Research progress on the application of feed additives in ruminal methane emission reduction: a review

Background Ruminal methane (CH4) emissions from ruminants not only pollute the environment and exacerbate the greenhouse effect, but also cause animal energy losses and low production efficiency. Consequently, it is necessary to find ways of reducing methane emissions in ruminants. Studies have reported that feed additives such as nitrogen-containing compounds, probiotics, prebiotics, and plant extracts significantly reduce ruminant methane; however, systematic reviews of such studies are lacking. The present article summarizes research over the past five years on the effects of nitrogen-containing compounds, probiotics, probiotics, and plant extracts on methane emissions in ruminants. The paper could provide theoretical support and guide future research in animal production and global warming mitigation. Methods This review uses the Web of Science database to search keywords related to ruminants and methane reduction in the past five years, and uses Sci-Hub, PubMed, etc. as auxiliary searchers. Read, filter, list, and summarize all the retrieved documents, and finally complete this article. Results Most of the extracts can not only significantly reduce CH4 greenhouse gas emissions, but they will not cause negative effects on animal and human health either. Therefore, this article reviews the mechanisms of CH4 production in ruminants and the application and effects of N-containing compounds, probiotics, prebiotics, and plant extracts on CH4 emission reduction in ruminants based on published studies over the past 5 years. Conclusion Our review provides a theoretical basis for future research and the application of feed additives in ruminant CH4 emission reduction activities.


INTRODUCTION
Methane (CH 4 ) is the world's second most abundant greenhouse gas after carbon dioxide (CO 2 ), accounting for 16% of total greenhouse gas emissions (De Visscher & Van Cleemput, 2003). The potential global warming effect of CH 4 is 28-fold higher than that of CO 2 (Stocker et al., 2013). In addition, rumen CH 4 emissions from ruminants account for 13% to 19% of the global CH 4 emissions (Liu & Whitman, 2008); therefore, ruminant feeding is a major factor in exacerbating global warming. Therefore, reducing rumen CH 4 emissions could decrease the rate of global warming, which would be of great significance to efforts to reduce global greenhouse gas emissions. CH 4 emissions also represent energy losses during ruminant farming. On average, approximately 8-12% of the energy consumed in feed is wasted in the form of CH 4 emissions (Johnson & Johnson, 1995).
Accordingly, to remedy the low production efficiency and mitigate the potential damage caused by livestock CH 4 emissions to the environment, researchers have begun to explore the roles of different feed additives in reducing ruminant CH 4 emissions. Among them, N 2containing compounds, probiotics, prebiotics, and plant extracts, which are feed additives that are not harmful to animal health, have been the first subjects of research and are expected to become ideal CH 4 inhibitors in the future. This article reviews the mechanism of CH 4 emission production in ruminants and the potential influence of nitrogenous compounds, probiotics, prebiotics, and plant extracts on ruminal CH 4 production.

SURVEY METHODOLOGY
In this review, keywords related to additives, ruminants, methane emission reduction in the past five years were searched through the Web of Science database, and Sci-Hub, PubMed, etc. were used as auxiliary searchers. Perform a rough reading of all the retrieved documents; screen out documents related to the effects of additives on ruminant methane in ruminants; then list the documents related to the effects of different additives on ruminant methane in ruminants according to the type of additives; finally, classify different categories Make a summary and finally complete this article.

Methane production mechanism in ruminants
After ruminant ingestion, the nutrients (proteins, lipids, and carbohydrates) in feed are degraded by rumen microorganisms to produce hydrogen (H 2 ) and primary fermentation products that contain methyl groups such as formic acid, acetic acid, methanol, and methylamine. Afterward, methanogens convert the primary fermentation products into CH 4 and energy is obtained. There are three pathways of ruminal CH 4 production (Thauer et al., 2008) (Fig. 1), including (1) the CO 2 -H 2 reduction pathway; (2) synthesis pathways using short-chain fatty acids such as formic acid, acetic acid, and butyric acid as substrates; (3) and synthesis pathways using methyl compounds such as methanol and ethanol as substrates. Among the three, the CO 2 -H 2 route is the primary pathway (Ellis et al., 2008) because the growth rates of Methanococcus that exploit acetic acid are low (Liu & Whitman, 2008), and acetic acid-producing bacteria have a low affinity for H 2 (Morgavi et al., 2010). In addition, only methanogens of Methanosphaera use methanol to produce CH 4 (Liu & Whitman, 2008).

Effects of nitrogenous compounds on methane production in ruminants
N-containing compounds are used as ammonium-N (NH 4 + -N) supplements in ruminant diets. Extensive research has revealed that N-containing compounds can reduce CH 4 production via their influence on rumen microorganisms, for example, by reducing the Figure 1 Schematic diagram of methane production. There are three basic pathways of ruminal methane production: (1) represents the CO 2 -H 2 reduction pathway, (2) represents the synthesis pathway of short chain fatty acids such as formic acid, acetic acid, and butyric acid as substrates, and (3) represents the synthesis pathway with methyl compounds such as methanol and ethanol as substrates. Among these, route (1) is considered to be the primary route of methane production.
Full-size DOI: 10.7717/peerj.11151/ fig-1 activity of participating CH 4 -producing enzymes and competing for hydrogen (Table 1), in addition to supplementing NH 4 + -N. Among them, nitrate-N (NO − 3 -N) is considered to be a urea substitute; it can not only meet the requirements of rumen microorganisms for NH 4 + -N, but can also decrease CH 4 production substantially (Adejoro et al., 2020;Adejoro, Hassen & Thantsha, 2018;Alvarez-Hess et al., 2019;Wu et al., 2019). The mechanism of action is linked to the competitive effects of NO − 3 -N over H 2 consumption and the inhibitory effect of the generated nitrite (NO − 2 -N) on methanogen proliferation. However, large doses of NO − 3 -N may cause the accumulation of toxic NO − 2 -N (Jeyanathan, Martin & Morgavi, 2014). Therefore, it is necessary to control NO − 3 -N dosages, or supplement feed with NO − 2 -N reducing agents, to minimize nitrite toxicity (Jeyanathan, Martin & Morgavi, 2014); NO − 2 -N capsules could also be used (De Raphelis-Soissan et al., 2017). A novel N-containing compound, 3-nitrooxypropanol (3-NOP), has recently been introduced; it can continuously reduce CH 4 production without adversely affecting animal growth or development (Romero-Pérez et al., 2016). It is an ideal CH 4 inhibitor. The structure of 3-NOP is similar to that of methyl-coenzyme M, which is associated with the last step of CH 4 production, and 3-NOP can inhibit the activity of the reductase. Notes. *** The additive has a significant effect on methane inhibition. ** The additive has a general effect on methane inhibition.
Nitroethane (NE), 2-nitroethanol (NEOH), and 2-nitro-1-propanol (NPOH) can also inhibit methanogenic bacteria and significantly reduce the expression of the methylcoenzyme M reductase gene (Zhang et al., 2020a). In addition, these compounds could reduce the content of coenzymes F420 and F430, reducing ruminal CH 4 production in turn (Zhang et al., 2020a). There are numerous other N-containing compounds that inhibit methanogen activity and alter the structure of rumen microbial flora, the activity of enzymes involved in CH 4 production, and the distributions of volatile fatty acids, leading to the consumption of H 2 and reduction of CH 4 production in turn.

Effects of probiotics on methane production in ruminants
Probiotics are a class of beneficial active microorganisms or their cultures. Probiotics could reduce CH 4 emissions in ruminants (Table 2). There are many types of probiotics, and different strains have different inhibitory effects on CH 4 emissions. For example, the GA03 strain of Acetobacter is more effective at inhibiting CH 4 production than other isolated strains (Kim et al., 2020b). Most probiotics reduce CH 4 production by influencing the activities of ruminal microorganisms, with no adverse effects on animals. In addition, probiotics enhance ruminal fermentation. Lactic acid bacteria, which have been used as feed additives for a long time, not only reduce CH 4 emissions per unit volatile fatty acid (VFA) output, but also improve the fermentation quality and fiber digestibility of silage (Guo et al., 2020). In addition, the denitrifying bacterium Bacillus 79R4 could prevent NO − 2 -N poisoning and microbial ecosystems from impairing fermentation efficiency (Latham et al., 2019). Furthermore, Bacillus licheniformis reduces CH 4 production and increases feed energy and protein utilization (Deng et al., 2018). However, the inhibitory mechanism of lactic acid bacteria on CH 4 is still unclear; therefore, in the future, more research will need to be conducted on the influence of lactic acid bacteria on rumen microbes and hydrogen competition to elucidate the mechanism of inhibiting CH 4 production.

Effect of prebiotics on methane production in ruminants
Prebiotics are substances that are not easily digested or absorbed by the host. They selectively stimulate the growth and activity of one or several ruminal microorganisms with a positive effect on ruminal fermentation (De & Schrezenmeir, 2002). Prebiotics suppress ruminal CH 4 production in ruminants. Prebiotics mainly reduce rumen CH 4 production by altering the bacterial community structure, influencing the permeability of the cell walls of methanogenic archaea, and stimulating other bacteria to compete with methanogens for H 2 (Table 3). According to Tong et al. (2020), the prebiotic chitosan can influence bacterial community structures by altering microbial population compositions, for example, by replacing fibrinolytic enzyme-producing microbes (Firmicutes and Fibrobacteres) with amylolytic enzyme-producing microbes (Bacteroides and Proteus); in turn, reducing CH 4 production.
According to Seankamsorn, Cherdthong & Wanapat (2020), chitosan could influence the ruminal fermentation process by altering VFA distributions and increasing propionic acid concentrations, which reduces CH 4 production in turn. However, according to some Probiotic products of Ruminococcus flavefaciens *** 2 g probiotic products in powder; 1.2 ml/g of dry matter 10 ml probiotic products in liquid; 1.2 ml/g of dry matter Reduce the number of rumen protozoa Notes. *** The additive has a significant effect on methane inhibition. ** The additive has a general effect on methane inhibition. *The additive has no obvious effect on methane inhibition.

Inhibition mechanism References
Chitosan *** 3000 (molecular weights) dry matter; 22.9% ml/day (Tong et al., 2020) 2% Chitosan + 21% of crude glycerin; 53.67% (Seankamsorn, Cherdthong & Wanapat, 2020) (1) Alters microbial community structure (Tong et al., 2020) (2) Alters fermentation pathway (Seankamsorn, Cherdthong & Wanapat, 2020) ( Yeast products *** researchers, the reduction in CH 4 is associated with the degree of chitosan deacetylation, which could alter the permeability of the methanogen cell wall. In addition, Vallejo-Hernández et al. (2018) observed that various yeast products could reduce CH 4 emissions by stimulating acetic acid-producing bacteria to compete with methanogens or metabolize hydrogen. Overall, compared with other feed additives, prebiotics are still relatively less applied in feed, and their types are limited. Therefore, future research should target strategies to promote the adoption prebiotic feed additives. For example, when conducting scientific research, it is necessary to strengthen contact with breeding companies so that companies can see the effects of prebiotic additives and reduce penalties due to pollution. It is also necessary to enforce the guidelines and requirements of the national environmental protection department for low environmental pollution and exploit consumers' demand for healthy food to promote the widespread use of prebiotic additives.

Effects of plant extracts on methane production in ruminants
In recent years, the effects of plant-derived feed additives on rumen microbial fermentation, rumen CH 4 production, and ruminant performance have been increasingly recognized. Many previous studies have demonstrated that natural plant-derived compounds are promising anti-CH 4 -generation compounds, including tannins, essential oils, and saponins (Table 4). Although plant extracts have potentially significant effects on CH 4 emission reduction in ruminants, most of the inhibition mechanisms are not clear. According to research findings, the effects of plant-derived feed additives on ruminant methane emission reduction are mainly based on competition for hydrogen and rumen microbes. Competing for hydrogen is manifested in the form of increased propionic acid contents in fermentation products. Effects on rumen microbes are manifested in the number and activity of protozoa, methanogens, and total bacteria, and the results vary based on types of plant-derived feed additives. Medicinal plant extracts (for example: patchouli, atractylodes, and honeysuckles), tannins, and essential oils have all been shown to suppress the production of CH 4 by altering ruminal microbial structure and abundance (Kim et al., 2016). The inhibitory effects of tannins on CH 4 reduction are influenced by their molecular weight (Petlum et al., 2019;Piñeiro Vázquez et al., 2018a). However, if the molecular weights of tannins are too high, the palatability of the diet would be adversely affected, and, in turn, the performance of animals. Therefore, it is critical to determine the optimal tannin supplementation levels. The effects of plant essential oils on rumen microorganisms could be linked to their antibacterial, antiviral, antifungal, and insecticidal properties. Plant essential oils contain various active ingredients that can regulate rumen fermentation and reduce CH 4 emissions (Soltan et al., 2018). As antibiotic substitutes, medicinal plant extracts could have unique influences on rumen microbes due to their equally unique medicinal properties, including CH 4 emission reduction (Kim et al., 2016;Yadeghari et al., 2015).
Generally, plant extracts have a significant effect on reducing methane emissions from ruminants, but most of its mechanism of action is still unclear. Almost all tests are in vitro tests, which are short-term tests. At present, research on plant extracts in animals is still lacking, and the effect of long-term use of plant extracts on animals is still unclear.

Types of probiotics Inhibitory effects
Addition amount; maximum suppression methane amount

Inhibition mechanisms References
Papaya leaf extract *** Methane production (mL/250 mg dry matter ) decreased with increasing levels of Papaya leaf extract.
(2) Hydrogen consumption Wastes of tomato fruit *** Therefore, in the future, we should focus on using plant extracts in animals and study the effects of long-term use on animals.

CONCLUSIONS
Considering the results of the studies that have been published over the past 5 years, the application of nitrogenous compounds, probiotics, prebiotics, and plant extracts has been shown to reduce ruminal CH 4 emissions. There are three main ways of reducing CH 4 production: (1) reducing the number of rumen protozoa and inhibiting methanogen activity; (2) increasing propionic acid production to compete with methanogens for hydrogen; (3) inhibiting the activity of enzymes involved in methanogen activity. However, the mechanisms of action of most plant extracts remain unclear; and almost all studies are based on in vitro fermentation tests. In addition, most plant extracts have no adverse effects on animals, and they are rich in resources. Consequently, research on the effects of plant extracts in animals and their mechanisms of action should be the main research direction in the future, to enhance their application in animal production and the mitigation of the adverse effects of global warming.

ADDITIONAL INFORMATION AND DECLARATIONS Funding
This study was supported by the National Natural Science Foundation of China (31860657). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Grant Disclosures
The following grant information was disclosed by the authors: National Natural Science Foundation of China: 31860657.

Competing Interests
The authors declare there are no competing interests.

Author Contributions
• Kang Sun conceived and designed the experiments, performed the experiments, analyzed the data, authored or reviewed drafts of the paper, and approved the final draft.
• Huihui Liu and Huiyu Fan conceived and designed the experiments, prepared figures and/or tables, and approved the final draft.
• Ting Liu and Chen Zheng conceived and designed the experiments, authored or reviewed drafts of the paper, and approved the final draft.

Data Availability
The following information was supplied regarding data availability: This is a review article; there is no raw data.