Methane Synthesis as a Source of Energy Loss Impacting Microbial Protein Synthesis in Beef Cattle—A Review

Simple Summary

This review explores the relationship between ruminal methanogenesis and microbial protein synthesis (MPS) in beef cattle, highlighting methane (CH₄) as a significant energy sink that can divert up to 12% of dietary gross energy from MPS. Methane synthesis, crucial for maintaining redox balance in the rumen, competes with propionate production, an efficient pathway supporting microbial growth. Dietary factors, including carbohydrate sources and fermentability, nitrogen synchronization, and passage rate, simultaneously influence MPS and CH₄ synthesis and production. Despite forage-based diets tending to increase methane production while exhibiting reduced MPS efficiency, an analysis of real data might suggest that such a relationship might not be inverse. Plant secondary metabolites, methane inhibitors, and optimized forage-to-concentrate ratios, show promise in reducing CH₄ emissions while improving nutrient utilization. This review emphasizes the need for integrated approaches, including dietary strategies, feed additives, and predictive models, to enhance MPSE and reduce the environmental footprint of beef production.

Abstract

Ruminal methanogenesis represents considerable energy loss within the fermentative processes mediated by microbial populations, by means of which up to 12% of gross energy intake is driven away from microbial protein synthesis (MPS). This review explores the relationship between methane (CH₄) synthesis and emission with MPS in beef cattle, focusing on the nutritional, biochemical, and microbial factors modulating these processes. The synthesis of CH₄ by ruminal archaea is essential for maintaining redox balance during the fermentation of carbohydrates. This process diverts metabolic H₂ from energy-efficient pathways like propionate synthesis, which could otherwise enhance microbial growth. Dietary factors, including carbohydrate fermentability, N synchronization, and passage rate, modulate MPS. Diets based on roughage might enhance CH₄ synthesis while impairing MPS efficiency by reducing diet digestibility and promoting microbial shifts towards methanogenic populations. Potential mitigation strategies, including plant secondary metabolites, CH₄ inhibitors, and controlled forage-to-concentrate ratios, demonstrate the potential to reduce CH₄ emissions while enhancing nutrient utilization. This review underscores the need for integrated approaches combining dietary strategies, advanced feed additives, and improved prediction models to optimize ruminal fermentation, enhance MPS, and reduce the environmental footprint of beef cattle systems.

1. Introduction

1.1. Global Significance of Methane Emissions in Ruminant Production Systems

Besides representing a loss of up to 12% of gross energy from the diet [1], methane (CH₄) from enteric fermentation [2,3] makes beef production, a notable contributor to global warming [4,5] particularly in the United States [6]. Animal scientists globally aim to reduce these emissions due to their implications on climate change the necessity to sustainably meet the demand for animal protein [7] and the current and future goals of limiting global warming [8]. Global warming has been associated with significant increases in greenhouse gas (GHG) emissions derived from economic activities, including livestock production [9]. Methane exhibits an elevated global warming potential (about 30 times higher than carbon dioxide (CO₂), representing a major threat to environmental sustainability [10]. Enteric CH₄ from ruminal fermentation represents 24% of GHG from livestock production [3,11], out of which 61% come from beef cattle production [12,13] particularly from cow-calf and backgrounding operations, which emitted around 83% of CH₄, of all beef cattle systems, given their grazing-based characteristic [14,15,16]. Several studies have shown that an elevated intake from forage and roughage has a direct link to CH₄ emission [17,18,19] and poor productive performance in beef cattle [20,21] and dairy cattle [22]. Thus, CH₄ mitigation is a desirable goal to guarantee the sustainability of beef production and achieve climate neutrality goals worldwide [23]. The interest in mitigating CH₄ has increased dramatically in recent decades by around 2400% since 1990 [24]. Enteric emissions of CH₄ from ruminants, particularly beef and dairy cattle, are significant contributors to global greenhouse gas outputs. While dairy cows produce more methane per animal (an average of 408 g/d) due to their higher feed intake for milk production [9], beef cattle contribute substantially at a global scale (average of 270 g/d for an adult steer) due to the large populations raised under extensive production systems based in grazing fibrous pastures, often characterized by lower feed efficiency and higher methane emissions per unit of product [15].

Enteric CH₄ emission from cattle are recognized as a significant energy sink in the process of ruminal fermentation of carbohydrates (CHS) [25,26,27,28]. Methanogenesis acts as a recipient of hydrogen (H⁺⁾ from oxidative reactions [29,30] during the fermentation of CHS into volatile fatty acid (VFA) synthesis [31,32,33,34], which is linked to carbon (C) derived from ruminal fermentation of CHS [34]. Thus, CH₄ synthesis is essential for maintaining ruminal fermentation by disposing of metabolic H⁺, reducing the pressure derived from its accumulation [7,35].

Besides representing an energy loss in the fermentation process mediated by ruminal microbes, CH₄ synthesis could simultaneously impact ruminal efficiency by impairing MPS in the rumen. This assumption has been previously addressed by [36], who observed that an excess of non-structural carbohydrates (NSC; 40% of DM) and soluble protein in the diet, resulted in an increment in CH₄ emission, and at the same time led to a reduction in MPS efficiency (MPSE).

1.2. Overview of Microbial Protein Synthesis (MPS) in Beef Cattle

The process of MPS is modulated by through the delicate fine-tuning of three key dietary elements; energy availability, nitrogen (N) content, and rate of passage [37,38,39]. Bacterial synthesis primarily relies on energy derived from CHS sources like cellulose, starch, and glucose [40]; however, an excess of readily fermentable carbohydrates can lead to a drop in ruminal pH [41], reducing MPSE [42]. Likewise, increases in the rate of passage, especially in wet diets, can also limit MPS due to incomplete cellular cycles before ruminal washout [43]. Roughage-based diets may reduce MPS by reducing the turnover rate of ruminal passage, which reduces the need for the growth of new bacteria [44]. Coincidentally, previous evidence has shown that beef cattle consuming roughage-based diets exhibit a greater enteric CH₄ emissions [45,46], which suggests an association between methanogenesis and MPS.

The efficiency of MPS is often defined as the amount of MPS per kg [47] or 100 g of organic matter (OM) digested in the rumen [48]. This process is paramount because it provides around 50% of the amino acids required to synthesize milk and meat protein in ruminants [49] making it a key factor in the global production of animal protein. Thus, considering the potential correlation between methanogenesis and microbial growth, the objective of this review is to explore the evidence connecting CH₄ synthesis as a hydrogen and energy sink in the ruminal environment and how it could impact MPSE. This review offers a unique perspective by focusing on the interaction between CH₄ production and MPS in beef cattle, highlighting how methanogenesis acts as an energy sink that impacts microbial efficiency, and how several dietary, management, and animal factors overlap in the modulation of these parameters. The scope of this review is limited to research products focused on enteric CH₄ methane emission and/or MPS in beef cattle. The literature on the role of methanogenesis in energy metabolism in ruminants was also considered. The review also considered scientific papers assessing CH₄ mitigation strategies, such as dietary modifications, plant secondary metabolites, and microbial manipulation.

2. Microbial Protein Synthesis in the Rumen

2.1. Relevance of MPS in Ruminant Nutrition

The microbial mass synthesized in the rumen represents the greatest and most efficient source of protein for beef cattle [50], representing a vital portion of the protein requirements of ruminants [51]. Such requirements for cattle are typically expressed in terms of metabolizable protein (MP) per day, divided into rumen degradable protein (RDP) and undegradable protein (RUP) [47]. Although amino acid (AA) requirements would be ideal for diet formulation, limitations in predicting microbial growth mean most diets are still formulated based on crude protein (CP) [52]. Adjustments to MP requirements significantly reduce RDP requirements, which are largely supplied by MPS contributing to more than half of the amino acids reaching the small intestine [53]. Understanding MPS and RDP components of feeds is crucial for efficiently meeting the microbial RDP needs and overall MP requirements in growing cattle [54]. A plethora of studies have proposed that MPS is affected by external factors, including N content, forage: concentrate ratio, N sources, energy: N synchrony, sulfur availability, and microminerals, as well as internal factors including ruminal pH, protozoa population, and the fractional rate of passage [55,56,57,58,59,60,61,62].

Bacterial protein synthesis primarily relies on energy derived from CHS sources like cellulose, starch, and glucose [40]; however, excessively fermentable CHS can lead to a drop in ruminal pH [41], reducing MPSE [42]. Likewise, the increase in the rate of passage, especially in wet diets, can also limit MPS due to incomplete cellular cycles before ruminal washout [43]. In the same sense, previous studies [63] showed how increases in dilution rate (from 2 to 12%/h) resulted in a greater production of bacterial dry matter per mol of ATP derived from ruminal fermentation. While a lower MPSE in grazing animals has been associated with the elevated level of structural CHS and a reduced ruminal outflow [53,57]. Intermediate levels of fermentable carbohydrates combined with a high RDP:RUP proportion has shown greater MPS, indicating the relevance of N-energy synchrony on MPSE [64,65]. Moreover, diets with elevated concentrations of crude protein (CP) and readily fermented CHS may exhibit reduced MPSE when AA (as the main source of CP) are replaced by non-protein nitrogen (NPN) as a source of RDP [36]. In addition, the effect of including NPN varies according to the source of CHS in the diet. Thus, in backgrounding diets characterized by reduced CP and high roughage inclusion, an elevated NPN supply in the diet can improve the MPSE observed in fibrolytic populations [66]. This evidence highlights the importance of synchrony between the source, type and rate of N and CHS to be fermented leading to microbial growth (i.e., MPS). Similar to the contrasting effects on MPS by different N sources, excessive dietary energy due to a high inclusion of rapidly fermented CHS may negatively impact bacterial synthesis, promoting the deposition of reserve CHS by ruminal microbes using the surplus of glucose in ruminal media [67].

2.2. Key Factors Influencing MPS

Bacterial growth relies on two simultaneous processes, the denaturalization of lysis of dietary protein and MPS, which are affected by ruminal pH [60,61], sulfur availability [36] microminerals [57], protozoa population [56], and forage:concentrate ratio [58,59,68,69]. Beyond the source of CHS, the central aspects regulating MPS are N content, energy availability [38,70,71], and digesta rate of passage [37,39,72].

2.2.1. Energy Availability and Nitrogen Balance

Imbalances in the supply of energy, especially those rich in NSC, promote the rapid growth of amylolytic bacteria while fibrolytic populations may be suppressed [73]. This can lead to an imbalance in the microbial community, altering fermentation dynamics and nutrient digestion. Indeed, several pieces of evidence suggested that either a deficiency or an excess of total digestible nutrients (TDN) in the diets might impair MPSE [74,75]. Likewise, an elevated neutral detergent fiber (NDF) content in forage-based diet has often been associated with a reduction in dry matter intake (DMI) [76,77,78], which has been negatively correlated with MPS [53,79]. Confirming these observations, previous studies have observed a positive and significant relationship between DMI and ruminal MPS in bovine [61,76,77,78,80]. Thus, MPS might be promoted via increasing DMI. Indeed a previous study [81], observed significant increases in MPS after supplementing starch (25% of steam-flaked corn) in adult dairy cows receiving a fibrous diet (35% of corn cobs), leading to a greater DMI (extra 1.3 kg of DM/d) and digesta passage rate (5% greater). In contrast, an elevated proportion of NSC (55%) in diets containing elevated CP levels (16%) fed to ruminally cannulated steers negatively impacted MPS [36]. Moreover, these authors observed that the highest MPS was registered when intermediate levels of fermentable CHS were combined with a high proportion of RDP:RUP (60:40), demonstrating the effect of N: energy synchrony on MPS.

2.2.2. Nitrogen Content and Sources

Regarding the modulation of MPS by soluble N, the sources of N in a cattle diet includes peptides [82], AA [83], branched AA [84], and NPN [85,86,87]. Despite most bacteria using peptides and or AA [66,88] as a source of N, most fibrolytic bacteria have the ability to use NPN sources to synthesize the AA required for their cellular structures [89,90,91]. Nitrogen sources incorporated by ruminal microbes may influence MPSE. For example, while Ruminococcus albus growth has a greater affinity for NPN to sustain MPS [66], an amylolytic bacteria, such as Streptococcus bovis, increased MPS by about 100% following AA supply in an NPN culture [92]. Replacing AA with NPN in the dietary RDP will reduce EMPS, especially in diets with elevated concentrations of CP (>16%) and readily fermented CHS [93,94]. Conversely, reducing NPN from 65 to 40% of the total N increased MPS in sheep-fed alfalfa silage [95]. Similarly [87], working with dairy cows and replacing urea and cottonseed meal, observed an increase in MPS from 26 to 29 g of microbial N/kg of organic matter (OM) truly digested in the rumen. Taken together, these findings might suggest that a greater AA availability from true protein sources will increase MPS. Such a scenario might not be usual in backgrounding diets, characterized by moderated CP and high roughage inclusion, in which NPN in the ruminal fluid may enhance EMPS for fibrolytic populations. Indeed, beyond the N source, energy-N synergy should be considered since under a similar CHS supply, MPS was impacted to a greater extent when AA was added compared to NPN [91]. This response may be derived from an increased maintenance requirement (energy spilling) of ruminal bacteria. Such a hypothesis was later confirmed by [67], who evaluated the addition of excessive NSC and registered increasing energy spillage, glucose intake, heat production, and CHS deposition (glycogen) in ruminal bacteria but not cellular growth. Alongside a sufficient level of N, bacterial synthesis entails the availability of energy derived from OM fermentation, particularly from readily fermentable CHS such as starch and glucose [40]. These biomolecules supply C skeletons and energy (ATP, and reduced cofactors) to bacteria to build the electric bonds of AA in their cellular structures [70]. Energy imbalances derived from an excess of rapidly fermentable CHS lead to a drop in ruminal pH [41], which has been correlated with reduced MPSE [60], a useful parameter associating gram of synthesized bacteria per gram of fermented OM. While ruminal pH values below 5.5 are not common under forage-based conditions, they may be registered when an elevated proportion of concentrated feed is included in the diet of beef cattle [76,77,78]. Despite the transition towards high-grain diets in a feedlot setting may enhance protein degradation but not MPS [90] because excessive dietary energy promotes microbial energy spilling, reducing bacterial growth and MPS [41,67].

2.2.3. Rate of Passage and Synchronization of Nutrient Availability

Regarding the modulation of MPS from the rate of passage, accelerated rates in diets with elevated moisture levels will lead to a moderate reduction in MPS [51] due to the impossibility of the renewal of cellular cycles to generate new microbial cells before ruminal washout [43,58]. Similarly, high-fiber rations (i.e., backgrounding diet) with elevated NDF content, diminish the availability of OM to be fermented, while also increasing the ruminal outflow of partially digested particles [57]. Under backgrounding diets, a high NDF content impairs MPS by reducing the rate of CHS digestion and slowing the turnover rate of liquid and particulate phases [44]. In contrast, recent studies showed that a greater starch: NDF proportion in the diet results in greater MPS in dairy cows [61] due to an increase in the passage of smaller particles [37], reducing OM digested in rumen, and ruminal microbial turnover. This process leads to a greater release of growth-related enzymes by ruminal bacteria, enhancing MPS [96]. Previous studies observed how under a greater dilution rate of ruminal fluid, bacteria flow out of the rumen at a faster rate, reducing their maintenance requirements and increasing the MPSE [67]. Such an increase is the result of a reduced time of bacterial retention in the rumen [97] and a faster passage of ruminal microorganisms to the small intestine, which decreases bacterial recycling [49,98]. Interestingly, some plant secondary metabolites (PSM) like saponins and tannins, may alter the rate of passage, and increase the dilution rate, which may lead to an enhanced MPS [65,70]. However, beyond attributing changes in MPS to a sole factor, most studies agree that EMPS is a result of the synchrony between N and energy available in the rumen [64,65]. It could be theorized that a high concentration of rapidly fermentable carbohydrates in the absence of sufficient N might lead to excess hydrogen production, which could drive methanogenesis as a hydrogen disposal mechanism. Indeed, the apparent lack of synchrony between degradable protein and CHS in the diet of goats seems to affect CH₄ emissions [99].

3. Methane Synthesis in Rumen Fermentation

3.1. Biochemical Pathways of Methane Production

Carbon dioxide (CO₂) is a byproduct of CHS fermentation by ruminal microbes [29,100,101]. This molecule is linked to H⁺ or formate to synthesize CH₄, an important metabolic reaction performed by hydrogenotroph archaea (Methanobacterium, Methanomicrobium and Methanobrevibacter spp.) [102]. Besides these main species, methylotrophs (Methanosphaera spp.) are able to utilize methyl compounds to synthesize CH₄, while acetoclastic organisms (Methanosphaera spp.) are able to uptake acetate to do so [103,104]. The metabolic process of CH₄ synthesis is rate-limited by methyl coenzyme M reductase (MCMR), which mediates the final step of the methanogenesis pathway [105,106,107,108] as shown in Figure 1.

Hydrogenotrophic methanogenesis is the main pathway where methanogens utilize H₂ to reduce CO₂ into CH₄, which has been characterized by Methanobrevibacter and Methanosphaera, consuming H₂ produced during fermentation and preventing its accumulation [90]. In contrast, during acetoclastic methanogenesis genera like Methanosarcina and Methanothrix (formerly Methanosaeta transform acetate into CH₄ and CO₂. While this process does not directly involve H₂, it plays a significant role in methanogenesis since ruminal environment possess abundant acetate [103]. Some methanogens from the Methanolobus genera can utilize methylated compounds, such as methanol or methylamines, to produce CH₄ using the methylotrophic pathway. Interestingly in certain cases, these genera can also use H₂ in the reduction of methyl compounds to CH₄, thereby contributing to hydrogen utilization in the rumen [104].

In addition to C and the rate-limiting enzyme, the hydrogen necessitated for CH₄ formation comes from the total pool of hydrogen atoms that can be exchanged among molecules within the microbial cellular structure and also from microbe-microbe interaction [34]. More than 80% of the methane synthesized by Archaea depends on the uptake of the dissolved H₂ form available in the rumen environment [109]. Similar to CO₂, hydrogen is a byproduct of CHS microbial fermentation, which maintains strict metabolic control on the balance between H⁺ produced and consumed [110] promoting proper rumen fermentation, as partial pressure in the rumen depends to a great extent on the dissolved H₂ concentrations [29]. Interestingly, this same author highlighted how the concentration of H₂ affects the thermodynamics of ruminal fermentation to such extent that its accumulation might reduce the thermodynamic of the pathways producing H₂ while promoting the H₂-incorporating pathways, such as propionate synthesis. Increments in propionate production after probiotic addition (M. elsdenii suspension) led to an increment of 10% of MPS in lambs [111]. Moreover, a greater propionate availability and diet fermentability were associated with enhanced microbial dynamics in the rumen [112]. Thus, in addition to representing an energy loss, CH₄ synthesis might be inversely related to some bacterial growth processes, and therefore for MPS.

3.2. Contribution of Methanogenesis to Energy Losses

At a productive level, methanogenesis in the rumen significantly contributes to energy losses in beef cattle, representing 6–12% of the gross energy (GE) intake [25]. While methanogenesis is essential for maintaining the redox balance during fermentation, it redirects valuable metabolic hydrogen away from alternative pathways like reductive acetogenesis, which could otherwise be used for microbial growth and energy conservation [113]. The extent of energy loss due to CH₄ production is evident in forage-based diets, where fermentation favors acetate production, a pathway closely associated with methanogenesis [1,29]. Conversely, diets higher in fermentable carbohydrates can shift hydrogen utilization toward propionate synthesis, a more energy-efficient pathway that reduces methane emissions [114]. Methanogenesis not only limits the availability of energy for MPS but also contributes to GHG underscoring the need for strategies that mitigate its impact to improve ruminal energy efficiency and sustainability [3,11].

3.3. Relationship Between Rumen Microbes, Hydrogen Utilization, and Methane

The relationship between rumen microbes, hydrogen utilization, and methanogenesis is inherently linked to the metabolic processes of ruminal fermentation. Methanogenic archaea utilize hydrogen to reduce CO₂ into CH₄, a process that plays a critical role in maintaining the redox balance within the rumen [34]. By consuming hydrogen, methanogens help prevent the accumulation of dissolved H₂, which could otherwise inhibit microbial fermentation by altering the thermodynamics of hydrogen-dependent pathways [34,115]. The availability and partial pressure of H₂ directly affect the Gibbs free energy of fermentation pathways, influencing the microbial ecosystem’s overall efficiency [116]. Therefore, diet composition and feeding practices significantly impact hydrogen dynamics, with higher dissolved H₂ concentrations observed in rapidly fermenting, high-digestibility diets [117]. Nonetheless, beyond digestibility and forage: grain as cited factors leading to hydrogen and CH₄ dynamics, the ruminal passage dynamic heavily impacts methanogenesis. Indeed, previous experiments [118], showed how in sheep receiving either forage-based or pellet-based diets, the retention time of solid particles drives CH₄ emissions.

4. Impact of CH₄ on Energy Efficiency and MPS

4.1. Energy Redirection from Microbial Growth to Methane Synthesis

The production of CH₄ in the rumen represents a significant diversion of metabolic energy from processes that support MPS. Methanogenesis redirects hydrogen and associated metabolic energy away from pathways that could be utilized for microbial biosynthesis [32]. Instead of contributing to ATP generation for microbial cell growth and protein synthesis, a substantial portion of energy is lost as methane [119]. The re-direction of available energy also impacts the fermentation profile. Methanogenesis is associated with acetate production, which generates more hydrogen compared to propionate production, a pathway that consumes hydrogen [113]. As a result, ruminants receiving diets that favor acetate over propionate production (i.e., forage-based diets) tend to exhibit greater CH₄ emissions [120] and lower MPS [121]. This imbalance reduces the overall energy available for MPS, which is critical for providing AA to the host animal.

Moreover, energy redirection through methanogenesis can be exacerbated by dietary imbalances. For instance, an excess of rapidly fermentable carbohydrates without sufficient N (or vice versa) leads to hydrogen accumulation and increased methane production [122]. similarly, the depicted scenario of asynchrony has been previously associated with a compromised energy available for microbial growth, resulting in a reduction in MPS [36,63,65]. Similarly, high protozoal populations, which host symbiotic methanogens, amplify CH₄ production while competing with bacteria for nutrients [123,124]. Likewise, elevated counts of ruminal protozoa have been associated with the uptake of readily fermentable substrates diverting energy from bacterial populations [35], which are the primary contributors to MPS. Furthermore, protozoa populations play a significant role in glycogen synthesis within the rumen [125,126]. They store large amounts of fermentable CHS as glycogen rather than utilizing them for microbial growth [124]. This glycogen synthesis represents an energy sink, increasing ATP expenditure on protozoal and other microbes’ maintenance and reducing the energy available for bacterial protein synthesis [126,127].

4.2. Implications of Methane-Related Energy Loss on Nutrient Use Efficiency

The evidence exhibited here suggests how methane-related energy loss impacts nutrient use efficiency by reducing the available energy for critical microbial metabolic processes. The gas produced during methanogenesis (up to 12% of gross energy intake), represents a substantial inefficiency in the conversion of energy from the diet into metabolites used as an energy source by cattle [121]. Such gas has no nutritional value for the host, and when re-directed, might represent a valuable portion of the energy that could support MPS and VFA synthesis [35,113]. Such inefficiency further limits the availability of key nutrients and animal performance (growth, reproduction, and maintenance), particularly in forage-based diets that promote acetate and methane production over propionate, a more energy-efficient pathway [29,120]. The resulting decrease in nutrients use efficiency and metabolizable energy availability due to methanogenesis, would likely translate into a higher feed intake necessity, to achieve the same level of animal performance, increasing the cost and environmental footprint of ruminant production systems [128].

Methane synthesis mitigation has been shown to be an effective way to promote ruminal efficiency. Thus, additives like monensin utilized to mitigate up to 12% of methane promote a population shift towards propionate-producing bacteria, associated with a greater efficiency of nutrients’ fermentation [129,130]. This response demonstrates that the diversion of hydrogen and energy toward methanogenesis also disrupts the delicate balance of fermentation pathways in the rumen, affecting microbial populations and fermentation dynamics [131]. Thus, a roughage-based diet promotes CH₄ emission and protozoa populations, which host methanogens and compete with bacteria for nutrients [124], reducing the bacterial populations responsible for MPS [132], and thereby decreases the flow of microbial protein to the host animal. Likewise, inefficiency in N utilization increases ammonia accumulation, increasing the requirement of available energy to metabolize dietary N inputs or to process those products in the liver [40,62] While grazing is a fundamental component of beef cattle diets in cow-calf and backgrounding operations [46] its impact on CH₄ emissions is largely dependent on forage maturity and fiber composition rather than its mere inclusion [17,18]. The primary concern lies in including overly mature pastures with elevated acid detergent fiber (ADF) content in the diet, which has been consistently linked to increased methane production due to lower digestibility and prolonged ruminal retention time [130,133,134]. High-ADF forages results in greater acetate production, which enhances hydrogen availability for methanogenesis, thus promoting higher enteric CH₄ emissions [29]. Conversely, feeding less mature, higher-quality forages has been demonstrated to enhance nutrient digestibility, reduce CH₄ yield per unit of product, and improve microbial protein synthesis (MPS) by optimizing ruminal fermentation dynamics [8,132]. Therefore, rather than eliminating roughage, a strategic approach that prioritizes less mature, digestible grasses represents an effective means to reduce product-based methane emissions while maintaining rumen function and productivity in beef cattle [8].

4.3. Correlation Between Methane Production and MPS

The relationship between CH₄ synthesis and MPS varies significantly with diet composition, particularly the forage-to-concentrate ratio. Previous reports have shown that beef cattle fed low-quality fresh-cut ryegrass produce approximately 10.6 g CH₄/kg of DMI, which is 73% higher than those on high-concentrate diets based on corn and barley. This increase is attributed to the higher fiber content in forages, leading to increased acetate production and hydrogen availability, which methanogens utilize to produce methane [135]. However, the impact on MPS efficiency in high-forage diets is less clear and warrants further investigation. In contrast, high-concentrate diets have been associated with reduced CH₄ emissions as the percentage of the gross energy intake ranged from 0.9% to 6.9% on a low Forage:Grain diet and from 0.7% to 4.9% on a high Forage:Grain diet [46,136,137]. It has been hypothesized that the lower CH₄ emissions in high-concentrate diets are due to increased propionate production, which competes with methanogenesis for hydrogen, thereby reducing methane output [115,138]. Additionally, high-concentrate diets may enhance MPS efficiency by providing more readily fermentable carbohydrates available for microbial growth [40]. These observations underscore the importance of diet composition in managing CH₄ emissions and optimizing MPS in beef cattle, and the relevance of considering both measurements (in vivo CH₄ emission and MPS determination) in beef cattle nutritional studies.

There are few studies simultaneously measuring MPS under in vivo conditions and methane emissions, and despite the theoretical impact of methanogenesis (as a metabolic loss of energy) on MPS, practical data might indicate that this association is not as clear nor direct as previously thought. Based on an interesting meta-analysis recently published [139], we include new in vivo data on methane emission and MPS from beef steers [140] and analyze productive data from a total of 224 bovines receiving 50 different diets in 17 studies [141,142,143], In these studies, in vivo CH₄ emission were determined using four techniques including respiration chamber, hood, SF₆, and GreenFeed technology (C-Lock, Inc., Rapid City, SD, USA). Diets varied from 100% forage, hay, corn silage, and alfalfa silage, as well as 50 or 60% inclusion of corn covering a wide range of corn processing procedures. In order to explore the potential association between in vivo CH₄ emissions and MPS using real available data, we calculated the MPS values from each diet evaluated in those studies by using the widely accepted mathematical model from [54]. This model (see Figure 2) predicts MPS for cattle based on the intake of total digestible nutrients intake (TDNI) and fat-free TDN intake (FFTDNI). These calculations are the foundation for estimating the requirements of MP by predicting MPS in equations 6-1 and 6-2 of the latest NASEM (2016) guidelines [46]. Data on the dietary content of fat (%), DMI (kg/d), CH₄ emissions (g/d), bodyweight (BW), and TDN from each diet were extracted (or calculated) from each of the selected studies to calculate TDNI and FFTDN. Considering the profound effect of BW, physiological stage, and breed type on DMI and therefore on CH₄ emission and MPS, all parameters were analyzed by kg of metabolic body weight (BW^0.75).

The average value for in the vivo CH₄ emissions rates was 338 ± 115 g/d, when corrected by metabolic BW of 2.7 ± 0.3 g of CH₄/kg BW^0.75 (see Supplementary Material Tables S1 and S2). The average predicted value for MPS was 998.82 and 989.33 g of microbial CP/d when equations 6-1 and 6-2 were calculated. The correlation between MPS predictors (either TDNI or FFTDNI) and CH₄ emissions (as g CH₄/kg BW075) was high (77%, n = 50). Thus, despite the suggested thermodynamic effect of methanogenesis on microbial growth and therefore on MPS, the data analyzed here showed that greater values of in vivo CH₄ emissions seem to be positively correlated with a higher values of MPS. However, this preliminary analysis only evaluated one model to predict MPS, thus only the intake of the estimated TDN was utilized to predict the microbial production on a given day. Similarly, the predictors considered by [54] are directly affected by DMI [144] and therefore by physiological stage and the composition of the evaluated diet, which varied across the studies considered. Additional factors affecting MPS include rate of passage, which modulate the amount of OM flowing out of the rumen, including microbial mass [37,38,39]. Indeed, recent data [140] showed that the bacterial mass flowing out of the rumen mixed with the fluid portion is notably higher in beef steers receiving a high-grain diet. The latter study showed how the liquid phase of the ruminal fluid exhibited a higher rate of passage (%/h) in finishing diets (11.1%) compared to forage-based diets (9.9%), resulting in greater values of MPS (18.9 vs. 14.7 g microbial N/kg of OM truly digested in rumen) and MPSE (11.9 vs. 9.2 g microbial protein/100 g of OM truly digested in rumen). Simultaneously, during this study, ruminally cannulated steers receiving the same diets exhibited a notorious reduction in in vitro CH₄ concentration (20%), and production (38%). It is essential to highlight that this preliminary analysis only includes adjustment for DMI and metabolic bodyweight. However, no adjustment for N or energy intake were considered for the final regression, which was only based on the parameters required by the model proposed by Galyean and Tedeschi, 2014 [54]. Thus, it is recommended to enhance the database and explore the effect of these potential adjustments on the real association between CH₄ emission and MPS. Nonetheless, in vivo CH₄ emissions were not quantified in the experimental steers in which MPS was determined. Thus, a meta-analysis should include the rate of passage for the specific diets evaluated in the studies to confirm the nature of the association between these parameters. Likewise, further studies should consider the simultaneous quantification of in vivo CH₄ emissions and MPS to confirm the analysis presented in the present study and establish the potential dual-effect of a diet or a specific additive to mitigate CH₄ emissions, while improving MPS.

5. Mitigation Strategies for Methane and Enhancement of MPS

5.1. Dietary Interventions

A broad variety of mitigation strategies are focused on the reduction of CH₄ yield (g CH₄/kg of DM consumed), intensity (g CH₄/L of milk or kg of gain) or emissions (g/head/d) [133]. Reductions in CH₄ intensity can be achieved by increasing the feeding level [145], allowing the grazing of fresh forages [146], and increasing the inclusion of concentrates in the diet [8,34]. Absolute values (g of CH₄/d) have been reduced through CH₄ inhibitors [147], tannins-rich forages [148], fumaric acid and nitrates [34,149], fats and oils [150,151,152], and oilseeds [153]. A recent review classified the strategies as (a) animal and feed management, (b) changes in diet formulation, and (c) additives for manipulating rumen function [8]. The increase in grain inclusion and concentrate has been associated with a greater rate of passage and MPS [61,62]. In this sense, there are reports showing the sensitive reduction of CH₄ production in beef cattle receiving a diet based on red clover silage compared to ryegrass silage [154]. The authors reported a reduction of CH₄ yield in 21%, which was associated with greater rates of passage from the rumen and shifts in the site of digestion. Similarly, an interesting simulation on data from 130 dairy cows grazing annual rye-grass or ryegrass + red and white clover reported a reduction of CH₄ yield (g kg⁻¹ fat corrected milk at 3.5%) of 45% [155]. Likewise, an additional research [156] indicated that the inclusion of 25% of DMI in a ryegrass-based grazing system of sheep, resulted in a reduction on CH₄ yield (g/kg DM) of 31%, which was accompanied by a significant reduction in Methanobrevibacter abundance. Diverse mechanisms cited to mitigate ruminal CH₄ include the stimulation of reductive acetogens [157] and the addition of non-methanogenic hydrogenotrophic to ruminal fluid, such as fumarate reducers [158]. More recently, the use of a synthetic molecule called nitroxy-propanol (3NOP) gained interest due to its ability to link the active site of MCRM, inhibiting the last step of CH₄ synthesis [159] and shifting the metabolic fate of H⁺ towards propionate synthesis [136,160,161].

In addition to commercial additives, natural extracts from fruits and plants containing PSM, particularly saponins and tannins, have been extensively studied under both in vitro [162] and in vivo conditions [163,164], resulting in indirect CH₄ mitigation via protozoa reduction, [165]. Protozoa are the natural hosts of archaea and serve as their major H⁺ source [123,124]. Decreasing ruminal protozoa has been positively associated with a reduction in archaea population, especially Methanobrevibacter [166], as well as CH₄ mitigation [167]. Moreover, anacardic acid contained in the Cashew nutshell extract (CNSE), directly affects the cellular structure of archaea, reducing CH₄ and increasing propionic acid proportion without affecting total VFA [168]. A recent review [107] found that among the additives used to manipulate rumen function, 3NOP, essential oils (i.e., oregano, garlic), and tannins were among the most effective additives accounting for an average CH₄ reduction of 60%.

5.2. Plant Secondary Metabolites (PSM) as a Methane Mitigation Strategy

The antibiotic effect of saponins is derived from its triterpene glycoside structure, which is able to form bonds and link lipids [169,170], similar to the sterol molecules in the protozoa membrane [171]. Reductions in protozoal populations led to a reduction in the ruminal NH₃ concentration [124,148], CH₄ emission [133], and fiber degradability [134]. Interestingly, defaunation reduces bacteria predation [172], potentially leading to an increase in MPS [173]. The reduction in ruminal degradation of fiber may lead to decreased intake due to physical ruminal filling, downregulating DMI [174] and, thereby limiting MPS. Addressing the contradiction in the proposed mechanisms, Ref. [171] suggested that the potential effect of saponins on MPS could be negligible when compared to their broader microbiological effects [175]. A more specific effect may be observed under a moderate concentration of starch in the medium (0.1 w/v) in which saponins appear to preferentially target cellulolytic populations [171,176], potentially enhancing propionic acid production, leading to a greater H⁺ uptake, and reducing its flow towards methanogenesis.

Similar to saponins, tannins, especially the condensed type, directly mediate protozoal reduction by targeting their membrane [177]. These results are relevant, considering that around 37% of total CH₄ comes from protozoa-associated archaea [178]. Likewise, tannins mediate an indirect reduction in CH₄ synthesis by affecting ruminal fermentation [179] and have been negatively associated with NDF and OM degradation in the rumen [174]. Besides bacterial predation, protozoa compete with amylolytic bacteria for starch molecules, affecting amylolytic population and tipping the balance towards acetate instead of propionic acid production in the rumen [134,180]. Hence, tannin-mediated protozoa reduction may lead to an enhanced propionate proportion by the unaffected amylolytic microbes [181]. Moreover, by decimating protozoa, a greater amount of dietary energy will be fermented towards propionate, which ultimately translates into around 10% more [182]. Taking this evidence together, various mechanisms of tannins mitigating energy losses are suggested. Thus, by reducing CH₄ synthesis (via protozoa reduction), tannins may reduce losses of dietary gross energy. Likewise, the reduction in protozoa could reduce bacterial predation and, therefore, N recycling, resulting in increasing MPS efficiency. Moreover, since OM fermentation in the rumen could be influenced by the addition of tannins, it is likely that MPS efficiency may be affected.

Among PSM, anacardic acid, cardanol, and cardol present in cashew nutshell extract (CNSE) have been proposed as feed additives for ruminants [183]. In contrast, to saponins or tannins, CSNE exhibits a direct CH₄ mitigation effect via the reduction of mcrA gene expression in archaea, a gene codifying for the rate-limiting reaction of methanogenesis [184]. In addition, CNSE mediates CH₄ mitigation by limiting H⁺ and formate by reducing the populations producing these electron donors used for archaea in CH₄ synthesis [108,138]. Evaluating a low in vitro addition of liquid CSNE (5 mL) in a batch culture incubation, Ref. [185] observed a linear reduction in OM digestibility from 66 to 59 % and, at the same time, an increment in total VFA, suggesting improvements in dietary energy use and microbial efficiency. Likewise, in vitro, the addition of CNSE may generate a switch in ruminal populations, promoting the abundance of structural CHS degraders (i.e., Butyrivibrio and Succinivibrio) at the expense of those specialized in degrading NSC and dietary protein (i.e., Prevotella and Treponema) [186]. Various In vitro studies associated CH₄ mitigation with a ruminal shift in population rather than a reduction in microbial synthesis [186,187,188]. Indeed, working with continuous culture fermenters [189], showed that any change in MPS after the addition of vegetable fat might be related to a reduction in NDF digestibility observed under in vitro conditions. Therefore, by shifting populations and reducing NDF digestibility, it is possible to suggest that additives like CNSE could reduce CH₄ while maintaining the MPS unaffected. Recently, Cuervo et al. [190] proposed the use of invasive pigweed (Amaranthus spinosus) based on its elevated content of PSM (polyphenols) to mitigate CH₄ production. In vitro evidence showed how the inclusion of low levels (2.5 and 5% of DM) of seeds, leaves, and the whole plant of pigweed, reduced acetate proportion, elevated propionate concentration and led to a reduction of up to 60% of CH₄ production without significantly compromising digestibility.

5.3. Grain and Forage Management to Optimize Carbohydrate Fermentation

The fine tuning of grain and forage inclusion is critical for enhancing CHS fermentation in beef cattle by promoting the efficiency of ruminal microbes in utilizing dietary organic matter [191]. Incorporating grains with higher levels of non-structural carbohydrates (e.g., starch) improves the availability of fermentable substrates, supporting microbial protein synthesis (MPS) while promoting propionate production, a more energy-efficient pathway [40]. However, the excessive inclusion of grains can lead to a drop in ruminal pH and potentially ruminal acidosis [41], reducing fiber digestion and therefore, MPS efficiency [42]. Balancing the forage-to-grain ratio is essential to maintaining rumen pH and fiber degradability while providing sufficient energy for microbial growth. In this sense, excessive NSC diverts energy away from microbial growth to maintenance and carbohydrate storage [127,192]. Additionally, the inclusion of high-quality forages with improved digestibility can complement grain-based diets by supplying structural CHS that maintain optimal rumen function.

Strategic supplementation with grains in forage-based systems also enhances dry matter intake and microbial growth rates, particularly in diets with low protein or high fiber content [193]. Likewise, the transition from high to low forage inclusion in dairy cows deeply affects microbial diversity reducing the abundance of Bacteroidetes and increasing Firmicutes [194] which is associated with the elevated inclusion of starch in grain and concentrates [195]. Thus, it has been suggested that after the inclusion of more than 80% of diet DM, MPS could be affected [193]. Besides specific orders and family of microbes being affected by such proportions, including grains and concentrates of up to 70% in diets based on high-quality forages increases protozoa (48% higher counts) but not in those based on low-quality forages, suggesting protozoa populations are not a linear (negative) function of increasing levels of concentrate but rather depend on the chemical and nutritional content of basal forage [196]. In contrast, greater inclusions of roughage in forage-based diets could lead to a greater synthesis of fibrolytic bacteria, as previously reported by [197,198]. This factor might promote greater synthesis of bacteria compared to protozoa in steers receiving a forage-based diet. By carefully balancing carbohydrate sources, grain and forage management effectively supports microbial fermentation, reduces energy losses like methane production, and improves overall feed efficiency in beef cattle.

5.4. Defaunation-like Effect of PSM to Mitigate Methane Synthesis and Improve MPS

A reduction in absolute CH₄ involves using methane inhibitors, forages high in tannins, electron sinks (i.e., nitrates), fats, and oils [147,150,151,152,153]. This category of CH₄ mitigation (referred to as rumen manipulation strategies) aims to stimulate reductive acetogens [158], reducing protozoa populations [162] through the inclusion of, among others, enzymatic additives (i.e., 3NOP), flavonoids, saponins and tannins, which are all widely studied PSM targeting specific microbial population in the rumen environment [199,200]. Saponins affect protozoa membranes, reducing NH₃ concentration and CH₄ emission [165,166], potentially increasing MPS. Nonetheless, complete defaunation negatively affects fiber degradation and dry matter intake (DMI), limiting MPS [174]. Similar to saponins, tannins directly reduce protozoa populations and indirectly reduce CH₄ methane synthesis by affecting ruminal fermentation [179], leading to increased propionic acid production and reduced energy losses [181]. Similar to defaunation, tannins impair protozoa viability by reducing bacterial predation, which may enhance MPS efficiency and decrease losses of dietary gross energy [182]. Similar to tannins and saponins, anacardic acid, a PSM from cashew (Anacardium occidentale), is present in cashew nutshell extract (CNSE), a byproduct that has been proven to downregulate genes associated with methanogenesis in archaea [183], resulting in the reduction of microbial populations serving as electron donors for archaea [108,138], and improving dietary energy use and microbial efficiency. Overall, the inclusion of 3NOP and PSM represent two different CH₄ with particular advantages and limitations. While the widely studied mechanism (methyl-coenzyme M reductase inhibition) of 3NOP reduces enteric CH₄ emissions by up to 30% in a consistent mode with no adverse effects reported [201]. The high cost, regulatory approval requirements, microbial adaptation over time, and scarce performance improvement pose challenges for widespread adoption [161]. In contrast, PSM (tannins, saponins, and essential oils), offer a natural alternative with additional benefits beyond methane mitigation, including the modulation of nitrogen metabolism and potential antimicrobial effects on specific rumen microbes [165]. However, PSM’s effects can be highly variable, depending on plant source, concentration, and interactions with other dietary components [65,141,151], making standardization difficult. Additionally, some PSMs may reduce feed palatability or negatively impact fiber digestion if included at high levels [140]. While 3NOP provides a more targeted and predictable CH₄ reduction, PSMs offer a holistic approach with broader metabolic effects, but with greater variability and formulation challenges. A combination of both approaches may provide a more sustainable and adaptable methane mitigation strategy for ruminant production systems.

Mediating similar effects to defaunation without the negative impact on fiber degradation, the addition of CNSE has demonstrated numerous benefits in beef and dairy cattle, including reduced gas production and methane emissions [202,203,204], improved milk production, and enhanced nutrient absorption [186]. However, reports of CNSE affecting OM and NDF digestibility suggest that its effects depend on dietary composition, especially CHS type and proportion [186,200,205]. Therefore, evaluating CNSE’s effects on forage-based (backgrounding) and grain-based (finishing) diets could shed light on eventual dietary interactions affecting CNSE mitigation effects. Moreover, few studies have investigated the combined effects of CNSE with other PSM in mitigating methane.

6. Future Directions and Research Opportunities

6.1. Emerging Technologies and Practices to Improve MPS

Novel techniques using the meta-omics approach have opened new avenues to enhance our understanding of how to modulate MPS in ruminants [206]. Meta-omics techniques have been increasingly employed in the CH₄ research field to understand how some dietary strategies mediate CH₄ mitigation in ruminants. Recently, an interesting review [206] highlighted the potential of employing an integrative approach including metagenomics to analyze the diversity and abundance of ruminal microbes, meta transcriptomics (RNA), metaproteomics and metabolomic in biological samples (ruminal fluid, blood, and tissue). This approach allows the identification of key genomic regions and genetic markers like host genes that modulate the composition of the ruminal microbiome. Such markers linked to CH₄ production can then be leveraged in selective breeding programs to cultivate traits associated with lower emissions. Building on this approach, studies in dairy cattle that combined metagenomics, metatranscriptomics, and metaproteomics have identified specific microbial taxa and functions associated with improved feed efficiency—an outcome inversely related to CH₄ emissions—which could also be explored to enhance microbial protein synthesis (MPS) [207]. Recent reports suggest that integrating these technologies to evaluate CH₄ mitigation strategies and feed efficiency has revealed certain microbial genera (e.g., Succinivibrionaceae) and phyla (e.g., Fibrobacteres, Ciliophora, Euryarchaeota) that exhibit strong genetic correlations with CH₄ emissions and appear to be partially regulated by the host genotype [208].

While metagenomics analyses may identify profiles of ruminal microbial populations, metatranscriptomics evaluate gene expression derived from dietary interventions, metaproteomics identify key proteins (i.e., enzymes) of interest involved in methanogenesis and MPS and metabolomics evaluates and predicts how MSP and methanogenesis metabolic pathways might be overlapped. These tools can identify microbial strains or consortia with high MPS potential, aiding in the development of precision microbiome management strategies. In addition, real-time monitoring of rumen fermentation parameters through sensors [209] and automated feeding systems allows for dynamic dietary adjustments, improving synchronization of energy and nitrogen supply, the use of novel additives based on amino acids [210] and the maximization of MPS efficiency under diverse production systems. Finally, further statistical models should consider alternative approaches to predict MPS in beef and dairy cattle, including alternative predictors (other than TDN) such as rate of passage, OM truly digested in rumen, OM intake, and CP intake [211].

6.2. Microbiome Association Between CH₄ and MPS

Hydrogenotrophic methanogens, including Methanobrevibacter and Methanosphaera, play a crucial role in CH₄ synthesis by utilizing hydrogen to reduce CO₂ into CH₄ [102]. These archaea establish syntrophic relationships with protozoa, which produce hydrogen as a byproduct of carbohydrate fermentation [149]. Thus, the elevated concentration of protozoal populations has been linked to increased CH₄ emissions in beef cattle [124] while simultaneously competing with bacteria for nutrients, potentially reducing bacterial populations responsible for MPS [172]. The fermentation profile of the rumen is heavily influenced by diet composition, which at the same time affects microbial populations and their hydrogen utilization strategies [29]. Fiber-degrading bacteria promote acetate production, which generates hydrogen and supports methanogenesis, whereas amylolytic bacteria (i.e., Succinimonas amylolytica, Selenomonas ruminantium) favor propionate production, competing for hydrogen, reducing CH₄ synthesis and enhancing energy efficiency and microbial growth [113,138,181]. Supporting the notion of the impact of diet on the microbial associations in the CH₄-MPS axes, roughage-based diets promote higher CH₄ emissions by supporting fibrolytic bacteria and protozoa, which enhance hydrogen availability for methanogenesis [193]. Conversely, diets with increased fermentable carbohydrates and higher passage rates favor amylolytic bacteria, leading to greater MPS efficiency and reduced methane losses. Although reducing CH₄ emissions can improve energy utilization, certain strategies may negatively impact MPS efficiency [121]. For instance, some 3NOP reduce hydrogen availability, which could decrease the abundance of firmicute, proteobacteria, and Bacteroidetes [201], likely impacting the microbial community structure and decreasing bacterial protein synthesis. Additionally, excessive reductions in methanogenesis may disrupt the ruminal redox balance, affecting fermentation efficiency and microbial growth [29]. A deeper understanding of microbial interactions is needed to simultaneously optimize CH₄ mitigation and MPS efficiency. As previously discussed, advanced meta-omics approaches can provide insights into microbial adaptations to CH₄ mitigation strategies and identify potential microbial targets for improving protein synthesis while maintaining a stable rumen environment.

6.3. Development of Novel Feed Additives and Management Practices

The assessment of alternative additives (i.e., PSM from invasive weeds) poses the necessity of a continuous exploration of novel approaches, along with the evaluation of lipids, enzymes, supplements, and commercial additives that might mediate both effects, enhancing MPS while mitigating CH₄ emissions. Evaluating the novel additives to replace soybean meal with NPN nitrogen sources like slow-release urea technologies might represent an alternative to the controlled release of N that coincides with MPS dynamics, enhancing nutrient utilization and reducing the risk of ammonia toxicity [212]. The use of Taurine to upregulate the gene expression of enzymes involved in MPS-promoting pathways represents a plausible alternative to enhance microbial growth [210].

Additives based on PSM mixtures represent a promising approach, as they may provide enhanced control over Archaea [213]. However, the search for additives to exert a consistent effect without being compromised by the adaptation of the ruminal microbiome remains a persistent challenge for ruminant nutritionists. In this sense, effective additives mitigating CH₄ and enhancing MPS must modulate ruminal fermentation by reducing protozoal populations, shifting microbial communities affecting archaea, or redirecting hydrogen from methanogenesis toward microbial anabolism. Given the complexity of ruminal ecology, achieving all these effects simultaneously with a single feeding strategy or additive appears to be as elusive as discovering the philosopher’s stone. Long-term studies are needed to evaluate whether interventions like defaunation, PSM, or synthetic inhibitors (i.e., 3NOP) mediate shifts in ruminal microbiome diversity and metabolic pathways. Such changes could impact fiber digestion, fermentation pathways, and nutrient utilization. Research into microbial adaptation and the potential trade-offs associated with methane reduction will ensure that mitigation strategies are sustainable and do not compromise overall productivity or animal health.

While increasing the inclusion of concentrate in beef cattle diets improve MPS and reduce methane emissions, maintaining an appropriate proportion of forages (55–75% of DMI) ensures optimal rumen function, provides structural CHS that support fibrolytic populations [214], a healthy rumen environment and pH [215], and sets the proper conditions to enhance MPS. Including high-quality forages (particularly with elevated digestibility) reduces CH₄ emissions by decreasing the time fiber remains in the rumen [216], thus minimizing methane-producing fermentation. High-quality forages can also provide a synchronized release of energy and protein, enhancing nutrient utilization [217]. However, supplementing a grazing-based diet with concentrates (at levels below 70%) remains a well-established strategy to improve overall microbial protein synthesis efficiency (MPSE).

6.4. Validation of Findings Through On-Farm Trials

In order to determine, rather than estimate, the nature of the MPS-CH₄ emission association, in the near future trials must simultaneously evaluate the impacts of dietary strategies on MPS and CH₄ emissions, as well as on animal performance. Updated studies based on established and recognized techniques to determine and quantify in vivo MPS (Triple marker technique, purines determination, ¹⁵N, 16S and 18S molecular markers) are necessary to validate classic studies and models. Future experiments may include a mix of classic and newer approaches to determine CH₄ emissions particularly under grazing conditions including the GreenFeed technology, SF₆ technique [218], individual spirometry masks [219], and even drones [220]. Later, translating laboratory findings into practical applications is essential to validate such findings under real-world conditions. Thus, on-farm trials may provide external insight into the efficacy of feed additives, management practices, and methane mitigation technologies in diverse production settings. As discussed in this review, increasing concentrate feed in the diet of beef cattle is a well-documented strategy to mitigate CH₄ emissions by shifting ruminal fermentation toward propionate production [113,138], which serves as an alternative hydrogen sink. However, there are some trade-offs and limitations that should be considered including rumen health risks [92], economic feasibility, and broader environmental impacts.

One major concern is rumen health and acidosis risk [41]. High-concentrate diets reduce ruminal pH, which can suppress fiber-degrading microbial populations, potentially compromising fiber digestion and overall gut health [215]. This may negatively affect long-term animal performance, particularly in forage-based systems where efficient fiber utilization is essential. Besides the impacts on ruminal and animal performance, the improved feed efficiency and CH₄ mitigation mediated by concentrated feed inclusion, relies on cereals and grains, leading to higher feed costs and competition with human food production. This raises economic and sustainability concerns, particularly in regions where grain availability is limited or costly [221]. In this sense, a broader environmental consideration is the carbon footprint of concentrate production. Producing and processing grains such as corn and cereals for cattle feed requires significant energy inputs, including fuel for cultivation, harvesting, transportation, and processing. Increased concentrate use could lead to higher CO₂ emissions from agricultural machinery, fertilizer application, and feed processing facilities, potentially offsetting the methane reduction achieved in the rumen [222]. In contrast, well-managed pasture-based systems rely more on natural forages, reducing the need for fossil fuel-intensive feed production. Thus, while increasing concentrate feed can be an effective methane mitigation strategy, balancing dietary fiber, optimizing feed costs, and considering the full carbon footprint of grain-based feeding systems are crucial to achieving truly sustainable methane reduction in beef production.

6.5. Gaps in Research and Future Directions

Despite significant advancements in CH₄ mitigation and MPS improvement strategies, several research gaps remain unaddressed. As mentioned, long-term studies are essential to assess the sustainability of current mitigation strategies, as microbial adaptation could reduce their effectiveness over time. In addition, there are relevant trade-offs between CH₄ emissions reduction and MPS efficiency that deserve further exploration. Thus, while some interventions have shown effective reduction in CH₄ and enhanced propionate synthesis. Often, such results are obtained at the expense of fiber digestion or nitrogen utilization. Dietary interactions with methane mitigation additives must also be considered, as the effectiveness of different additives and inhibitors depends on forage quality, protein sources, fiber digestibility, and even the genome of the host. While controlled studies have provided valuable insights, more on-farm validation is necessary to determine the practical applicability of these strategies under commercial conditions over sustained periods of time. Emerging meta-omics technologies can be consolidated as powerful tools to better understand the dynamic relationships between ruminal microbes, upregulated fermentation pathways, and energy utilization in the rumen. However, their integration into research on understanding the relationship between CH₄ mitigation and MPS remains limited and/or unexplored. Furthermore, economic feasibility must be carefully evaluated, as certain mitigation strategies may be impractical due to feed costs, regional availability, or producer acceptance.

Designing, performing and publishing in vivo studies with beef cattle considering the simultaneous determination of CH₄ emissions and MPS are essential to building a greater and more reliable empirical database with valuable information to confirm the correlation observed in the preliminary association analyzed here. Addressing these research gaps will be critical for developing sustainable, scalable, and effective methane mitigation strategies in beef cattle production.

7. Conclusions

Ruminal methanogenesis by Archaea is a relevant energy sink, estimated to be as high as 12% of the gross energy intake in beef cattle, diverting energy away from being used for MPS and contributing to GHG emissions. Promoting the efficiency of MPS is essential for providing a reliable and biologically-cheap source of protein for ruminants to support vital functions. Fine-tuning of the forage: concentrate ratio, feeding high-quality forages, and including additives such as PSM or commercial CH₄ inhibitors are some of the strategies that have shown promise in mitigating methane emissions with a potential increase in MPS. Nonetheless, the theoretical association between reducing methanogenesis to promote propionate synthesis and other anabolic pathways leading to a greater MPS and MPSE seemed not to coincide with real data from the current meta-analysis and established MPS prediction models. However, since enteric CH₄ emissions and inefficiencies in MPS are closely associated, integrated approaches are urgently needed. Optimizing ruminal fermentation and microbial efficiency through dietary strategies, advanced feed additives, and new evaluation technologies such as meta-omics and precision feeding systems should be combined. Collaboration between researchers, on-farm test, and industry stakeholders is critical to identify scalable solutions focused on mitigating GHG emissions, climate change, and the environmental footprint of livestock production.