isoflavones. Polyphenols can modulate rumen fermentation by binding molecules such as proteins and carbohydrates and altering microbial populations in the rumen (Egea et al., 2016). These types of compounds are also present in some abundant by-products generated by the agroforestry industry worldwide. In Chile, the potential use of the polyphenol-containing by-product pine (Pinus radiata) bark and grape (Vitis vinifera) marc as enteric CH₄ mitigation ingredients in ruminant feeds has been extensively investigated at Universidad de Concepción.

Vera et al.(2018) found that up to 6% pine bark extract added to rumen batch cultures caused modest, numerical decreases in CH₄ production but decreased ammonium concentration extensively (Table 2), which was attributed to the binding of proteins by polyphenols contained in the bark extract. In another rumen batch culture study, Vera et al.(2022) compared pine bark with quebracho extract, and found a large decrease in CH₄ production and ammonium concentration with both by-products (Table 2). However, apparent DM disappearance and total volatile fatty acids (VFA) were decreased by both extracts. The long-term effects of pine bark extract were studied in vitro with a 70:30 and 30:70 forage to concentrate ratio substrate in a semi-continuous Rusitec system (Table 2) (Vera et al., 2025a). Pine bark extract decreased ammonium and VFA concentrations with both substrates, but CH₄ per gram of organic matter (OM) disappeared decreased only with high forage.

Grape marc is the residue left after making wine, and represents between 20 and 30% of the original grape weight (Beres et al., 2017). Grape marc contains both oils and tannins and has been reported to decrease enteric CH₄ production when fed to dairy cows (Moate et al., 2014; Akter et al., 2024). In Chile, wine production exceeded one billion liters in 2020, resulting in the generation of 260,000 tons of grape marc in that year (Buzzetti Horta, 2021). Grape marc is mainly deposited in landfills, causing environmental damage. Therefore, it is of interest to promote its use in ruminant feeding (Beres et al., 2017). Suescun-Ospina et al.(2021) studied the effects of four levels of an extract obtained from grape marc ranging from 0 to 2.1% of the substrate DM on CH₄ production and fermentation in semi-continuous rumen cultures in Rusitec. Methane production and ammonium concentration decreased by 29 and 30%, respectively, without affecting gas production or DM digestibility, although total VFA concentration was not determined in that study. In another study, Suescun-Ospinaet al. (2022a) found that the inclusion of grape marc at 0, 10, or 20% DM in batch cultures in replacement of hay in predominantly forage or concentrate substrates moderately decreased CH₄ production by up to 16%, but there was also a decrease in DM digestibility and total VFA concentration. There was also a strong decrease in the concentration of ammonium and branched-chain fatty acids, indicating diminished fermentation of amino acids (Table 2) (Suescun-Ospina et al., 2022a).

Further work in semi-continuous cultures found a severe decrease in the abundance of total methanogens when grape marc was included in the substrate (Suescun-Ospina et al., 2022b). Suescun-Ospina et al.(2023) reported that oven drying at 60 °C for 48 h resulted in lower in vitro CH₄ production than lyophilization or oven drying at 40 °C for 72 h. However, oven-drying at both temperatures resulted in lower in vitro digestibility and total VFA compared to lyophilization. Suescún-Ospina et al.(2024) evaluated combinations of grape marc extract and grape marc oil at 0, 2.5 or 5% DM in semi-continuous cultures. Both grape marc extract and oil decreased CH₄ production; however, their effects were not additive. Both grape marc and oil decreased or tended to decrease DM digestibility and ammonium concentration, and increased propionate molar percentage. Palacios et al.(2024) replaced grass silage with two types of grape marc (sweet or fermented) at 10, 15, 20, or 25% in batch cultures. Sweet and fermented grape marc included at 25% DM decreased CH₄ production at 24 h of incubation by 22 and 20%, respectively. However, total gas production was also decreased by 18% for both types of grape marc at the same timepoint.

Overall, the two types of polyphenol-containing by-products generated in Chile that have been evaluated to date, pine bark extract and grape marc, have shown interesting effects at decreasing ammonium concentration in vitro, suggesting their potential to decrease rumen protein degradation and improve N utilization efficiency when fed to ruminants. However, as decreases in digestion and fermentation have been noted in some studies, it is important to consider doses of these by-products that, while exerting favorable effects on N metabolism and CH₄ production, do not impair digestion and performance when fed to ruminants. In a subsequent in vivo study supplementing pine bark extract to lambs at 1 or 2% DM, no detrimental effects on performance, carcass quality, or blood circulating variables were observed. Methane production and N excretion were not measured, but pine bark improved the fatty acid profile of subcutaneous fat by increasing the content of fatty acids beneficial to human health (Vera et al., 2023). Therefore, further digestion and metabolism studies, including CH₄ and N balance measurements at different doses, are of interest.

Supplementation of algae

Macroalgae contain bioactive compounds and their supplementation to ruminants is thus being investigated as an anti-methanogenic strategy (Beauchemin et al., 2022). Beltran et al.(2022) replaced in rumen batch cultures 10% of the cereal portion of a substrate composed by 80% grass pasture and 20% of a cereal-based concentrate in the DM with one of three Chilean seaweeds: the brown seaweeds Macrocystis pyrifera and Lessonia spicata, and the red seaweed Agarophyton chilensis, with each alga collected both in the winter and summer. On average, M. pyrifera produced 22 and 11% less CH₄ at 24 and 48 h of incubation, respectively, compared to the control treatment without algae. However, it produced 11 and 9% less total gas at 24 and 48 h of incubation, respectively, suggesting that the decrease in CH₄ production could be explained, at least partially, by less digestion and fermentation. Considerably larger in vitro screening of macroalgae for their anti-methanogenic capacity identified very few candidates, most notably the bromoform-containing red algae Asparagopsis (Machado et al., 2014; Wasson et al., 2023); thus, it is to an extent expected that smaller screenings do not yield promising candidates recommendable for further progress to in vivo research.

A follow-up study investigated the inclusion of M. pyrifera and L. spicata replacing 0, 1, 3, 6, and 8% of the concentrate portion of the substrate in 48 h in vitro rumen batch cultures (Stolzenbach et al., 2022). Again, M. pyrifera linearly decreased CH₄ production by 30 and 24% after 24 and 48 h of incubation, respectively. However, it also decreased asymptotic total gas production by up to 10%, suggesting that the anti-methanogenic effect might have been partially mediated by less digestion and fermentation. A separate report indicated that the inclusion of M. pyrifera linearly increased the molar percentage of propionate and decreased the ammonia concentration in rumen batch cultures (Ruiz-Tagle et al., 2023). While the authors indicated as P > 0.05 that organic matter and N digestibility as well as total VFA concentration were not significantly decreased by the algae, it is not possible to discern the closeness of these important response variables to significance, as treatment means and exact P values for digestibility and total VFA were not provided. The information provided in the conference summary by Ruiz-Tagle et al.(2023) precludes then concluding whether the evaluated algae might have negatively affected digestibility and fermentation. While the 0.05 threshold is considered acceptable for declaring an effect as significant, a P value greater than 0.05 does not confirm that a response variable is unaffected. One should be hesitant about recommending a particular additive or anti-methanogenic strategy for further research, and even more for adoption, if digestion and/or fermentation variables are numerically compromised. Researchers conducting in vitro studies should consider numerical decreases in key digestion and fermentation variables, such as apparent OM digestibility, total VFA concentration, or total gas production, as a warning sign (Durmic et al., 2025). Likewise, a numerical and non-significant (P > 0.05) decrease in milk production or growth in an in vivo experiment should not prompt researchers to recommend an additive or technology because significant detrimental effects were not found at P < 0.05 (Hristov et al., 2025).

The red algae Asparagopsis exerts the strongest inhibition of methanogenesis reported for any feed additive to date (Almeida et al., 2021). Asparagopsis inhibition of CH₄ production in batch culture was first reported in 2014 (Machado et al., 2014). Since then, numerous in vitro (Machado et al., 2015a; Vucko et al., 2017; Roque et al., 2019a; Kinley et al., 2021; Kinley et al., 2022; Romero et al., 2023; Terry et al., 2023; Thorsteinsson et al., 2023; Nunes et al., 2024) and in vivo (Kinley et al., 2016; Li et al., 2016; Roque et al., 2019b; Kinley et al., 2020; Muizelaar et al., 2021; Roque et al., 2021; Stefenoni et al., 2021; Krizsan et al., 2023; Indugu et al., 2024; Romero et al., 2024; Fennessy et al., 2025) studies with Asparagopsis, and more recently, various Asparagopsis preparations (Alvarez-Hess et al., 2023; Cowley et al., 2024; George et al., 2024), have consistently confirmed profound inhibition of methanogenesis with this red algae.

The inhibitory effect of Asparagopsis on methanogenesis is due to its haloalkane content, mainly bromoform (Machado et al., 2016). The Asparagopsis genus comprises two species, A. taxiformis and A. armata, and its use for enteric CH₄ mitigation has been patented since 2015 (Machado et al., 2015b) and licensed by FutureFeed (FutureFeed, 2021). Locally, Marin et al.(2024) evaluated in vitro three mixtures of undefined brown and red algae collected in Chile included at 2.0 or 5.0% of substrate DM, and reported greater than 98% inhibition of methanogenesis. Because all three mixtures of algae that they used contained a minimum of 50% DM of their red alga in the algal mix, the observed anti-methanogenic effects were ascribed to the red alga. The red alga evaluated by Marin et al.(2024) was collected in Antofagasta, where A. armata has been reported to grow as an invasive species (Ramírez et al., 2007; Villaseñor-Parada et al., 2018), and the authors speculated about the presence of halogenated compounds in the red alga. Although the specific algal species were not reported, it thus appears that the red alga evaluated by Marin et al.(2024) was likely A. armata collected in Antofagasta. The omission of an explicit identification of the algae evaluated is problematic as it affects the reproducibility of the findings of this study. It would be expected that the effects of Chilean A. armata on methanogenesis are consistent with previous results reported for Asparagopsis from other parts of the world, as Chilean A. armata does not constitute a separate subspecies or taxon and falls into the same genetic cluster as A. armata collected from places as diverse as the United Kingdom, Ireland, the Channel Islands, France, Spain, Italy, San Diego, and Australia (Chualáin et al., 2004).

Oyarzun et al.(2023) supplemented 150 g/d of an undefined red algae to dairy cows once daily at milking. The algal additive caused a 12% decrease in absolute CH₄ emissions pulse-measured at the parlor three times daily using the GreenFeed equipment (C-Lock Inc., Rapid City, SD, US). However, as DM intake and milk production and composition were not reported, it is unknown whether CH₄ production decreased at the expense of less DM intake and/or digestibility, and if animal performance was impaired by the additive. The effects of the algae evaluated by Oyarzun et al.(2023) on CH₄ intensity and CH₄ yield are similarly unknown. The lack of identification of the algae evaluated and of fundamental performance results that should be reported in in vivo trials evaluating feed additives inhibiting CH₄ production (Hristov et al., 2025) makes unconvincing the authors’ claim about the potential impact of this algal additive to mitigate the environmental impact of dairy production.

Inhibitors of methane production

Apart from the red alga Asparagopsis, supplementation with pure chemicals that inhibit rumen methanogenesis appears to be the most potent strategy to mitigate CH₄ emissions from ruminants (Almeida et al., 2021; Arndt et al., 2022). Garcia et al.(2022) hypothesized that inhibiting rumen methanogenesis with 3-nitrooxypropanol (3-NOP, Bovaer®) would stimulate the microbial synthesis of amino acids and long-chain fatty acids as an alternative electron sink to CH₄ in the rumen of dairy cows. Although an unintended dietary change precluded the authors from testing their hypothesis, the study showed that 3-NOP was highly effective in inhibiting rumen methanogenesis with diets containing both solely plant protein and urea providing part of the N (Table 1).

While confined ruminants emit a relatively minor portion of global enteric CH₄, most applied research on the inhibition of rumen methanogenesis using feed additives, whether pure chemicals or of natural origin, has been conducted with total mixed rations. However, research on the abatement of enteric CH₄ emissions from grazing ruminants has lagged behind (Ungerfeld, 2022). A search of the PubMed database for “3-nitrooxypropanol” or “3-nitroxypropanol” retrieved 93 records, 48 of which corresponded to in vivo experiments with ruminants fed total or partial mixed rations, 34 corresponded to literature reviews, meta-analyses, deterministic simulations and LCA, biochemistry and other studies, and only two studies with grazing ruminants, both with dairy cows (Costigan et al., 2024; Muñoz et al., 2024), one of which was conducted in Chile. A similar search for “Asparagopsis AND methane” yielded 50 records, with 15 records corresponding to original research with ruminants fed total or partial mixed rations and only one with ruminants on pastures (Meo-Filho et al., 2024).

In a 2 × 2 factorial arrangement study, Muñoz et al.(2024) evaluated the supplementation of 3-NOP mixed with a concentrate with or without whole cottonseeds. Supplementation of 3-NOP moderately decreased CH₄ emissions by 13% with the non-cottonseed-containing concentrate and had no effect when it was offered with cottonseed (Table 1). This mild CH₄ mitigation with grazing dairy cows is in sharp contrast to an average 33% CH₄ decrease obtained in a meta-analysis of 14 experiments quantifying the effects of 3-NOP offered to dairy cows on total mixed rations (Kebreab et al., 2023). Muñoz et al.(2024) concluded that supplementing 3-NOP mixed with concentrates offered at milking and before grazing introduced a time gap between the peak of 3-NOP concentration in the rumen and grass ingestion, which considerably decreased the effectiveness of 3-NOP. The study by Costigan et al.(2024) in Ireland, who also evaluated the supplementation of 3-NOP to grazing dairy cows, was published shortly after the Muñoz et al.(2024) study. Costigan et al.(2024) also found a mild decrease of about 5% in daily CH₄ production in grazing dairy cows supplemented 3-NOP. In contrast, Meo-Filho et al.(2024) reported a 38% decrease in CH₄ production in grazing steers fed pellets containing Asparagopsis. Meo-Filho et al.(2024) estimated an average total DMI of about 8.5 kg/d, of which about 0.5 kg corresponded to the Asparagopsis-containing pellet. Thus, it seems that the considerable CH₄ mitigation achieved in the Meo-Filho et al.(2024) study was obtained with grass constituting most of the DMI. Meo-Filho et al.(2024) offered Asparagopsis-containing pellets in GreenFeed units three times daily, with at least 6 h between each offering, whereas Muñoz et al.(2024) and Costigan et al. (2024) supplemented 3-NOP twice daily.

Frequent supplementation of anti-methanogenic additives seems therefore important to maximize their effectiveness in pasture-based systems, although this appears difficult in extensive ruminant production systems. In addition, while bromoform is rapidly metabolized by in vitro rumen cultures, its reduction product, dibromomethane, would prolong the effect of Asparagopsis (Glasson et al., 2022; Romero et al., 2023). This said, 3-NOP is metabolized in the rumen to 1, 3-propanediol and nitrite, with nitrite also inhibiting methanogens (Duin et al., 2016).

Studies evaluating the supplementation of grazing ruminants with anti-methanogenic feed additives are scarce, which contrasts with the global importance of pasture-based systems in enteric CH₄ emissions (FAO, 2022). Solutions for pasture-based systems, including slow-release feed additives and optimal grazing and supplementation management to enhance their effectiveness, are urgently required. Unfortunately, developing feed additives that inhibit CH₄ production in pasture systems has not been a priority for the industry developing feed additives (Hegarty et al., 2021).

Khurana et al.(2024) supplemented a garlic and citrus extract (Mootral Ltd., Abertillery, UK) to grazing dairy cows in a commercial dairy in the south of Chile. They measured milk production and composition, estimated DMI based on an equation derived for Brazilian tropical conditions (Souza et al., 2014), and estimated the total output of CO₂e based on the Global Livestock Environmental Assessment Model-interactive tool (GLEAM-i) (FAO, 2025). They concluded that garlic and citrus extracts increased energy-corrected milk production and decreased CO₂e emission intensity. Whilst GLEAM-i can be used to estimate CO₂e emissions of commercial herds for carbon accounting purposes (e.g., national inventories), using GLEAM-i in original research intended to demonstrate the efficacy of an anti-methanogenic feed additive is grossly inadequate. Demonstrating mitigation of total CO₂e output at the farm level requires, among other aspects, direct measurements of enteric CH₄ production (Hristov et al., 2025), mathematical models derived from various trials demonstrating the efficacy of feed additives at decreasing CH₄ production (Dijkstra et al., 2025), and an LCA tailored for a specific farm (del Prado et al., 2025). The response in milk production reported by Khurana et al.(2024) is of much interest, but their claim of a lower CO₂e intensity is dismissed because of lack of CH₄ measurements demonstrating the effects of the feed additive and because of lack of a farm-specific LCA.

Future perspectives

As the role of CH₄ in global warming has gained attention worldwide, the volume of research on enteric CH₄ mitigation conducted in Chile has been steadily increasing. A total of 18 peer-reviewed papers (10 in vitro and 8 in vivo, with 50% of the in vitro and 88% of the in vivo reports published in top impact factor quartile (Q1) journals) have been published in this period, apart from numerous conference presentations. However, as evidenced in this review, many results are only available as conference papers, often with incomplete information, and only available at the national level. The limited number of scientific publications and the time lag between study completion and publication in a scientific journal appear to be a common challenge for Chilean research groups working in this field. This is of concern as it implies that a significant portion of the research has not yet been evaluated under rigorous scientific standards, potentially limiting its reach and influence. In addition, all in vivo research in which CH₄ production was measured has been conducted with dairy cows. This is also a limitation, and future research also needs to address enteric CH₄ mitigation in beef cattle, sheep, and goats.

It is highly important that research on enteric CH₄ mitigation in Chile accelerates, not only because of the existing urgency to mitigate global warming, but also because most of the applied research on enteric CH₄ mitigation conducted elsewhere took place with total mixed rations and may often not be applicable to Chile’s (and vast areas of the world) pasture-based conditions, as illustrated by the Muñoz et al.(2024) study supplementing 3-NOP to grazing dairy cows. Even with supplementation, diets in predominantly pasture-based systems are imbalanced, as the amount and composition of supplements cannot be adjusted quickly enough to balance for changes in the composition and digestibility of pastures. In addition, depending on the season and pasture, DMI may be constrained by grass availability. These are inherent characteristics of the diets ingested by grazing ruminants in comparison with ruminants fed total mixed rations. Therefore, it is important to consider whether these conditions inherent to pasture-based systems might interact with the treatments being evaluated for DMI, performance, and CH₄ production. This highlights the need for Chilean research to develop and adapt solutions feasible for implementation by Chilean producers, considering biological, economic, and societal aspects and constraints.

Moving forward, it is important to develop and establish reliable verification methods that can be applied by producers to demonstrate their efforts to mitigate enteric CH₄ emissions. This, in turn, should be reflected in the national greenhouse gas inventory to support Chile’s commitment to reducing greenhouse gas emissions. Most importantly, any credible mitigation claims must be backed by scientific literature. Addressing these challenges is crucial for ensuring the accountability of the ruminant production industry to the national climate goals.

It is challenging to find solutions for enteric CH₄ mitigation that are both technically and economically feasible, safe to use, and not compensated for by increased emissions of other greenhouse gases. Research on enteric CH₄ mitigation in Chile has primarily focused on evaluating nutritional strategies, including supplementation with concentrates, use of more digestible forages, inclusion of oils via oilseeds, presence of secondary compounds in by-products from the national agricultural industry, and introduction of anti-methanogenic additives, both chemical and contained in algae. Solutions based on the intensification of ruminant production and improvement of production efficiency are considered straightforward and economically attractive ways to decrease CH₄ intensity. Production intensification strategies to decrease CH₄ production evaluated in vivo in Chile include supplementation with concentrates and improvement of forage quality. Of these, only the improvement of forage quality was effective in moderately decreasing CH₄ intensity, but at the same time likely increased N voided to the environment. Increasing the supplementation of concentrates to dairy cows did not decrease CH₄ intensity and increased total CH₄ emissions. A specific antimethanogenic strategy, supplementation with cottonseeds, moderately decreased CH₄ intensity and total CH₄ emissions, but also increased N voided to the environment in urine. The latter aspect highlights the need to conduct LCA of CH₄ mitigation strategies proven effective in assessing their potential environmental impacts in terms of total greenhouse gas emissions. To date, no LCA of agricultural greenhouse gases from ruminants conducted in Chile have been published in peer-reviewed journals.

Currently, only 3-NOP can be recommended for practical application in Chile, and solely for confined cattle. This limits its potential contribution to reducing national CH₄ emissions, as only 1.22% of beef and dairy cattle are managed in confined systems in Chile with controlled diets that allow precise mixing and feeding of the additive (ODEPA, 2019). Research on optimizing the supplementation of feed additives that inhibit CH₄ production in the rumens of grazing ruminants is urgently required. Other anti-methanogenic strategies cannot yet be endorsed due to insufficient evidence of effectiveness, potential increases in the emissions of other greenhouse gases (which decrease the net impact of enteric CH₄ mitigation on total emissions of CO₂e), or negative externalities on production, such as reduced feed intake or product yield, as well as concerns about the quality and safety of the final product; all of these aspects still need to be addressed by local research. Some anti-methanogenic strategies that are potentially applicable to pasture-based systems and have not been investigated in Chile include the genetic selection of ruminants producing less CH₄ and vaccination against methanogens. Selecting ruminants based on residual CH₄ can allow moderate (~10%) decreases in CH₄ output per animal. This requires measuring CH₄ production in large numbers of animals or discovering reliable markers that are easy to measure and can accurately predict CH₄ production. Progress has been made in basic research on the development of vaccines against methanogens, but so far, there are no preparations effective at decreasing CH₄ production in vivo (Beauchemin et al., 2022). While these research lines are of great interest because of their potential to be applied in pasture-based systems, even in extensive ones, beginning long-term research in these areas in Chile might mean diluting already scarce human and financial resources. It is important to consider that there are several constraints that limit research productivity in Chile: lack of funding from government and the private sector (0.36 % of GDP investment in R&D, which is considerably lower than the 2.7% average of OCDE countries), limited availability of facilities and equipment, limited access to trained personnel, low appreciation of peer-reviewed research outside of the academic community, among others.

Importantly, the lack of solutions for enteric CH₄ mitigation that are ready for implementation by producers is a global problem. Even in intensive production systems, solutions that are technically feasible face economic constraints and require the adoption of financial incentives. An example are the subsidies in Denmark for producers who use 3-NOP or incorporate fat in dairy cows’ diets (Danish Dairy Board, 2025). Pilot projects on the incorporation of 3-NOP into the diet of confined cows have been privately financed in Chile (Vargas García, 2024). Possible policies to be implemented to favor the adoption of anti-methanogenic strategies should be discussed by scientists in conjunction with the government, industry, consumers, and other decision makers.

Conclusions

Considerable research on enteric CH₄ mitigation, reviewed in this paper, has been conducted in Chile in the last 10 years; however, much of this research remains as conference summaries and still needs to be published in peer-reviewed journals. There was ample variation in CH₄ production among ryegrass genotypes in vitro. However, given that the least methanogenic genotypes were the most digestible, it is possible that these more digestible genotypes allow greater DMI in vivo, compensating for the lower CH₄ per gram of organic matter disappeared, and result in no differences in total CH₄, as found by one of the in vivo studies conducted in Chile evaluating forage quality. Forage rape and chicory did not decrease total CH₄ but increased milk production and decreased N excretion.

There is interest in further in vivo evaluation of pine bark extract and grape marc at different doses to assess whether they can decrease CH₄ production and urine N excretion without impairing performance and digestibility. Supplementation with cottonseed moderately decreased CH₄ production in the majority, but not in all studies. An increase in N excretion highlights the need to conduct LCA to estimate the effects of anti-methanogenic strategies on CO₂e emissions. The in vitro and in vivo evaluation of Chilean algae showed no promise at decreasing CH₄ production without compromising digestion and fermentation. In other instances, the information provided in conference summaries was insufficient to conclude about the effects of the algae evaluated on digestion, fermentation, and performance, or the algal species was not informed, casting doubt about the novelty of the results.

The chemical inhibitor 3-NOP was highly effective at inhibiting CH₄ production in dairy cows fed a total mixed ration; however, the inhibition was mild in grazing dairy cows. At present, there is no strategy to mitigate enteric CH₄ emissions proven consistently effective under pasture-based conditions in Chile, demonstrated to have no negative effects on performance, economically attractive, and not offset by greater emissions of other greenhouse gases. Future research should focus on optimizing the supplementation of anti-methanogenic feed additives to maximize their effectiveness, as well as evaluating in vivo anti-methanogenic by-products containing secondary compounds at different doses, seeking to lower CH₄ emissions and N excretion without negatively affecting performance and health. Other lines of research, such as tannin-containing forages and genetic selection of low CH₄-producing animals may be considered. Life cycle assessments and research on ruminants other than dairy cows are needed. Despite the many challenges in conducting research in Chile, after one decade of progress, research on enteric CH₄ mitigation in Chile has reached a stage of maturity.

Declarations

Competing interests statement

The authors declare that they have no competing interests.

Author contributions

Conceptualization: EMU, NLU, CM; Visualization: EMU, NLU, CM; Writing – Original Draft: EMU, NLU, CM, JA, JPK; Writing – Review & Editing: EMU, NLU, CM, JA, JPK.

Acknowledgements

Agencia Nacional de Investigación y Desarrollo, Projects FONDECYT N° 1240264 and N° 1240525.