There is a need for rigorous and scientifically-based testing standards for existing and new enteric methane mitigation technologies, including antimethanogenic feed additives (AMFA). The current review provides guidelines for conducting and analyzing data from experiments with ruminants intended to test the antimethanogenic and production effects of feed additives. Recommendations include study design and statistical analysis of the data, dietary effects, associative effect of AMFA with other mitigation strategies, appropriate methods for measuring methane emissions, production and physiological responses to AMFA, and their effects on animal health and product quality. Animal experiments should be planned based on clear hypotheses, and experimental designs must be chosen to best answer the scientific questions asked, with pre-experimental power analysis and robust post-experimental statistical analyses being important requisites. Long-term studies for evaluating AMFA are currently lacking and are highly needed. Experimental conditions should be representative of the production system of interest, so results and conclusions are applicable and practical. Methane-mitigating effects of AMFA may be combined with other mitigation strategies to explore additivity and synergism, as well as trade-offs, including relevant manure emissions, and these need to be studied in appropriately designed experiments. Methane emissions can be successfully measured, and efficacy of AMFA determined, using respiration chambers, the sulfur hexafluoride method, and the GreenFeed system. Other techniques, such as hood and face masks, can also be used in short-term studies, ensuring they do not significantly affect feed intake, feeding behavior, and animal production. For the success of an AMFA, it is critically important that representative animal production data are collected, analyzed, and reported. In addition, evaluating the effects of AMFA on nutrient digestibility, animal physiology, animal health and reproduction, product quality, and how AMFA interact with nutrient composition of the diet is necessary and should be conducted at various stages of the evaluation process. The authors emphasize that enteric methane mitigation claims should not be made until the efficacy of AMFA is confirmed in animal studies designed and conducted considering the guidelines provided herein.

Both condensed and hydrolysable tannins (CTs and HTs, respectively) have the ability to reduce enteric CH₄ production in ruminants. However, the precise mechanism of action is not fully understood. Among the proposed hypotheses are the reduction of ruminal digestibility, direct control action on protozoa, reduction of archaea, and a hydrogen sink mechanism. In this in vitro study, which simulated rumen fermentation, two additives, one containing CTs (70% based on DM) from quebracho and one with HTs (75% based on DM) from chestnut, at four levels of inclusion (2, 4, 6, 8% on an as-fed basis) were added to the fermentation substrate and tested against a negative control. Both types of tannins significantly reduced total gas (GP) and CH₄ (ml/g DM) production during the 48 h of incubation. The lower GP and CH₄ production levels were linked to the reduction in dry matter digestibility caused by CTs and HTs. Conversely, no significant differences were observed for the protozoan and archaeal populations, suggesting a low direct effect of tannins on these rumen microorganisms in vitro. However, both types of tannins had negative correlations for the families Bacteroidales_BS11 and F082 and positive correlations for the genera Prevotella and Succinivibrio. Regarding the fermentation parameters, no differences were observed for pH and total volatile fatty acid production, while both CTs and HTs linearly reduced the NH₃ content. CTs from quebracho were more effective in reducing CH₄ production than HTs from chestnut. However, for both types of tannins, the reduction in CH₄ production was always associated with a lower digestibility without any changes in archaea or protozoa. Due to the high variability of tannins, further studies investigating the chemical structure of the compounds and their mechanisms of action are needed to understand the different results reported in the literature.

Plant secondary metabolites (PSMs) can potentially reduce ruminal methane formation. However, related to differences in their molecular structures, it is not yet clear what causes an anti-methanogenic effect. In an in vitro system simulating rumen fermentation, we investigated the impact of eight compounds with distinct chemical characteristics (gallic and salicylic acids, tannic acid, catechin, epicatechin, quercetin, rutin, and salicin) when added to a basal feed (maize silage) at a concentration of 12% of the feed dry matter. After 48 h of incubation in buffered rumen fluid, methane production was significantly lowered by quercetin (43%), tannic acid (39%) and salicylic acid (34%) compared to the control (maize silage alone) and without changes in total volatile fatty acid production during fermentation. No other PSM reduced methane formation as compared to control but induced significant differences on total volatile fatty acid production. The observed differences were related to lipophilicity, the presence of double bond and carbonyl group, sugar moieties, and polymerization of the compounds. Our results indicate the importance of distinct molecular structures of PSMs and chemical characteristics for methane lowering properties and volatile fatty acid formation. Further systematic screening studies to establish the structure–function relationship between PSMs and methane reduction are warranted.

Condensed tannins (CT) are plant polyphenols that can affect feed digestibility and are potentially able to reduce enteric CH₄ emissions in ruminants. In this in vivo trial with 8 lactating goats, we investigated the effects of 4 levels of inclusion of a commercial CT extract from quebracho (0%, 2%, 4%, and 6% on dry matter basis; CON, Q2, Q4, and Q6, respectively). The experimental design was a repeated 4 × 4 Latin square with 28-d periods (24 d of diet adaptation and 4 d of sample collection) using metabolic cages and 4 open-circuit respiration chambers. The inclusion of CT in the diets did not affect the dry matter intake (DMI) but caused a linear decrease in diet digestibility, with reductions up to -11% for dry matter, -21% for crude protein (CP), -23% for α-amylase- and sodium sulfite-treated neutral detergent fiber corrected for insoluble ash (aNDFom), and -13% for gross energy, when comparing the Q6 and CON diets. However, ruminal total volatile fatty acids (VFA) concentration was not affected by CT, although there were changes in VFA proportions. Milk yield was highest for Q4 (3,371 g/d) and lowest for Q6 (3,066 g/d). In terms of milk composition, CT induced a linear reduction of fat and CP concentrations. The reduction in CP digestibility resulted in a linear reduction in the milk urea level, up to -37% with Q6. Positively, CT linearly reduced the somatic cells count expressed as linear score. The feed efficiency was linearly decreased by CT inclusion. Furthermore, a shift from urinary to fecal nitrogen excretion was observed with CT. The retained nitrogen was always negative (on average -1.93 g/d). The CH₄ yield (on average 19.2 g of CH₄/kg DMI) was linearly reduced by CT inclusion, up to -18% with Q6. Regarding the CH₄ intensity, CT induced a linear reduction when expressed per kilogram of milk, but not per kilogram of fat and protein-corrected milk. Moreover, the CH₄ production per kilogram of digestible aNDFom was linearly increased by CT. The metabolizable energy intake (MEI) was not affected by the treatments, but the metabolizability (q = MEI/gross energy intake) was reduced as CT inclusion increased. From the results of the present study, it turned out that CT have a negative impact on feed digestibility and feed use efficiency. Condensed tannins can lower CH₄ emissions from ruminants; however, the main mechanism of action is likely the decrease in feed digestibility. Furthermore, CT did not improve the N use efficiency. According to these findings, the positive environmental impacts of CT are only related to the shift from urinary to fecal N excretion.

The effect of whole linseeds or hemp seeds on milk production, energy and nitrogen balance, and methane emission was studied in 12 Alpine goats using respiration chambers. Diets tested were a control diet (C) and two diets supplemented with whole linseeds (L) or hemp seeds (H) at 9.3% on a dry matter (DM) basis. DM intake was similar among treatments, whereas DM and organic matter digestibility were lower for L compared to C. Milk yield (2.30 kg/d on average) and rumen fermentation profile were not affected by treatments. Treatment also did not affect the milk composition, with the exception of fat, which was higher in H and L compared to C (4.21, 3.94, and 3.20%, respectively). Oilseed supplementation caused a reduction in the concentration of de novo fatty acids (FA) (41.1, 48.8, and 64.1% of FA, for L, H, and C, respectively). Moreover, L and H diets reduced the sum of saturated FA, and increased monounsaturated FA, whereas only the L diet increased the concentration of polyunsaturated FA. Regarding methane production, and nitrogen and energy balances, no differences were registered among the diets. Our research indicates that including whole linseeds and hemp seeds in the dairy goat diet is an effective strategy for increasing milk fat content and positively modifying the milk FA composition, without a change in nitrogen and energy balances, but also without a reduction in enteric methane emission.