Integrating Technological Innovations and Sustainable Practices to Abate Methane Emissions from Livestock: A Comprehensive Review

Abstract

Livestock farming is a vital component of global food security, yet it remains a major contributor to greenhouse gas (GHG) emissions, particularly methane (CH₄), which has a global warming potential 28 times greater than carbon dioxide (CO₂). This review provides a comprehensive synthesis of current knowledge surrounding the sources, biological mechanisms, and mitigation strategies related to CH₄ emissions from ruminant livestock. We first explore the process of methanogenesis within the rumen, detailing the role of methanogenic archaea and the environmental factors influencing CH₄ production. A thorough assessment of both direct and indirect methods used to quantify CH₄ emissions is presented, including in vitro techniques (e.g., syringe method, batch culture, RUSITEC), in vivo techniques (e.g., respiration chambers, Greenfeed, laser CH₄ detectors), and statistical modeling approaches. The advantages and limitations of each method are critically analyzed in terms of accuracy, cost, feasibility, and applicability to different farming systems. We then examine a wide range of mitigation strategies, organized into four core pillars: (1) animal and feed management (e.g., genetic selection, pasture quality improvement), (2) diet formulation (e.g., feed additives such as oils, tannins, saponins, and seaweed), (3) rumen manipulation (e.g., probiotics, ionophores, defaunation, vaccination), and (4) manure management practices and policy-level interventions. These strategies are evaluated not only for their environmental impact but also for their economic and practical viability in diverse livestock systems. By integrating technological innovations with sustainable agricultural practices, this review highlights pathways to reduce CH₄ emissions while maintaining animal productivity. It aims to support decision-makers, researchers, and livestock producers in the global effort to transition toward climate-smart, low-emission livestock farming.

1. Introduction

Livestock farming plays a vital role in global food security, serving as a cornerstone of protein supply, rural livelihoods, and agricultural economies. However, the sector faces growing scrutiny due to its considerable environmental footprint, particularly its contribution to climate change. Livestock, especially ruminants such as cattle, buffalo, sheep, and goats, are responsible for substantial greenhouse gas (GHG) emissions, primarily methane (CH₄) and nitrous oxide (N₂O), which significantly contribute to global warming [1].

Methane is predominantly released during enteric fermentation in the rumen and via anaerobic decomposition of manure [2]. Despite its relatively short atmospheric lifetime (~12 years), CH₄ has a global warming potential approximately 28 times greater than that of carbon dioxide (CO₂) over a 100-year period [3]. Nitrous oxide, typically arising from manure and fertilizer applications, is 265 times more potent than CO₂ and remains in the atmosphere for over a century [4]. Together, livestock-related CH₄ and N₂O emissions account for approximately 14.5% of global anthropogenic GHGs [1]. Data from 2020 show that dairy cattle alone contributed around 72% of methane emissions within the livestock sector, followed by buffalo (8.7%) and small ruminants (6.7%) [5,6]. These figures underscore the disproportionate impact of ruminants on climate forcing and highlight the urgent need for mitigation strategies that address their specific digestive physiology. As the global demand for animal-source foods continues to rise, livestock intensification exerts increasing pressure on land, water, and feed resources, amplifying emissions and threatening food system sustainability [1]. Beyond their biological sources, GHGs emitted by livestock are part of a broader suite of climate-relevant gases, including CO₂, CH₄, N₂O, hydrofluorocarbons (HFCs), perfluorocarbons (PFCs), and sulfur hexafluoride (SF₆), as recognized by the United Nations Environment Program and further described by Anderson et al. [7]. Of these, CH₄ and N₂O are especially relevant in the context of animal agriculture, as they result directly from enteric fermentation, manure storage, and nitrogen cycling in soils [2,8]. The environmental implications of these emissions extend well beyond agriculture, affecting climate systems, ecosystems, and human livelihoods [9]. Although several studies and reviews have addressed aspects of methane mitigation in livestock systems, most focus on isolated topics such as feed additives, genetic selection, or microbial interventions [10,11]. What remains lacking is a comprehensive and integrative perspective that connects methane measurement methodologies with practical, scalable, and sustainable mitigation solutions. Furthermore, few existing works sufficiently address the policy and implementation contexts needed to translate scientific innovation into meaningful emissions reductions on the ground.

This review is developed in response to that gap. We hypothesize that a holistic integration of advanced methane measurement techniques with sustainable, technology-driven mitigation strategies can significantly reduce CH₄ emissions from livestock without compromising animal productivity or system efficiency. To explore this hypothesis, we ask: What are the most commonly used and emerging methods for quantifying methane emissions in livestock systems? Which mitigation strategies are biologically effective, economically feasible, and practically scalable? How can these tools be implemented in a manner that aligns with broader climate and sustainability goals? And finally, where are the remaining gaps in knowledge, practice, and policy? In addressing these questions, the review sets out to: (1) summarize and compare methane measurement methods—spanning in vitro, in vivo, and indirect models; (2) evaluate a range of sustainable mitigation strategies, from dietary formulations and microbial modulators to manure management and biotechnological innovations; (3) assess the trade-offs and applicability of different techniques across diverse production contexts; (4) identify critical research and policy gaps; and (5) propose integrated pathways for climate-smart livestock transformation.

To ensure methodological rigor and reduce bias, the literature reviewed was systematically selected from peer-reviewed publications indexed in Scopus, Web of Science, and PubMed, covering the period from 2010 to 2024. The selection prioritized high-impact research, systematic reviews, and meta-analyses focused on GHG emissions from livestock, methane quantification, and mitigation technologies. Non-English articles, non-peer-reviewed sources, and outdated studies were excluded. Through this evidence-based synthesis, we aim to support science-informed policies, encourage investment in low-emission livestock innovations, and contribute to a resilient and environmentally sustainable future for the animal agriculture sector.

2. Methodology

To ensure methodological rigor and transparency, this scoping review was conducted following the PRISMA 2020 Extension for Scoping Reviews guidelines (see Supplementary Material). A systematic literature search and selection process was carried out to identify relevant peer-reviewed studies on methane emissions, measurement techniques, and mitigation strategies in livestock systems.

2.1. Literature Search Strategy

A comprehensive search was performed using three major electronic databases: Scopus, Web of Science, and PubMed. The search covered publications from January 2010 to April 2024. Keywords and Boolean operators included combinations of: “methane emissions”, “ruminants”, “livestock”, “GHG mitigation”, “methane measurement”, “in vitro”, “in vivo”, “indirect methods”, “feed additives”, “rumen microbiota”, “sustainable livestock”, and “climate-smart agriculture”.

2.2. Inclusion Criteria

• Peer-reviewed journal articles
• Published in English
• Focused on ruminant livestock (cattle, buffalo, sheep, goats)
• Addressed methane measurement or mitigation
• Included original data, systematic reviews, or meta-analyses
• Published between 2010 and 2024

2.3. Exclusion Criteria

• Non-English articles
• Editorials, conference abstracts, or opinion pieces
• Studies on non-ruminant species
• Non-peer-reviewed publications
• Duplicate or retracted articles

2.4. Screening and Selection Process

Titles and abstracts were first screened for relevance. Eligible full texts were then reviewed independently by two authors. Discrepancies were resolved by discussion or consultation with a third reviewer. The study’s overall screening and selection process is presented in the PRISMA flow diagram (Figure 1).

2.5. Data Charting and Extraction

Relevant information from the included studies was systematically extracted and charted in tables. Data included study objectives, species, measurement techniques, mitigation strategies, outcomes, and limitations. The review synthesized findings thematically and organized the discussion into categories aligned with the review objectives.

2.6. Quality Control

Although formal risk-of-bias assessments were not required for this scoping review, emphasis was placed on selecting studies from high-impact journals and those with clear experimental designs, methodologies, and reproducibility of results.

3. Overview of GHGs and Potential Global Warming

The Sun produces energy at extremely short wavelengths, which affects the Earth’s environment. About one-third of this solar energy is reflected back into space by the atmosphere [12]. The rest of the energy is absorbed by the atmosphere and the Earth’s surface. To keep things balanced, the planet emits longer-wavelength energy to counteract the absorbed energy. The atmosphere captures this energy and re-releases it back to Earth, a process known as the greenhouse effect. This effect helps warm the Earth because certain gases in the atmosphere trap solar radiation [13]. These gases, called GHGs, are essential for making the Earth warm enough to support life. Key GHGs include CO₂, CH₄, N₂O, and several other fluorinated gases [13], with carbon dioxide being responsible for about 80% of the greenhouse effect. Human activities, such as burning fossil fuels and agriculture, notably increase CO₂ and other gas emissions, leading to GW [14,15]. This results in significant yearly temperature increases, which disrupt climate patterns and pose risks like the spread of diseases [16]. In 2020, global temperatures were 1.27 °C higher than in the late 19th century [17], with projections suggesting a future rise of 5.4 °C by 2100 [18]. GW threatens many aspects of society, including environmental degradation and changes in agriculture, and increased severe weather events like hurricanes, droughts, landslides, floods, fires, and heat waves are expected to intensify [19].

4. Contribution of Livestock on GHGs Emissions

Agriculture, and by extension, livestock, stands out as a major source of GHGs emissions, thereby playing a substantial role in CC [4]. These emissions, primarily stemming from physiological processes and the production of animal-derived products, account for 14.5% of total anthropogenic GHGs emissions, equivalent to 7.1 gigatons of CO₂ equivalent (Gt CO₂e) [6]. Within this total, production and processing of feed contribute roughly 45% or 3.2 Gt CO₂e, about 39% (2.8 Gt CO₂e) comes from enteric fermentation, and roughly 10% (0.71) comes from manure management. The processing and transportation of animal products is responsible for the remaining 6%, or 0.42 Gt CO₂e [6]. Alternatively, focusing solely on direct CH₄ and N₂O production from rumen fermentation, manure, and the estimation of distribution indicates the contribution to be 5.4 Gt CO₂e [8]. In the European Union (EU), Perez [20] showed that agriculture is responsible for up to 10.3% of total GHGs production. Notably, CH₄ produced from rumen fermentation represented about 32%, while manure contributed an additional 16%. Among livestock species, with 4.6 Gt CO₂e, or 61% of total emissions, cattle production leads the way, with other categories contributing far less: poultry 0.7 Gt CO₂e (8%), sheep and goats 0.474 Gt CO₂e (6%), and pigs 0.7 Gt CO₂e (9%) [6,8].

5. Understanding of Methane (CH₄) Mitigation in Livestock Sector

The importance of reducing CH₄ emissions from farm animals is discussed in two main areas: environment and economy. CH₄ is much more effective than CO₂ in trapping heat, being 28 times more potent, and it stays in the atmosphere for a shorter time, with an 8.6-year half-life compared to CO₂’s 120 years [21]. This makes it a key target for quick action against GW, allowing for immediate positive effects. Researchers are trying to enhance animal production while cutting CH₄ emissions, especially from ruminants. In developed countries, removing low-yielding animals is suggested, but this faces challenges in developing nations due to economic, cultural, and religious factors [22]. Ruminant producers must find cost-effective ways to reduce emissions while meeting food and environmental needs. The atmosphere has about 1. 9 ppm of CH₄, while cattle exhale around 1000 ppm [23]. Recently, atmospheric CH₄ levels reached 1.898 ppm, showing a 162% increase from pre-industrial times. The levels of atmospheric CH₄ have increased 2.5 times over the last 300 years, reaching the highest levels in 650,000 years [24]. Domestic ruminants produce CH₄ through a process in their stomachs called rumen fermentation [25].

The rumen environment, with temperatures around 39 °C and pH between 6.5 and 6.8, allows specific microorganisms to create CH₄. Most of this gas is expelled through burping, making up 87% of total emissions, while some is absorbed into the bloodstream. CH₄ emissions not only impact climate change but also create an economic burden for small farmers due to energy loss from feed. Sheep and goats emit 10 to 16 kg of CH₄ annually, while dairy cows emit between 60 to 160 kg [26]. Conversely, when these cattle were fed a highly digestible, high-concentrate diet, they produced only 0.07 kg of CH₄/animal/day, representing a conversion of merely 1.9 to 2.2% of feed energy into CH₄ [27]. Research shows cattle fed low-grade feed produce significantly more CH₄ compared to those on high-quality diets.

6. The Sources of CH₄ Emission

6.1. Methanogenesis Process

Methanogenic archaea, which are anaerobic microorganisms responsible for CH₄ production for adenosine triphosphate (ATP) production, encompass approximately 155 species identified in nature. There are 29 genera, 14 families, 6 orders, and 4 classes for these species [28]. The majority of these microorganisms survive liberally in rumen contents or as biofilms attached to feed [29], with a small proportion forming symbiotic relationships with protozoa [30]. Among the rumen methanogens, Methanobrevibacter (Mbb) is the most prevalent, constituting 63.2% of all separates, followed by Methanosphaera (9.8%) and Methanomicrobium (7.7%) [31]. The remaining methanogens pertain to subspecies like Methanobacterium, Methanosarcina, and Methanimicrococcus [31]. Based on previous studies, methanogens that are hydrogenotrophic predominate in terms of both CH₄ formation and community structure [32]. Rumen archaea, due to their limited substrate range, appear to exhibit less diversity compared to rumen bacteria. Mbb ruminantium and Mbb gottschalkii, which constitute 74% of the rumen methanogen population, have been observed across different animals, locations, diets, and environmental conditions [32]. Until now, just 13 ruminal methanogen species have been effectively identified and cultured in pure forms (including Methanobacterium beijingense, Methanoculleus bourgensis, Mbb boviskoreani, Mbb bryantii, Methanosarcina barkeri, Mbb formicicum, Methanoculleus marisnigri, Methanoculleus olentangyi, Mbb millerae, Methanomicrobium mobile, Methanosarcina mazei, Mbb olleyae, Methanoculleus ruminantium, and Mbb formicicum) [31,33].

6.2. Rumen Fermentation Processes

In ruminants, enteric fermentation and manure storage are the main sources of CH₄ [34]. The rumen microbial fermentation provides volatile fatty acids (VFAs) and microbial proteins for the host animal while also releasing CH₄ into the atmosphere. Simultaneously, this fermentation occurs with various microorganisms, including bacteria, protozoa, and fungi, that anaerobically transform their substrates into fermentation end-products. In the context of methanogenesis, methanogens in the rumen mainly convert CO₂ into CH₄ using the available H₂ in the rumen environment. Consequently, methanogens can serve as significant H₂ scavengers in the rumen, lowering H₂ levels and preventing potential inhibition of rumen digestion [35]. Two types of methanogens, Methanomicrobium and Methanobrevibacter, thrive at temperatures of 39 and 40 degrees Celsius, respectively, and contribute significantly to CH₄ production, accounting for about 82% of all CH₄ production in the rumen [36]. This is expressed by stoichiometric equations: 4H₂ + CO₂ → CH₄ + 2H₂O and HCO³⁻ + 4H₂ → H⁺ + CH₄ + 3H₂O [37]. Additionally, about 15–20% of CH₄ comes from formate, which methanogens can also use. Formate dehydrogenase processes formate to produce CO₂ and CH₄, aiding the overall CH₄ generation in the rumen, as expressed by the stoichiometric equation 4HCOOH → 3CO₂ + CH₄ + 2H₂O [38]. Methanogens, exemplified by Methanosarcinales and Methanobacteriales, engage in the oxidation of methyl groups, like those found in methanol and methylamines, into CO₂. This process yields electrons utilized to reduce methyl groups to CH₄, as depicted in the stoichiometric reactions: 4CH₃OH → 3CH₄ + CO₂ + 2H₂O, CH₃NH₂ + H₂ → CH₄ + NH₃, CH₃OH + H₂ → CH₄ + H₂O [39]. Furthermore, in acetoclastic methanogenesis, Methanosarcina and Methanosaeta use acetate as a substrate [40]. In this process, acetate undergoes splitting to generate carboxyl compounds that are subsequently oxidized to CO₂. The stoichiometric equivalence signifies that CH₃ groups enter the hydrogenotrophic pathway to produce CH₄: CH₃COOH → CO₂ + CH₄. However, CH₃ groups and CH₃CO₂ play a negligible role in rumen methanogenesis due to the slow growth of methanogens relying on these conversions in vitro.

7. Measuring CH₄ Emission from Enteric Fermentation

7.1. General Background

Given the significance of CH₄ emission, it is crucial to address methods for measuring these emissions before delving into diverse strategies aimed at mitigating GHG releases from ruminants, with CH₄ being the primary component. It is essential to acknowledge that while direct control over emissions is limited, managing the factors that influence them, such as diet, animal genetics, and manure handling, is key to effective mitigation. Several techniques have been developed to assess CH₄ emissions in the fermentation process. These methods include direct measurements of the generated gas, conducted either in vitro or in vivo. To determine the most suitable scientific method, factors like accuracy, dimensions, cost, and the experimental model must be considered. Awareness of the feasibility and limitations of each technique is essential. The pursuit of rapid, precise, and straightforward methods for measuring CH₄ and other products of ruminal fermentation has been a focal point in studies on ruminal nutrition. Various techniques have been developed, by different countries, to quantify CH₄ emissions from ruminants across diverse experimental conditions (Figure 2 and

Table 1. Summary of Methane Measurement Methods.

Method Type	Technique	Description	Advantages	Limitations
In vitro	Syringe technique	Measures gas volume or pressure from fermentation in gas-tight syringes	Simple, low-cost, widely used	Limited data resolution; not automated
	Batch culture	Static fermentation using rumen fluid and feed to measure CH4 and fermentation end-products	Scalable, suitable for screening	Lacks continuous feeding and product removal
	RUSITEC (Rumen Simulation Technique)	Multi-vessel continuous system mimicking rumen environment over long periods	Reproducible, long-term studies, microbial adaptation possible	Complex setup, labor-intensive
	Semi-automated gas production technique	Uses water displacement and pressure sensors for periodic gas measurements	Reduces manual handling, higher data resolution	Requires operator supervision; not fully continuous
	Automated wireless gas production technique	Uses sensors with wireless data transmission to record gas production in real-time	Real-time data, reduced human error, scalable	Requires calibration and controlled environment
	Fully automated wireless gas production technique	Fully integrated system for gas, pH, and temperature monitoring with remote control	High throughput, real-time monitoring, minimal labor	High initial cost, needs technical setup
In vivo	Respiration chambers	Sealed chambers monitor animal respiration gases (CH4, CO2, O2)	Highly accurate; gold standard	Expensive, limited throughput, animal confinement
	GreenFeed system	Portable feeding unit measures CH4 during short-term visits	Field-usable, less invasive, allows for multiple animals	Depends on animal behavior and access
	SF6 tracer technique	Uses sulfur hexafluoride as a tracer gas to estimate CH4 in breath samples	Applicable to grazing animals	Technical complexity, calibration required
	Laser CH4 detector (LMD)	Infrared laser detects CH4 concentration near the animal’s muzzle	Non-invasive, portable, instant readings	Measures concentration, not volume; influenced by wind
	Sniffer, face mask, ventilated hood	Devices to capture and analyze breath CH4 directly from animal’s headspace	Easy to deploy, moderate cost	Variability due to animal movement, moderate accuracy
Indirect	IPCC Tier 1–3 models	Statistical models estimating CH4 based on emission factors and activity data	Useful at national or regional scales	Tier 1 is highly generalized; accuracy improves with data quality

). These techniques, rooted in animal nutrition, aim to assess energy losses in ruminants that are fed different diets and prove valuable in assessing the environmental efficiency of the production system. Each method comes with its own set of advantages and disadvantages concerning the comprehensive depiction of ruminal fermentation.

7.2. In Vitro Techniques

These techniques, including the incubation of rumen contents with tested substrates, have been widely utilized for assessing the nutritional evaluation of ruminant diets in conjunction with standard laboratory analyses of chemical composition and present a rapid and cost-effective alternative for determining nutrient digestibility compared to in vivo assessments [41]. The application of these techniques offers an advantage in minimizing in vivo animal trials, and ones testing a huge number of treatments are required. For developing countries with limited resources, utilizing the in vitro method frequently becomes the primary choice for exploring potential agents for CH₄ reduction. According to the experiment objectives, in vitro trials serve as useful tools for testing and providing insight into appropriateness for further in vivo assessment. However, it is crucial to note that a positive outcome in vitro does not necessarily ensure the same effect in vivo. In many cases, results for diet evaluation and CH₄ reduction can be misleading if the limitations of batch culture systems, such as lack of microbial adaptation or fixed incubation conditions, are not properly considered during interpretation [42]. Additionally, the objectives, design, findings, and conclusions of in vitro trials need careful and certain clarification. Results from in vitro experiments often lack relevance to commercial situations due to factors such as the tested additive being too expensive for use, effectiveness being unverified in vivo, or potential harmful effects on animal performance.

7.2.1. Syringe Technique

Czerkawski and Breckenridge [43] developed a method involving the movement of a piston by pressure generated (gases) throughout feed fermentation by rumen fluid within a glass syringe. This concept laid the foundation for the Hohenheim method as established by [44]. Originally designed for determining the endpoint fermentation of feeds after a 24-h incubation, the ‘syringe technique’ underwent modification by [45]. This adjustment involved placing syringes in a water bath instead of a rotating incubator. By recording gas production at more frequent intervals, this modification enabled the analysis of fermentation kinetics.

7.2.2. Semi-Automated Gas Production Technique

An alternative method was introduced to assess fermentation kinetics in vitro [46]. In this method, fermentation takes place within a sealed container containing rumen fluid, buffer, and substrate. A pressure transducer is utilized to measure the accumulation of gas in the headspace of the vessel. The basic setup of this system was outlined by [47], where headspace pressure is manually measured. Samples of gas are gathered for the assessment of concentrations of CO₂, CH₄, N₂O, and/or H₂ upon the release of gas pressure [48].

7.2.3. Automated Wireless Gas Production Technique

Cornou et al. [49] reported the findings of a ring test assessing the application of a wireless system designed by Ankom (Ankom Technology, Macedon, NY, USA) for automated gas release. Although this method is currently utilized in multiple laboratories, it still depends on the manual sampling and analysis of gas.

7.2.4. Full Automated Wireless Gas Production Technique

A fully automated wireless gas production system that utilizes pressure sensors for real-time monitoring of gas production has been utilized [50]. This system automatically measures the proportions of CH₄ and H₂ in the released fermentation gases through computer-controlled gas chromatography. Notably, this approach differs from previous automated systems [49] as it involves collecting and analyzing fermentation gases using a computer-controlled gas chromatograph instead of releasing them into the air upon reaching a threshold pressure.

7.2.5. Batch Culture (BC) Techniques

The background of the BC model has been detailed previously by Muetzel et al. [50]. This method is employed for the extensive cultivation of microorganisms or substances produced by microorganisms, wherein, at a specific point (24 or 48 or 72 h), the fermentation process is halted, and the culture is processed. This fermentation approach, often referred to as a ‘closed culture’ system, involves the initial addition of nutrients and other supplements in specified quantities. Initially, microorganisms experience rapid growth due to the abundance of nutrients. Over time, their numbers increase, utilizing nutrients quickly and concurrently generating toxic metabolites. The current application of BC for assessing the evaluation of ruminant feeds or feed additives is primarily reliant on the in vitro method established by Tilley and Terry [51] and subsequently improved by Goering and Van Soest [52]. The digestion of samples is gauged after anaerobic breakdown by bacteria in the rumen. The BC facilitates the measurement of gas production kinetics (mostly CO₂, CH₄, and N₂O) to develop models predicting feed consumption, the production of microbial proteins, and metabolizable energy, along with gas patterns and the speed and the degree of substrate digestion.

7.2.6. Rumen Simulation Technique (RUSITEC)

The Rumen Simulation Technique (RUSITEC) is a continuous culture in vitro system developed to mimic ruminal fermentation processes under controlled and reproducible conditions. It addresses the limitations of batch culture systems by allowing longer experimental periods, continuous infusion of buffers and substrates, and removal of fermentation end-products [53]. The RUSITEC consists of a series of fermentation vessels (usually 4 to 6), each maintained at rumen temperature (39 °C), pH, and anaerobic conditions. Rumen fluid is typically collected from fistulated donor animals and mixed with buffer solutions. The system includes peristaltic pumps for inflow and outflow, and feed substrates are introduced in nylon bags suspended inside the vessels. Solid and liquid digesta are maintained and exchanged to simulate rumen turnover. This technique is particularly valuable for evaluating the effects of dietary interventions, feed additives, and methane mitigation strategies on gas production, including CH₄ and CO₂, VFA profiles, ammonia-N concentration, microbial protein synthesis, and shifts in microbial populations (via qPCR or sequencing).

The RUSITEC allows for extended experimental periods (up to 49–60 days), making it suitable for studying microbial adaptation over time. Compared to in vivo studies, it reduces variability and ethical concerns, though it still depends on fresh rumen inoculum and lacks some dynamic physiological interactions present in the live animal. Although features like automatic pH control, semi-permeable membranes, or ion-exchange columns can enhance system fidelity, they also increase complexity, and maintenance demands in multi-vessel setups. Despite these challenges, RUSITEC remains a widely accepted model for evaluating the impact of dietary changes on rumen fermentation and methane emissions [54,55].

7.3. In Vivo Techniques

7.3.1. Respiration Chamber (RC)

The RC is a recognized and reliable method for accurately measuring CH₄ emissions from rumen and hindgut fermentation; it is also known as the “gold standard” [56]. This technique involves placing an animal in a sealed chamber for 24 h, maintaining slightly negative atmospheric pressure to prevent gas leaks [57]. The emissions of CH₄ are calculated by measuring airflow and concentration differences between inlet and outlet air with the use of an infrared gas analyzer and automated sampling, plus measuring other GHGs and animals heat production [58]. The RC offers several advantages, such as describing diurnal CH₄ emission patterns, providing insights into underlying mechanisms, and investigating relationships between CH₄ production and various factors [59]. In addition, it enables precise assessments of emissions arising from fermentation in both the rumen and hindgut, measurements that may not be captured by several alternative methods. However, the RC technique has limitations, including the need for sophisticated equipment, high building and upkeep expenses, as well as limitations on the quantity of animals available for experimentation simultaneously [60]. However, measurements are made under artificial conditions, raising concerns about their reliability compared to animals actively grazing in natural environments. Calibration and recovery tests are crucial to minimize uncertainty associated with the RC system [61].

7.3.2. Portable Accumulation Chamber (PAC)

The PAC serves as a simplified and cost-effective alternative to the RC system, designed for estimating CH₄ emissions in sheep breeding values. Introduced by Goopy et al. [62], the PAC is a clear booth made of polycarbonate that has a volume of about 0.8 m³. During a maximum 2-h period, a sheep is enclosed in the PAC, allowing CH₄ to accumulate. The calculation of CH₄ emissions involves determining the concentration and multiplying it by the chamber’s net volume. Gas analyzers, including optional measurements of CO₂, O₂, and ammonia (NH₃), can be employed every 30 min, with corrections for ambient CH₄ concentration. The net chamber volume is determined by subtracting the animal-occupied volume, estimated as 1 L/kg body weight, from the PAC volume.

7.3.3. Sulfur Hexafluoride Tracer Technique (SHTT)

Sulfur Hexafluoride (SF₆) is a non-isotopic technique utilized to measure CH₄ production in both indoor and grazing ruminants [63]. Sulfur Hexafluoride, an inert gas, is dosed orally through a controlled-release permeation tube into the rumen, and its release rate is calibrated before and after insertion [61]. The CH₄ emissions are sampled around the mouth and nostrils, and the concentrations are analyzed using gas chromatography [64]. The SF₆ gas relies on the controlled release of a predetermined amount of SF₆ tracer gas (mg/d) from a pre-calibrated permeation tube inserted into the reticulo-rumen. The daily CH₄ production (g/d) is calculated using the ratio of gas concentrations ((CH₄) in ppm/(SF₆) in ppt) from breath gas samples collected around the animals’ nostrils. This ratio is adjusted for concentrations in background air (BG). The formula for calculating daily CH₄ production is as follows, where MWCH₄ is the molecular weight of CH₄ (14 g) and MWSF₆ is the molecular weight of SF₆ (146 g) according to Rochette et al. [65].

$${\mathrm{CH}}_4(\mathrm{g}/\mathrm{d})={\mathrm{SF}}_6 \mathit{release\; rate}(\mathrm{mg}/\mathrm{d})\times \mathrm{C}\mathrm{H}4 {\displaystyle \frac{\mathrm{C}\mathrm{H}4-\mathrm{C}\mathrm{H}4 Bachground }{\mathrm{S}\mathrm{F}6-\mathrm{S}\mathrm{F}6 Background}}\times {\displaystyle \frac{MW\mathrm{C}\mathrm{H}4}{MW\mathrm{S}\mathrm{F}6}}\times 1000$$

The SF₆ technique offers advantages, such as no animal confinement, cost-effectiveness, and non-invasiveness, making it suitable for studying CH₄ emissions from numerous grazing animals simultaneously, commonly in a nominal 24 h period [64]. However, challenges include assumptions about identical fluxes of SF₆ and CH₄ through animal nose and mouth, potential overestimation of CH₄ emissions with prolonged tube deployment, and the need for recovery tests [66]. Additionally, adjustments for ambient gas concentrations, consideration of wind conditions, and attention to SF₆ equipment characteristics are essential for accurate results and animal welfare.

7.3.4. Greenfeed Technique (GF)

The GF serves as a portable feeding station integrated with an automated head-chamber system intended for spot sampling of CH₄ emissions and gaseous exchange in ruminants [67]. The system measures CH₄ emissions from individual animals by integrating gas concentration data, airflow, bait feed intake, and radiofrequency identification. It automatically triggers gas sampling when an animal consumes bait feed, drawing air from the nose and mouth into an infrared gas analyzer to measure CH₄ levels. Average daily CH₄ output is calculated over several days. The GF is portable and automated, making it suitable for estimating individual CH₄ emissions under various conditions, provided that measurement timing is well controlled. However, the system has limitations, including high variability between days and animals, difficulty detecting effects of diet and animal factors, and potential interference from wind in grazing environments. Accurate estimates require strict control over the number and timing of animal visits to the GF unit.

7.3.5. Ventilated Hood (VH)

The VH, or breathing head box, is a basic version of the RC in which only the animal’s head is covered, maintaining principles of gas measurement [68]. It allows movement, access to feed and water, and minimizes air leakage through a neck sleeve. A system of air circulation within the hood connects to a multi-gas analyzer for concentration measurement, maintaining a slightly negative pressure. This approach reflects diurnal CH₄ emission changes and dynamic flux variations, providing an affordable option that is as accurate as RC. Agreement was observed between CH₄ production values in beef cattle dignified by the VH and their daily outputs by RC [69]. Additionally, the average output of CH₄ in dairy cows from a VH aligned with RC but with a 70% lower construction cost. Similar to RC, animals need restraint and training for adaptation to the VH or head box systems, measuring CH₄ emissions only from the mouth and nostrils, like SF₆, GF, and sniffing methods, without taking into account emissions of CH₄ from the hindgut.

7.3.6. The Sniffer Technique (ST)

The ST, introduced by Garnsworthy et al. [70], focuses on measuring gas concentration during the eructation of lactating dairy cows while they are being milked. It assumes a strong link between daily CH₄ production and its concentration and frequency in eructation. Gases are extracted into a tube in the feeding trough of an automated milking system and sent to an infrared analyzer for CH₄ measurement. Garnsworthy et al. [70] created a method to compute daily CH₄ emissions based on the average peak concentration and frequency of eructation. One of the ST’s benefits is the ability to quickly measure CH₄ concentrations from many cows in a commercial setting during milking. However, it shows greater variability among cows compared to other methods and has lower accuracy. Factors such as cow movements and feed trough designs can affect accuracy. The ST does not measure CH₄ production directly but uses prediction values based on regression equations developed from existing methods, which may require different formulas for various diets.

7.3.7. The Facemask (FM)

The FM uses a gas concentration analysis method similar to that of a VH and RC, focusing on spot sampling. A mask fitted on the animal’s muzzle connects to gas analyzers through a tube and is often used when an animal is in a squeeze chute. Sampling usually occurs for 30 min every 2–3 h, up to 7 times a day, although this can be less frequent if there is a strong correlation with total daily CH₄ emissions [71]. The FM is a cost-effective and portable option, is simpler than the SF₆ and GF techniques, and allows for checking more animals. Adding flow meters can enhance flux data. Short-term measurements with the facemask showed CH₄ levels similar to SF6 and RC in bulls, and comparable results were seen in dairy cattle. However, the FM requires more cooperation from the animal and limits eating and drinking during measurements. Pain from the squeeze chute may also disrupt the method. Measurement timing and frequency significantly affect results, with variability posing challenges in accurately assessing short-duration CH₄ mitigation strategies.

7.3.8. The Laser CH₄ Detector (LMD)

The LMD, created by Tokyo Gas Engineering Solutions Inc., is a portable device that measures CH₄ levels in the atmosphere using infrared absorption spectroscopy. A reference bar or reflective target is typically placed between the LMD and the animal’s muzzle to help guide the beam and ensure accurate CH₄ concentration readings in the animal’s exhaled breath [72], working effectively at a distance of 1 to 3 m. The measurement time is about 2 to 4 min, and CH₄ concentration is shown in parts per million-meter (ppm-m). It operates in temperatures from −17 °C to 50 °C and humidity levels from 30% to 90%. Originally designed for industrial areas like coal mines and landfills, the LMD is now a practical and affordable tool for measuring CH₄ in ruminants without disturbing them. Chagunda et al. [73] showed a positive correlation between LMD measurements and RC, indicating similar estimates of CH₄ emissions. The LMD can also detect changes in CH₄ concentrations from different cow activities. However, it only measures concentration, not flow, making it difficult to assess total CH₄ emissions because both concentration and airflow rate affect them. Factors like animal behavior and feeding regimens must be considered for accurate LMD evaluations. Environmental conditions can affect the LMD’s performance outdoors, limiting its use in pastures [74].

7.4. In Direct Methods (Statistical Models)

Several methods can estimate CH₄ produced by ruminants, either giving approximate values based on animal population or detailed information on factors like climate and nutrition. The IPCC [75] created national CH₄ emission inventories, classifying prediction methods into Tier 1, Tier 2, and Tier 3. Tier 1 uses animal population data for basic estimates. For better accuracy, some suggest country-specific values. Tier 2 provides more detailed estimations requiring in-depth data about energy use and key animals affecting emissions. The IPCC recommends refining Tier 2 and developing Tier 3 methods, which consider all factors influencing CH₄ emissions. Currently, many industrialized nations use Tier 2, but it overlooks significant factors affecting CH₄ energy losses. Ominski et al. [76] highlighted differences in CH₄ estimates between Tier 1 and Tier 2 methods, underlining the importance of improved accuracy using a comprehensive approach.

8. Sustainable Approaches to Reduce GHGs Emissions

To achieve the 1.5 °C reduction in global temperature target, it is essential to reduce agricultural CH₄ emissions by 11 to 30% of the 2010 level by 2030 and by 24 to 47% by 2050 [77]. This underscores the distinct obstacles that high-, middle-, and low-income countries must overcome [78]. Various recent technologies and practices have been harnessed in order to reduce GHG emissions associated with ruminants, especially CH₄, to achieve a greener and more sustainable future (

Table 2. Summary of CH₄ Mitigation Strategies in Livestock.

Strategy Type	Example Techniques/Additives	Mode of Action	Effectiveness	Challenges
Animal and Feed Management	Genetic selection, improved pasture, F:C ratio	Lower CH4 yield per unit product	Moderate–High (long-term)	Data-intensive, slow progress
Diet Formulation	Oils, tannins, saponins, seaweeds, microalgae	Inhibit methanogens, shift VFA profile	Variable (5–80%)	Cost, diet palatability
Rumen Manipulation	Probiotics, DFMs, ionophores, vaccines	Alter microbial fermentation, suppress CH4	Moderate–High	Regulatory limitations, consistency
Manure Management	Anaerobic digestion, composting, cooling, cover systems	Reduce CH4/N2O from waste storage	Moderate	Cost, infrastructure
Electron Acceptors	Nitrate, fumarate, sulfate	Compete with CH4 pathways	Moderate–High	Risk of toxicity (e.g., nitrate)
Emerging Technologies	Nanoclays, biochar, 3-NOP, halogenated compounds	Target methanogenesis enzymes or microbes	High (up to 90%)	Safety, acceptance, regulations

). These strategies included those focused on the animal itself or feed management, as well as others aimed at diet formulation, manure management, and rumen manipulation.

8.1. Animal and Feed Management Related Strategies

8.1.1. Genetic Selection

Numerous studies have shown that the heritability of CH₄ characteristics in dairy cattle ranges from 0.11 to 0.33. In sheep, the heritability of CH₄ yield is higher, between 0.24 and 0.55 [79]. Genetic selection is a long-term method to reduce CH₄ emissions, but it requires a multidisciplinary approach and large datasets on animals with CH₄ measurements, which only a few countries have. Progress is slow, but international groups are aiding in this research. Combining data globally could improve CH₄ trait accuracy. It is also essential to consider how genetic selection impacts productivity. Using residual CH₄ as a breeding trait may help select for lower emissions without affecting economic traits. The link between CH₄ emissions and feed efficiency is important; however, various factors can influence both [80]. Studies show heritability of CH₄ yield is under host control, leading to better physiological changes, such as ewes with low CH₄ yield weaning heavier, leaner lambs that produce more wool [81]. However, the challenge lies in measuring CH₄ in a sizable herd, even for industrial farms. Genetic selection programs require thousands of measurements, taken weekly, and the difficulty increases due to variations in grazing systems [82]. Animal selection necessitates the development of reliable biomarkers capable of estimating CH₄ production across different farm types. While pooling data from various studies could aid in creating a future genomic reference database, immediate action is crucial for reducing CH₄ and other GHGs from livestock [83]. If governments choose to implement animal breeding strategies for reducing enteric CH₄ production, there must be predictability, which can only be achieved through an adequate quantity of animals with genotypes and phenotypes, with the records being made open for free access.

8.1.2. Feed Management

• Forage management

Feed management measures are promising strategies for reducing CH₄ and overall GHG emissions [84]. Increasing cellulosic feed in animal diets raises enteric CH₄ formation, with variability influenced by forage type, digestibility, and chemical composition. Forage production systems also vary widely based on farm-specific factors such as location, climate, soil fertility, water availability, and management practices, creating opportunities for CH₄ mitigation through targeted forage strategies. Effective mitigation includes improving forage quality, optimizing harvest times, using highly digestible species, incorporating tannin-rich plants, and preserving nutrient content. To ensure effective results, these strategies should be evaluated using region-specific life cycle assessments that account for local forage and animal productivity.

• Forage-to-concentrate (F:C) ratio

Among the strategies to lower enteric CH₄ emissions, managing the F:C ratio is widely researched. In grazing systems, ruminants get concentrate feeds when pasture quality or quantity limits their performance [85]. Dairy cows on a high-forage diet produce 35% more CH₄ than those on a high-concentrate diet [86]. Fermenting cellulose in carbohydrates produces the most CH₄, while high-concentrate diets result in lower emissions [87]. Studies indicate that a higher concentrate ratio reduces CH₄ emissions and boosts growth in crossbred goat kids [88], with emissions decreasing as the concentrate ratio increases [89]. Rams on a high-concentrate diet also produced less CH₄ [90]. Adjusting the F:C ratio affects bacterial populations and CH₄ emissions, with lower ratios leading to less CH₄ [91]. Forage quality and higher concentrate levels help lower CH₄ production [92]. Research by Chagunda et al. [93] found that better-quality silage and more concentrate in diets lead to lower CH₄ emissions. Factors like forage type and maturity influence CH₄ emissions [94]. Balancing F:C ratios is important for maintaining production and animal health, as high concentrate can negatively impact milk quality and lead to metabolic issues [95]. Singh and Sharma [96] found that young goats gained weight with a diet that switched from concentrate to green fodder after weaning, emphasizing the importance of fodder quality and type.

• The pasture quality

Improving forage digestibility and increasing intake of digestible forage are widely recommended as key practices for mitigating CH₄ emissions [84]. The quality of fodder affects CH₄ production in animals. Feeding legumes can effectively lower CH₄ emissions [97]. Animals on high-quality pasture produced 14% less CH₄ based on energy intake and 11% lower per kg of dry matter intake (DMI) than those on lower-quality pasture. The tropical legume Desmanthus has also demonstrated a higher potential to reduce in vivo CH₄ emissions [85]. Vázquez-Carrillo et al. [98] investigated the impact of lemongrass, chamomile, and Mexican aster in cattle diets, reporting a 33% reduction in CH₄ yield with lemongrass and a 28% reduction with Mexican aster, while chamomile showed no effect. Common Egyptian forage legumes, such as leucaena (Leucaena leucocephala), acacia (Acacia saligna), prosopis (Prosopis juliflora), and atriplex (Atriplex halimus), have also shown promise in reducing CH₄ production. These legumes were tested in vitro and demonstrated a potential CH₄-reducing effect [99].

8.2. Diet Formulation

8.2.1. Feed Additives

• Fats and oils

Fats used as feed supplements can help reduce CH₄ emissions [33]. These substances can reduce the levels of certain microorganisms in the rumen, including methanogens and protozoa [100], which produce CH₄. However, for certain fats, the reduction in CH₄ elevated levels may be achieved at the cost of reduced diet digestibility. Lowering the rumen pH to between 5 and 6 can enhance the efficacy of lipid supplements in decreasing methanogenesis [101]. Fatty acids possess the capacity to damage the membranes of archaeal cells, which is vital for their metabolism, leading to less CH₄ production [22]. Adding fat sources to animal diets to achieve a fat content of 34 g fat/kg of dry matter in the diet resulted in an average reduction of 14% in long-term CH₄ emissions [102]. The effectiveness of fat supplementation in reducing CH₄ emissions depends on the type and amount of fat and the overall diet composition, with concentrate-based diets being more effective than forage-based ones [103]. When the fat content in the diet exceeds 6%, it can reduce feed digestibility, which could lead to more nutrients and organic matter being affected and potentially increase manure-related CH₄ emissions [104]. Additionally, fat supplements may decrease dry matter intake (DMI), fiber digestibility, rumen fermentation, and milk fat production [105]. In growing animals, oilseed supplementation reduces feed intake; weight gain might also decrease while maintaining similar milk yields [78]. Therefore, using oils and oilseeds might be more suitable for lactating animals than for growing animals. Certain oils, like sunflower oil, can reduce energy loss as CH₄ for cattle on high-forage diets, even if they also lower fiber digestibility [106]. Vegetable oils can reduce CH₄ emissions and increase the formation of propionic acid in ruminants [107]. Fats in ruminant diets help cut CH₄ emissions by inhibiting methanogen growth and decreasing fermentation capacity. Judy et al. [108] showed that supplements like corn oil and calcium sulfate can reduce CH₄ production while improving energy balance in lactating cows. There are five ways to reduce CH₄ through fat supplementation: (1) reduce fiber digestion, (2) decrease overall feed consumption if fat exceeds 6–7%, (3) lower methanogen levels, (4) decrease protozoan populations, and (5) accelerate biohydrogenation.

• Phytochemicals compounds

Several plant secondary composites, for example EOs, propolis, tannins, saponins, flavonoids, and organosulfur compounds, have been identified as possible modulators of ruminal microbial fermentation [109,110]. These plant secondary compounds represent natural phytochemicals that can influence rumen fermentation without inducing microbial resistance or leaving residual harmful effects on animal products [111]. In contrast to ionophores, the diverse active components present in plant extracts possess the capacity to influence ruminal microbiota via modes of action that are more potent, such as antimicrobial and antioxidant properties. This characteristic may mitigate the hazard of a decline in activity over time [112].

• Essential oils (EOs)

EOs are complex, aromatic compounds obtained from various plants through steam distillation. They can be extracted from different parts of plants and contain many active substances, mainly terpenoids and phenylpropanoids. These EOs can affect diverse rumen bacteria by interacting with their cellular membranes. Many EOs have antioxidant and antibacterial properties, helping to regulate ruminal fermentation. Unlike ionophores, EOs may work through mechanisms that last longer without losing effectiveness. Two mechanisms have been proposed to explain how the components of EOs work together to enhance antimicrobial activity [112]. First, phenolic compounds may increase cell membrane permeability, allowing terpene hydrocarbons to enter microbial cells and interact with proteins and enzymes. Second, phenolic compounds may alter the size or number of pores formed by terpene hydrocarbons in the cell membrane. Thus, the effects of EOs on rumen fermentation depend on their concentrations, types, diets, and adaptation periods, but most EOs can reduce CH₄ production. Patra and Yu [113] tested various EOs and found that all inhibited CH₄ production to different extents. Further research is needed to understand how these compounds interact with diet, their effects on CH₄ producers, and their impact on fermentation and fiber degradability. Attention is also needed for palatability, as some EOs may negatively affect this and DMI. There are questions about the suitability of commercially available encapsulated EOs as feed supplements, especially in developing countries.

• Propolis supplementation

Propolis is a blend of resinous materials gathered from deciduous tree buds, crevices in coniferous and deciduous tree bark, and secretions by honeybees [114,115]. Honeybees employ propolis to seal cracks, cover hive walls, and preserve intruding insects or small animals [116]. The composition of propolis exhibits significant variability depending on the bee collection site, influenced significantly by geographical location [117]. Its biological activity originates from its bioactive components, such as isoflavones, flavonoids, and fatty acids, which are known for their beneficial effects. Bee propolis has been highlighted as a natural substitute for antibiotics in the diets of ruminants [115]. In comparison to ionophores like monensin, various propolis sources demonstrate the ability to reduce CH₄ production while enhancing organic matter digestibility and VFAs both in vitro and in vivo [114,115]. This suggests that propolis has the potential to redirect degradation of ruminal organic matter away from CH₄ production toward microbial production and VFAs. In practical terms, propolis holds promise as a feed additive, particularly in regions where it is abundantly produced, such as Brazil.

• Saponins

Saponins constitute a category of plant secondary metabolites characterized by high molecular weight glycosides, wherein a sugar is linked to a hydrophobic aglycone. They are broadly categorized as steroidal and triterpenoid saponins [118,119]. Numerous studies have extensively examined the impact of saponins on modulating rumen fermentation [118]. The primary impact of saponins on biological entities occurs in the cell membranes of bacteria and protozoa. Saponins, especially lethal to protozoa, can create complexes with sterols found on the surface of protozoal membranes, resulting in the disturbance of membrane function [119]. This indirect impact extends to methanogenic archaea via their symbiotic association with ruminal protozoa [119]. However, certain sources indicate that the impact of saponins on rumen protozoa might be transient, as ruminal bacteria have the capability to break down saponins into sapogenins. The sapogenin compound lacks the capacity to influence protozoa [120].

• Tannins

Tannins are polyphenolic substances with molecular weights between 500 and 5000 Da, categorized into hydrolyzable tannins (HT) and condensed tannins (CT) [121]. CT forms strong complexes with dietary proteins and carbohydrates at ruminal pH, making them important in rumen modulation [99]. In addition, the effects of CT supplementation on CH₄ reduction vary in research. It is assumed that tannins might act as an H₂ sink, reducing H₂ available for converting CO₂ into CH₄ [122]. Additionally, CT directly impacts methanogens by binding with proteins or cell envelopes, disrupting the methanogens-protozoa complex and reducing H₂ transfer [122]. A secondary effect was indicated through anti-protozoal action, with some CT having a direct effect on rumen methanogenic archaea, regardless of protozoa. Tanniferous legumes and tree foliages are considered beneficial feeds for small ruminants, especially in drought-prone areas, to achieve CH₄ mitigation goals in developing nations [123,124]. Tannins may also help lower CH₄ production by forming complexes with proteins, which enhances animal productivity [123]. However, different studies show variability in results, indicating that tannin concentration, type, and molecular weight play roles in their effectiveness. However, the interaction of HT and other plant metabolites can also affect CT action [125].

• Flavonoids

Flavonoids, similar to tannins, are plant compounds known for their various biological activities, including antimicrobial effects [39]. Flavonoids may lower the populations of protozoa and methanogens in the rumen, which helps reduce CH₄ production by absorbing H₂ after breaking their carbon structures [33]. Using flavonoids in animal feed could boost productivity by increasing propionate production compared to acetate [126]. In vitro studies show that flavonoids like naringin and quercetin can cut CH₄ emissions and decrease certain microorganisms [127]. Commercial flavonoid extracts, like Bioflavex^®, have also shown promise in reducing CH₄ and increasing propionate levels when given at specific doses [128]. Other research suggests that luteolin-7-glucoside can lower CH₄ and NH₃ concentrations in rumen fluid without hurting fermentation efficiency. Many available flavonoid products are crude plant extracts, making it hard to assess their effects on rumen microbes. Advanced extraction methods are being explored to enhance efficiency and reduce environmental impact [129]. More in vivo studies are needed to fully understand flavonoids’ benefits in reducing CH₄ emissions.

8.2.2. Microalgae and Macroalgae (Seaweeds)

Algae can be used as an alternative to fish oil to reduce CH₄ production. Microalgae, like fish oil, have essential omega-3 fatty acids such as eicosapentaenoic acid and docosahexaenoic acid. Algae’s positive effects are linked to their secondary metabolites. Laboratory experiments show that microalgae can reduce methanogenesis, lowering acetate levels and increasing propionate levels due to their unsaturated fatty acids content, especially C22:6, n-3 [130]. Recently, Sucu [131] found that Chlorella Vulgaris and C. variabilis decreased acetate and increased propionate levels, along with less CH₄ production. Anele et al. [132] found significant CH₄ reductions when comparing different algae species to Micractinium reisseri and Chlorella vulgaris. Other studies indicated that Asparagopsis taxiformis and Dictyota bartayresii also reduced CH₄ compared to control [133]. Asparagopsis contains bromoform, which has antibacterial properties that help reduce CH₄ [134]. Adding Asparagopsis taxiformis to sheep diets resulted in an up to 81.3% reduction in CH₄ [91]. Similar results were seen in dairy cattle diets [135]. However, some species, such as Sargassum fulvellum, did not impact CH₄ production [136]. It is important to be cautious, as certain algae contain bromoform, a carcinogenic compound [137].

8.2.3. Enzymes

In a comprehensive analysis of the effect of supplemental fibrolytic enzymes in the diets of ruminants. Enzymes supplementation has the potential to enhance the productive performance of both beef cattle and dairy cows [138]. However, the effectiveness of this improvement is contingent upon maintaining an appropriate ratio of xylanases to cellulases in accordance with the diet’s composition. Dietary supplementation with hemicellulases and cellulases has been shown to have positive effects on fiber digestion and overall productivity in ruminants [139]. Consequently, these enzyme additions led to a substantial reduction in in vivo CH₄ production, with a notable decrease of 28% and 9%, respectively [140].

8.2.4. Chitosan (CHI)

CHI is a natural polymer that is non-toxic, biocompatible, and biodegradable, making it safe for humans and animals [141]. CHI is made up of units derived mainly from marine crustaceans and can also be found in fungi, crustaceans, mollusks, insects, and algae [142]. It offers various benefits, including antioxidative, antitumor, anti-inflammatory, and antimicrobial effects. Its antimicrobial properties target fungi, bacteria, and protozoa and have gained attention for modulating rumen fermentation [143,144], showing potential in reducing CH₄ emissions [141]. The antimicrobial action of CHI involves interaction with cell membranes, leading to cell destabilization and death. Its effectiveness depends on diet type and ruminal pH, being most effective in diets with grains at low pH levels. This can shift ruminal fermentation patterns, increasing propionogenesis and affecting specific bacteria [143,144,145], contributing to a reduction in CH₄. Furthermore, CHI supplementation has been shown to modify communities of bacteria in the rumen associated with fatty acid biohydrogenation, specifically affecting the Butyrivibrio group and Butyrivibrio proteoclasticus [146].

8.2.5. Chemical Modifiers Feed Additives

Various chemical additives are used to control rumen microbial activity to improve animal productivity. This includes defaunating agents and anti-methanogenic agents that specifically aim to lower CH₄ emissions. Studies confirm the effectiveness of nitrate in replacing CO₂ as electron acceptors leading to production of NH₃ instead of enteric CH₄ [147,148]. Meanwhile, nitrates can lower CH₄ emissions in different ruminants, such as sheep (23%), dairy cows (16%), and beef cattle (17%) [149,150,151]. Caution is needed with nitrate use due to possible toxicity at high amounts [33,148,152]. These agents work through different mechanisms, and when used together, they further lower CH₄ emissions. Anti-methanogenic agents include halogenated sulfonated compounds like 3-bromopropanesulfonate and 3-nitrooxypropanol (3NOP), which inhibit the activity of methyl-CoM reductase, the final step in methanogenesis. Other compounds, such as lovastatin, can inhibit enzymes critical in methanogen cell membranes. Nitrate is beneficial for reducing methanogenesis and provides nonprotein nitrogen (NPN), especially in low-quality diets. In the rumen, some microbes can convert nitrate into nitrite and then NH₃, creating competition with methanogenic archaea.

8.2.6. Nano Clays Additives

Geophagy is the practice of eating clays, observed in ruminants, with certain clay types like montmorillonite and zeolite considered safe for use by both animals and humans [153]. Montmorillonite clay, also known as microcrystalline kaolinite, is popular due to its availability, low cost, large surface area, high ion exchange activity, and tiny particle size [153]. It acts as a buffering agent that helps prevent acidosis, bloat, and diarrhea while also adsorbing heavy metals and aflatoxins [154]. Thus, it is frequently used as a feed additive for ruminants [155,156]. Recently, a promising in vivo investigation highlighted the potential of Arabic gum-nano montmorillonite as a natural candidate to replace monensin in enhancing ruminal VFA production, nutrient digestibility, feed efficiency, blood metabolites, and milk yield in dairy cows [157]. The application of montmorillonite as a rumen modifier to lower in vitro rumen CH₄ emissions, showing it inhibits methanogens, has been explored [153]. The ionic structure of montmorillonite allows it to engage in ion exchange reactions effectively [158]. It can be modified with cationic surfactants like quaternary ammonium salts to harm Gram-positive bacteria, or with anionic surfactants like sodium dodecyl sulfate (SDS), which enhances its cation exchange capacity and absorption of heavy metals [158]. A study by Soltan et al. [159] found that SDS-modified montmorillonite reduced CH₄ emissions by 38% at specific dosages. Grinding montmorillonite has also shown benefits in stability and antibacterial activity against Escherichia coli [159]. Recent work on zeolite at the nanoscale has shown improved chemical stability and reduced CH₄ and NH₃ emissions while aiding fiber degradation [160].

8.2.7. Other Strategies

• Biochar supplementation

The potential impact of administering biochar and potassium nitrate on CH₄ emissions has been assessed [161]. The inclusion of biochar at 0.6% of the diet’s DM and potassium nitrate at 6% of the diet’s DM resulted in a reduction of CH₄ emission by 22% and 29%, respectively. When combined, the effects were additive, leading to a 41% reduction in CH₄ production.

• Halogens

Halogenated derivatives like chloroform, 2-bromo-ethane sulphonate (BES), and bromochloromethane (BCM) are strong inhibitors of CH₄ emission in ruminants, allowing for significant reductions [162]. While some compounds, such as BCM, are banned, alternatives in macroalgae like Asparagopsis taxiformis also inhibit CH₄ production by blocking methanogenesis [163]. Studies show that adding 3% Asparagopsis taxiformis to sheep diets can reduce CH₄ emissions by 50% to 80% [163]. In dairy cows, a mixture containing 5% of this macroalgae led to a 95% reduction in CH₄ [135]. Other research has tested methods like 3-nitrooxypropanol (3NP) [164], which reduces CH₄ emissions by 24% in sheep [165] and 70% in cattle [166].

8.3. Rumen Manipulation Strategies

8.3.1. Direct Fed Microbials (DFMs) or Probiotics

DFMs are live cultures of organisms that enhance rumen microflora, benefiting animal health [167]. Rumen bacteria compete with methanogens for H₂, promoting propionogenesis pathways and reducing methanogenesis [168]. Propionic acid bacteria (PAB), like Propionibacteria, are naturally found in the rumen and aid in propionate production by utilizing available H₂. Since H₂ limits CH₄ production, adding these bacteria can potentially lower CH₄ formation process [169]. Various PAB strains, including Propionibacterium acidipropionici and others, are being studied for their capability to reduce CH₄ emissions [170]. Notably, Propionibacterium thoenii T159 showed a 20% reduction in CH₄ and a 21% increase in total VFAs in in vitro trial [171]. However, these bacteria face challenges in surviving high-starch diets in cattle, impacting their effectiveness [172]. Acetogens, a group of bacteria that produce acetate, are found in large numbers in the rumen and can utilize H₂ to reduce CO₂ or sugars for their proliferation [173]. Some studies suggest they could serve as alternative H₂ sinks, but their effectiveness is limited compared to CH₄-producing bacteria [174]. CH₄-oxidizing bacteria (MOB) can grow on CH₄ and oxidize it using specific enzymes, but research on using them as probiotics for cattle is limited [175]. Although some studies indicate that CH₄ oxidation can occur in rumen microbes, in vivo studies using MOBs as probiotics are still needed [170]. Methanotrophic bacteria could be utilized to reduce CH₄ and provide protein in livestock feed [176].

8.3.2. Ionophores

Ionophores are commonly used in beef cattle diets to reduce CH₄ emissions. These additives come from Streptomyces spp. and work by enhancing the permeability of certain bacterial membranes, slowing down bacterial growth, and changing how feed is fermented in the rumen. Several ionophores, such as lasalocid, monensin, salinomycin, laidlomycin, and narasin, are available and function similarly in the rumen. They promote specific bacteria that limit the food sources for CH₄-producing bacteria [177]. Monensin is the most widely used ionophore in cattle feed to improve feed efficiency and control coccidiosis. It is typically recommended at 20 to 50 mg/kg of complete feed and can reduce CH₄ emissions by up to 30% [178]. However, its effectiveness may decrease over time due to microbiota changes, and the optimal dosage can vary based on the health of the animal and herd [179]. Although ionophores are generally accepted, regulations differ by region, and there are growing concerns about their long-term use in livestock due to possible future regulations and consumer attitudes [180].

8.3.3. Vaccines for Limiting Methanogenesis

The development of vaccines to reduce CH₄ production focuses on enhancing the animal’s immune system to produce antibodies in saliva. These antibodies aim to inhibit the growth of methanogens in the rumen [181]. Another method involves using chicken egg antibodies, which allows for quick and inexpensive antibody production without altering agricultural systems [182]. This makes vaccination a promising approach to lowering CH₄ emissions, especially in pasture-based farms. For a vaccine to be effective, it must generate sufficient antibodies in saliva that can bind to various methanogen species. However, comparing studies on the effectiveness of these vaccines is complicated due to differing adjuvants, vaccination protocols, immunization methods, immunoglobulins used (IgG, IgA, and IgY), diverse approaches (in vivo and in vitro), and samples (blood, saliva, and rumen) [183]. In vitro studies consistently demonstrated a reduction in CH₄ release, ranging from 7 to nearly 70%, depending on antibody types and immunization protocols [183]. However, translating these positive in vitro results into significant in vivo changes has been less pronounced or even ineffective in certain cases [184,185,186]. A vaccine targeting protozoan antigens did not reduce the population in Merino sheep [29]. More research is necessary to assess the effectiveness and practicality of this vaccination strategy [183].

8.3.4. The Elimination of Protozoa from the Rumen Ecology (Defaunation)

Rumen defaunation is the process of removing protozoa from the rumen ecosystem, which play an important role in producing H₂ for CH₄ production. About 37% of rumen CH₄ emissions are linked to protozoa-associated methanogens [36]. Holotrich protozoa are especially effective at producing H₂ and have a stronger influence on CH₄ production compared to other types. Some studies show no change in CH₄ production among sheep with different protozoa communities [187], while others stress the importance of certain protozoa in methanogenesis [188]. Defaunation can lead to a 10–13% decrease in CH₄ emissions, an increase in propionate levels, and lower levels of acetate and butyrate in the rumen [189,190]. Although findings suggest little change in methanogen abundance [188], defaunation can enhance protein synthesis and nitrogen flow in the gut, improving livestock productivity and reducing CH₄ emissions. However, maintaining protozoa-free animals is challenging due to the need for rapid reinoculation to prevent animal cross-contamination. Defaunation is more complicated in cattle compared to sheep due to differences in rumen structures [191]. Furthermore, eliminating protozoa may lower the digestibility of organic matter and decrease DMI [190].

8.3.5. Electron Receptors (H₂ Sink)

In the rumen, archaea help break down food, producing CH₄ mainly by using H₂ and CO₂ [162]. alternative electron acceptors, including fumaric acid and myristic acid, have been investigated, demonstrating their potential to reduce CH₄ emissions [147]. Adding 2% fumaric acid to cattle silage can reduce CH₄ by 23% [192]. In lamb trials, a free-form fumaric acid provided at a specific amount led to a 62% drop in CH4 output, while the encapsulated form caused a 76% decrease [193]. Furthermore, myristic acid, when given to sheep, reduced CH₄ emissions by 22% in forage diets and 58% in concentrate diets [100]. However, the impractical high doses required make dicarboxylic acids economically prohibitive for widespread use [194].

8.4. Manure-Related Strategies

Animal manure is categorized into fluid, sludge, and solid forms and needs careful management to reduce GHG emissions, environmental harm, and health risks. This management includes collection, storage, treatment, transportation, and disposal. An effective system should prevent manure from affecting the environment while being cost-effective. Applying manure to fields for fertilization can lessen the need for nitrogen fertilizers and reduce nitrogen loss. Animal housing should enable easy collection of manure and safeguard against losses, with floors designed to keep rain off to prevent nutrient loss. Regular manure removal is crucial to avoid fermentation and GHG emissions, as keeping it too long can increase CH₄ emissions [195]. Storage time ranges from 3 to 10 months based on climate. Solid manure generates less CH₄; however, covering liquid manure storage helps maintain oxygen levels, which reduces N₂O and CH₄ emissions while controlling NH₃ odors [195]. Studies show varying CH₄ emissions depending on storage methods. Covering manure can lower NH₃ and CH₄ emissions but might increase N₂O emissions [196]. Cooling storage to below 15 °C can also cut CH₄ output [197]. Methods like anaerobic digestion convert livestock manure into biogas, which can be used as a renewable energy source for electricity, heating, or fuel, thereby reducing methane emissions and enhancing energy sustainability [198], while composting aids in odor control and pathogen elimination [152]. Improved timing and techniques for applying manure can lower emissions and costs for farmers, highlighting the need for effective manure management that considers environmental and economic factors.

9. Conclusions and Recommendations

This review has addressed the urgent need for effective mitigation of (GHGs) emissions, particularly methane, in livestock systems. Guided by the objectives of (1) summarizing methane measurement methods, (2) evaluating sustainable mitigation strategies, (3) assessing their trade-offs, (4) identifying research and policy gaps, and (5) proposing integrative approaches, we provided a comprehensive synthesis of the current scientific landscape.

The findings demonstrate that integrating innovative measurement technologies—such as in vitro fermentation models, in vivo analyzers, and indirect estimation tools—with sustainable practices like dietary reformulation, rumen microbiota modulation, and manure management holds substantial promise for reducing methane emissions in a practical and scalable manner. These strategies, though varied in complexity, collectively contribute to a greener and more climate-resilient livestock sector.

Despite these advances, challenges persist in ensuring wide-scale adoption, cost-efficiency, and system compatibility across different production contexts. Moreover, technological solutions must be supported by robust policy frameworks, stakeholder engagement, and continuous research to ensure long-term effectiveness.

To translate these insights into action, the following recommendations are proposed:

• Invest in innovation and scaling: Continued research and development in precision livestock technologies, such as automated feeding systems, CH₄ sensors, and microbiome-targeted feed additives, is essential to enhance emissions control and productivity.
• Promote knowledge transfer and capacity building: Establish farmer training programs and extension services focused on sustainable livestock management and methane mitigation.
• Enable supportive policy and incentives: Governments should offer financial incentives (e.g., subsidies, carbon credits) to encourage the adoption of climate-smart technologies and practices in livestock production.
• Foster multi-stakeholder collaboration: Strong partnerships between farmers, researchers, policymakers, and industry actors are needed to ensure practical implementation and continuous improvement of mitigation strategies.
• Enhance monitoring and accountability: Deploy standardized monitoring and reporting systems to evaluate environmental performance and ensure transparency in mitigation outcomes.
• Encourage sustainable consumption: Public awareness campaigns and policy nudges promoting responsible meat and dairy consumption can complement supply-side efforts to reduce emissions.
• In conclusion, this review contributes to the scientific foundation for designing and implementing integrated, data-driven strategies for methane mitigation in livestock systems. It provides decision-makers and practitioners with an evidence-based roadmap for advancing environmental sustainability in animal agriculture while maintaining productivity and food security.

10. Future Perspective

The Paris Agreement targets a 24–47% reduction in CH₄ emissions from animal husbandry by 2050 compared to 2010 levels. Livestock is responsible for 9–25% of global anthropogenic GHG emissions, with the variation due to different sources and models. While selective breeding offers long-term potential for CH₄ mitigation, immediate reductions can be achieved through dietary reformulation and feed supplements. However, effective strategies must be species-specific and take into account economic feasibility for producers and consumer perceptions of additives. Research continues to explore solutions that simultaneously lower CH₄ emissions and improve animal performance, with safety for both livestock and consumers remaining a priority. Innovative technologies like nanotechnology may also play a role in safe, effective feed additive development. The growing global population is driving increased demand for meat and dairy, leading to a rise in livestock numbers and associated CH₄ emissions from rumen fermentation. Meeting the Paris agreement goals requires coordinated, in vivo research that considers the economic limitations of developing nations and local livestock practices. Strategic implementation of nutritional and farm management innovations, along with breeder engagement and climate change awareness, is key to sustainable animal production.