A Meta-Analysis of 3-Nitrooxypropanol Dietary Supplementation on Growth Performance, Ruminal Fermentation, and Enteric Methane Emissions of Beef Cattle

: This study aimed to evaluate the effects of dietary supplementation with 3-nitrooxypropanol (3-NOP) on growth performance, ruminal fermentation, and enteric methane emissions of beef cattle using a meta-analytic approach. The final meta-analysis database included results from 15 scientific articles. The response variables were analyzed through random effects models, where the results were reported as weighted mean differences (WMD) between the treatments without 3-NOP and those supplemented with 3-NOP. The dietary inclusion of 3-NOP decreased ( p < 0.001) dry matter intake but did not affect ( p > 0.05) average daily gain and increased ( p < 0.05) feed efficiency. In the rumen, 3-NOP supplementation increased ( p < 0.01) the pH and ruminal concentration of propionate, butyrate, valerate, isobutyrate, and isovalerate. In contrast, dietary supplementation with 3-NOP decreased ( p < 0.001) the rumen concentration of ammonia nitrogen, total volatile fatty acids, acetate, and the acetate/propionate ratio. Furthermore, daily methane (CH 4 ) emission, CH 4 yield, and CH 4 emission as a percentage of gross energy ingested decreased ( p < 0.001) in response to 3-NOP dietary supplementation. In conclusion, dietary supplementation with 3-nitrooxypropanol can be used as a nutritional strategy to improve feed efficiency and ruminal fermentation in beef cattle and, at the same time, reduce enteric methane emissions.


Introduction
Beef cattle production systems are essential worldwide because they contribute to the supply of high-quality proteins required for human consumption [1].However, beef cattle and other ruminants produce enteric methane (CH 4 ) as one of the end products of digestion [2,3].According to Gerber et al. [4], enteric CH 4 emitted by ruminants contributes to approximately 40% of total livestock emissions and represents between 4 and 6% of global anthropogenic greenhouse gas (GHG) emissions.CH 4 is a GHG, which according to several authors [1,5], has a global warming potential 28 times greater than carbon dioxide (CO 2 ).Therefore, in recent years, there has been an increased interest in reducing enteric CH 4 emitted by ruminants [6,7].Several mitigation strategies for enteric CH 4 have been proposed and evaluated, among which are dietary supplementation with feed additives, such as ionophores, essential oils, macroalgae, tannins, and 3-nitrooxypropanol Fermentation 2024, 10, 273 2 of 14   [8,9].Among these additives, 3-NOP is one of the most effective in reducing enteric CH 4 in ruminants [7].
3-NOP is a small molecule of low molecular weight (121.09g/mol) with dual chemical functional groups: a primary alcohol and an organic nitrate ester [10].According to Duin et al. [3], 3-NOP can inhibit enteric CH 4 emitted by ruminants through the specific inactivation of the enzyme methyl-coenzyme M reductase (MCR), which is required to catalyze the last step of methanogenesis in methanogenic archaea [8].Various in vitro studies [11][12][13] suggest that the dietary inclusion of 3-NOP may be an effective strategy to reduce enteric CH 4 emissions in ruminants.Likewise, other studies found that dietary supplementation with 3-NOP successfully mitigated enteric CH 4 emissions in sheep [14] and dairy cows [6].
Particularly in beef cattle, to date, few studies have evaluated the effects of 3-NOP as a dietary additive on enteric CH 4 emissions [15][16][17], ruminal fermentation [18,19], and growth performance [1,5].Furthermore, the individual interpretation of these studies limits the obtainment of scientifically sound conclusions, since the results obtained in some of them are contradictory.For example, some authors [5,15] did not observe the effects of supplementation with low doses (30 to 75 mg/kg DM) of 3-NOP on growth performance, ruminal acid concentration volatile fatty acids, and enteric CH 4 emissions from beef cattle.In contrast, other authors found that high doses (100 to 200 mg/kg DM) of 3-NOP reduced CH 4 emissions [2] and improved beef cattle's feed efficiency [16] and rumen parameters [20].According to Yu et al. [7] and Dijkstra et al. [21], the type of diet, the dose, and the supplementation period are factors that influence the response of ruminants to 3-NOP supplementation.
Data reported in four previous meta-analyses [6,[21][22][23] show that 3-NOP supplementation successfully decreases enteric CH 4 emissions in ruminants without affecting productive performance.However, one of these meta-analyses [6] evaluated the effects of 3-NOP only in dairy cows.Likewise, the meta-analyses by Kim et al. [22], Dijkstra et al. [21], and Jayanegara et al. [23] only included four to seven beef cattle studies in their database.Furthermore, in the meta-analyses of Kim et al. [22], Dijkstra et al. [21], and Jayanegara et al. [23], the results for growth performance, ruminal fermentation, and enteric CH 4 were obtained through combining data from beef cattle and dairy cows since there were not enough studies available with beef cattle alone at that time.High flexibility in the design of a meta-analysis, such as using a low number of studies or mixing data from different experimental units (e.g., beef cattle and dairy cows), decreases the probability that the study findings are conclusive [24].In contrast, periodically updating meta-analyses using larger databases can increase the reliability of the results [6].In response to the increasing number of published studies on the use of 3-NOP in beef cattle, our study aimed to evaluate the effects of dietary supplementation with 3-nitrooxypropanol on growth performance, ruminal fermentation, and the enteric methane emissions of beef cattle through a metaanalytic approach.The hypothesis of the present study establishes that the inclusion of 3-NOP in beef cattle diets will benefit growth performance and ruminal fermentation while decreasing enteric CH 4 emissions.

Literature Search
To formulate the research question, the Population, Intervention, Comparison, and Outcomes (PICO) [25] strategy was used.Therefore, in the current study, the population was beef cattle, the intervention was the dietary supplementation of 3-NOP, the comparison was between diets with and without the addition of 3-NOP, and the results were the means of treatments obtained in growth performance, ruminal fermentation, and enteric CH 4 emissions.Subsequently, the PRISMA protocol [26] was used in the processes of identification, selection, election, and inclusion of scientific articles that tested the effects of the inclusion of 3-NOP in diets for beef cattle (Figure 1).To complete the process of identifying scientific documents, systematic searches were carried out in the PubMed, ScienceDirect,

Inclusion and Exclusion Criteria
Initially, 212 peer-reviewed manuscripts were identified through the searches.Duplicate documents were first removed from these manuscripts.Subsequently, from the remaining documents, those that had one or more of the following characteristics (exclusion criteria) were eliminated: (1) review articles, simulations, books, or conference proceedings; (2) studies that did not use beef cattle as experimental units; and (3) studies that did not use 3-NOP or combined 3-NOP with monensin or another additive.Finally, to be included in the final database of the meta-analysis, the remaining documents had to meet

Inclusion and Exclusion Criteria
Initially, 212 peer-reviewed manuscripts were identified through the searches.Duplicate documents were first removed from these manuscripts.Subsequently, from the remaining documents, those that had one or more of the following characteristics (exclusion criteria) were eliminated: (1) review articles, simulations, books, or conference proceedings; (2) studies that did not use beef cattle as experimental units; and (3) studies that did not use 3-NOP or combined 3-NOP with monensin or another additive.Finally, to be included in the final database of the meta-analysis, the remaining documents had to meet each of the following characteristics (inclusion criteria), similar to those reported in previous meta-analysis [27,28]: (1) peer-reviewed manuscripts published in the English language; (2) studies that used beef cattle as experimental units; (3) studies that measured and reported data on growth performance, ruminal fermentation, or enteric CH 4 emissions (measured with the sulfur hexafluoride "SF 6 " tracer technique, the Green-Feed system, or respirometry chambers); (4) studies that compared the effect of dietary inclusion of 3-NOP versus a control treatment (diet without 3-NOP) using the same basal diet; (5) studies that indicated the dose (mg/kg DM) of 3-NOP added to the experimental diets or had information necessary to estimate it; (6) studies that reported data on treatment means, standard error of the mean (SEM) and number of experimental units (n) of the experimental diets (diets added with 3-NOP) and control diets (diets without 3-NOP).

Data Extraction
The final meta-analysis database includes data from 15 scientific articles (Table 1).The following information was obtained from each selected manuscript: (1) dose of 3-NOP (mg/kg DM) added to the diets; (2) duration of the period of dietary supplementation with 3-NOP (days); and (3) amount of concentrate (g/kg DM) included in the experimental diets.The included response variables were grouped as follows: (1) dry matter intake (DMI), average daily gain (ADG), and feed efficiency (FE); (2)

Calculations and Statistical Analysis
Statistical analyses were performed through the "metaphor" and "meta" packages [35,36], available in the statistical software R (version 4.1.2).The effect size (ES) of 3-NOP supplementation was estimated by the weighted mean difference (WMD) between the treatments added with 3-NOP and control (without 3-NOP).WMDs were used in the current study because, according to Takeshima et al. [37], they allow the results to be easily interpreted and have greater statistical power.The weighting in the WMD was performed using the inverse of the variance following the methods and procedures described by Der-Simonian and Laird [38] for random effects models.

Heterogeneity and Publication Bias
The presence of heterogeneity was evaluated through Cochran's Q statistic (chi-square test), which was considered significant when p ≤ 0.05 [39].Additionally, the I 2 statistic was used to measure the percentage of variation attributed to heterogeneity [40].According to some authors [41,42], the degree of heterogeneity obtained with the I 2 statistic can be classified as follows: no heterogeneity (0 < I 2 ≤ 25%), low (25% < I 2 ≤ 50%), moderate (50% < I 2 ≤ 75%), and high heterogeneity (I 2 > 75%).On the other hand, the presence of publication bias in the meta-analysis was tested using two statistical tests: (1) Egger's regression asymmetry [43]; and (2) Begg's adjusted rank correlation [44].Publication bias was considered when p ≤ 0.05 in one or both tests (i.e., Egger and Begg).

Meta-Regression and Subgroup Analysis
Meta-regression analyses were performed to test the effects of 3-NOP doses, periods of 3-NOP supplementation, and the amount of concentrate included in the diets on the heterogeneity detected in the response variables.The method of moments proposed by Der-Simonian and Laird [38] was used in the meta-regression analyses since, according to Borenstein et al. [39], this method is well established for estimating the variance between studies.The response variables had to meet the following requirements to be evaluated with meta-regression analysis: (1) have I 2 > 50% and p < 0.05 in the Q test [39,45]; and (2) have p > 0.05 in the Egger regression asymmetry and Begg's adjusted rank correlation tests [43,44].Covariates were divided as follows: (1) 3-NOP doses 35-100 and 101-280 mg/kg DM; (2) 3-NOP supplementation period ≤ 60 and >60 days; and (3) amount of concentrate included in the diet ≤ 700 and >700 g/kg DM.When a significant effect (p < 0.05) of any covariate was detected, the WMD of the response variable was evaluated through subgroup analysis, as suggested by other authors [27,46].

Growth Performance
Table 2 shows that DMI decreased (p < 0.001) in response to 3-NOP supplementation.In contrast, the dietary inclusion of 3-NOP did not affect (p > 0.05) ADG and increased (p < 0.05) FE.

Heterogeneity and Publication Bias
Egger's regression asymmetry test and Begg's adjusted rank correlation were not significant (p > 0.05) for any of the response variables analyzed (Tables 2-4), indicating that there was no publication bias.On the other hand, Table 2 shows that there was significant (p < 0.05) heterogeneity (Q) for DMI.In contrast, no Q (p > 0.05) was detected for any of the response variables related to ruminal fermentation (Table 3).However, Q (p < 0.05) was observed in the daily CH 4 emission and CH 4 yield (Table 4).
Table 5 shows no significant relationship (p > 0.05) between DMI and the covariates included in the models used for the analysis.The 3-NOP dose explained (p < 0.001) 26.40 and 36.04% of the observed heterogeneity in the daily CH 4 emission and CH 4 yield, respectively.Likewise, the 3-NOP supplementation period explained (p < 0.001) 6.56 and 9.65% of the heterogeneity observed in the daily CH 4 emission and CH 4 yield, respectively.Furthermore, the amount of concentrate included in the diets explained (p < 0.05) 5.21% of the heterogeneity observed in the daily CH 4 emission.

Subgroup Analysis
Figure 2a shows that the daily CH 4 emission decreased (p < 0.001), regardless of the dose of 3-NOP included in the diet.However, the reduction in daily CH 4 emission was greater (WMD = −68.29 g/d) with high doses (101-329 mg/kg DM) of 3-NOP than with doses between 35 and 100 mg/kg DM (WMD = −34.75g/d).Similarly, CH 4 yield decreased (p < 0.001) regardless of the 3-NOP dose used (Figure 2b).However, the reduction in the CH 4 yield was greater (WMD = −6.82g/kg DM) when high doses (101-280 mg/kg DM) of 3-NOP were used than when the dietary inclusion of 3-NOP ranged from 35 to 100 mg/kg DM (WMD = −3.56g/kg DM).
Fermentation 2024, 10, x FOR PEER REVIEW 7 of 14 Table 5 shows no significant relationship (p > 0.05) between DMI and the covariates included in the models used for the analysis.The 3-NOP dose explained (p < 0.001) 26.40 and 36.04% of the observed heterogeneity in the daily CH4 emission and CH4 yield, respectively.Likewise, the 3-NOP supplementation period explained (p < 0.001) 6.56 and 9.65% of the heterogeneity observed in the daily CH4 emission and CH4 yield, respectively.Furthermore, the amount of concentrate included in the diets explained (p < 0.05) 5.21% of the heterogeneity observed in the daily CH4 emission.

Subgroup Analysis
Figure 2a shows that the daily CH4 emission decreased (p < 0.001), regardless of the dose of 3-NOP included in the diet.However, the reduction in daily CH4 emission was greater (WMD = −68.29 g/d) with high doses (101-329 mg/kg DM) of 3-NOP than with doses between 35 and 100 mg/kg DM (WMD = −34.75g/d).Similarly, CH4 yield decreased (p < 0.001) regardless of the 3-NOP dose used (Figure 2b).However, the reduction in the CH4 yield was greater (WMD = −6.82g/kg DM) when high doses (101-280 mg/kg DM) of 3-NOP were used than when the dietary inclusion of 3-NOP ranged from 35 to 100 mg/kg DM (WMD = −3.56g/kg DM). Figure 3a shows that the daily CH4 emission decreased (p < 0.001) regardless of the 3-NOP supplementation period.However, the reduction in daily CH4 emission was greater (WMD = −68.29 g/d) when supplementation periods lasted more than 60 days compared to supplementation periods of up to 60 days (WMD = −39.19g/d).Likewise, CH4 yield decreased (p < 0.001) regardless of the 3-NOP supplementation period used (Figure 3b).However, the reduction in CH4 yield was greater (WMD = −6.75g/kg DM) when supplementation periods lasted more than 60 days than with supplementation periods of up to 60 days (WMD = −4.25 g/kg DM).On the other hand, the daily CH4 emission decreased Figure 3a shows that the daily CH 4 emission decreased (p < 0.001) regardless of the 3-NOP supplementation period.However, the reduction in daily CH 4 emission was greater (WMD = −68.29 g/d) when supplementation periods lasted more than 60 days compared to supplementation periods of up to 60 days (WMD = −39.19g/d).Likewise, CH 4 yield decreased (p < 0.001) regardless of the 3-NOP supplementation period used (Figure 3b).However, the reduction in CH 4 yield was greater (WMD = −6.75g/kg DM) when supplementation periods lasted more than 60 days than with supplementation periods of up to 60 days (WMD = −4.25 g/kg DM).On the other hand, the daily CH 4 emission decreased (p < 0.001) regardless of the amount of concentrate included in the diets (Figure 3c).However, the reduction in daily CH 4 emission was greater when 3-NOP was included in diets with more than 700 g/kg DM of concentrate (WMD = −61.48g/d) than when 3-NOP was included in diets with up to 700 g/kg DM of concentrate (WMD = −47.31g/d).
Fermentation 2024, 10, x FOR PEER REVIEW 8 of 14 (p < 0.001) regardless of the amount of concentrate included in the diets (Figure 3c).However, the reduction in daily CH4 emission was greater when 3-NOP was included in diets with more than 700 g/kg DM of concentrate (WMD = −61.48g/d) than when 3-NOP was included in diets with up to 700 g/kg DM of concentrate (WMD = −47.31g/d).

Growth Performance
Lower DMI was observed in response to dietary supplementation with 3-NOP.In contrast, a previous meta-analysis [23] did not detect significant changes in the DMI of ruminants supplemented with 3-NOP.However, in that meta-analysis, the database only included four beef cattle studies.Another meta-analysis [22] using seven beef cattle studies reported only a trend toward decreased DMI in these animals.The database of the present meta-analysis used 15 studies, which explains the difference in the results obtained in DMI compared to previous meta-analyses.The exact mechanism through which 3-NOP modifies DMI in ruminants has not yet been studied [22].However, in beef cattle, previous studies [47,48] show that DMI decreases linearly when ruminal propionate increases.Therefore, the lower DMI detected in the present study could be partially explained by the increase in ruminal propionate concentration.
In the current study, 3-NOP supplementation did not affect ADG.Although an increase in ADG would be desirable in beef cattle, it should be considered that the main objective of dietary supplementation with 3-NOP is to reduce enteric CH4 emissions rather than improve growth performance [29].On the other hand, FE increased in response to 3-NOP supplementation.This effect could be directly related to the decrease in DMI and the lack of change in ADG.According to Johnson and Johnson [49], cattle lose up to 12% of the GE ingested due to enteric CH4 emissions.In the present meta-analysis, the emission of CH4 as a percentage of the ingested GE decreased by 30.0%, which could increase the metabolic energy available in the animals and partially explain the better FE observed.Likewise, supplementation with 3-NOP decreased (−10%) the ruminal NH3-N concentration, which is generally associated with increased microbial protein synthesis in the

Growth Performance
Lower DMI was observed in response to dietary supplementation with 3-NOP.In contrast, a previous meta-analysis [23] did not detect significant changes in the DMI of ruminants supplemented with 3-NOP.However, in that meta-analysis, the database only included four beef cattle studies.Another meta-analysis [22] using seven beef cattle studies reported only a trend toward decreased DMI in these animals.The database of the present meta-analysis used 15 studies, which explains the difference in the results obtained in DMI compared to previous meta-analyses.The exact mechanism through which 3-NOP modifies DMI in ruminants has not yet been studied [22].However, in beef cattle, previous studies [47,48] show that DMI decreases linearly when ruminal propionate increases.Therefore, the lower DMI detected in the present study could be partially explained by the increase in ruminal propionate concentration.
In the current study, 3-NOP supplementation did not affect ADG.Although an increase in ADG would be desirable in beef cattle, it should be considered that the main objective of dietary supplementation with 3-NOP is to reduce enteric CH 4 emissions rather than improve growth performance [29].On the other hand, FE increased in response to 3-NOP supplementation.This effect could be directly related to the decrease in DMI and the lack of change in ADG.According to Johnson and Johnson [49], cattle lose up to 12% of the GE ingested due to enteric CH 4 emissions.In the present meta-analysis, the emission of CH 4 as a percentage of the ingested GE decreased by 30.0%, which could increase the metabolic energy available in the animals and partially explain the better FE observed.Likewise, supplementation with 3-NOP decreased (−10%) the ruminal NH 3 -N concentration, which is generally associated with increased microbial protein synthesis in the rumen [50].In ruminants, greater microbial protein synthesis could increase the supply of amino acids to the small intestine and result in greater absorption and the metabolic availability of amino acids [51,52], which would partially explain the increase in FE.

Ruminal Fermentation
Supplementation with 3-NOP increased ruminal pH, which could be explained by the observed reduction in ruminal TVFA concentration [5].Likewise, the average ruminal pH values detected in the current study were within the range (5.8-6.5) reported as normal in beef cattle [53].This result suggests that 3-NOP does not alter microbial populations [51] or physiological functions of the rumen, such as absorption and motility [53].On the other hand, a lower ruminal concentration of NH 3 -N was observed in response to 3-NOP supplementation.The higher rumen concentration of butyrate observed with 3-NOP supplementation could partially explain the reduction in NH 3 -N since, according to Rémond et al. [54], ruminal NH 3 -N absorption is stimulated when ruminal butyrate concentration increases.In dairy cows, dietary supplementation with 3-NOP decreases (−33.9%) the relative rumen abundance of the microbial genus Clostridium [55], within which bacteria with high proteolytic and deaminative activity are found [56,57].A similar effect of 3-NOP consumption in beef cattle would partially explain the reduction in ruminal NH 3 -N concentration in the current study.
In dairy cows, a previous meta-analysis [22] reported similar changes to those obtained in the current study for the ruminal concentration of TVFA, acetate, propionate, butyrate, iso-butyrate, valerate, iso-valerate, and acetate/propionate ratio.The reduction in ruminal TVFA concentration observed in the present study could be explained by the lower DMI in beef cattle supplemented with 3-NOP, as suggested by Kirwan et al. [5].On the other hand, according to several authors [17,18,58], ruminal propionate synthesis is considered the main alternative pathway to eliminate H 2 when methanogenesis is inhibited.Therefore, the increase in ruminal propionate concentration detected in the present study could be closely related to the reduction in daily CH 4 emission.In beef cattle, dietary supplementation with 3-NOP increases the relative abundance of the microbial genus Succiniclasticum, which plays an important role in rumen propionate production through succinate utilization [59].A high ruminal concentration of propionate is positive because this volatile fatty acid is the main source of energy in ruminants [51,58].
Lopes et al. [55] reported that dietary supplementation with 3-NOP decreases (−20.7%) the abundance of Ruminococcus bacteria and increases (+33.3%) the abundance of Butyrivibrio bacteria in the ruminal fluid of dairy cows.Acetate-producing bacteria are found in the Ruminococcus genus [1], while the Butyrivibrio genus contains butyrate-producing microorganisms [55].Therefore, the similar effects of 3-NOP intake on the ruminal microbiota of beef cattle could explain the reduction in acetate and the increase in butyrate observed in the present meta-analysis.Likewise, the increase in the ruminal concentration of butyrate and valerate observed in the present meta-analysis can be explained by the lower emission of CH 4 since, according to several authors [5,58,60], the formation of butyrate and valerate can act as alternative H 2 sinks when methanogenesis is decreased or inhibited.On the other hand, Gruninger et al. [60] detected that 3-NOP increases (+22.5%) the ruminal relative abundance of Bacteroidetes microorganisms in beef cattle.This effect of 3-NOP could partially explain the increases observed in the ruminal concentration of isobutyrate and isovalerate in the current study since, according to Zhao et al. [61], the presence of Bacteroidetes in the rumen of cattle is positively correlated (r > 0.60) with the ruminal concentration of isobutyrate and isovalerate.

Enteric Methane Emissions
According to Gerber et al. [4], enteric CH 4 contributes a high proportion (43%) of the GHGs emitted in beef production systems worldwide.Some studies [28,62] indicate that reducing enteric CH 4 can improve environmental sustainability in beef cattle production systems.In the current study, daily CH 4 emission (−34.9%),CH 4 yield (−29.6%), and CH 4 emission as a percentage of ingested GE (−30.0%)decreased in response to dietary supplementation with 3-NOP.These effects suggest that dietary supplementation with 3-NOP could help improve environmental sustainability in beef production systems.Furthermore, the simultaneous reduction in the three types of CH 4 emission indicates that 3-NOP has a constant anti-methanogenic effect in beef cattle.Similarly, a recent meta-analysis [6] reported that dietary supplementation with 3-NOP decreases daily CH 4 emission, CH 4 yield, and CH 4 intensity by 32.7%, 30.9%, and 32.6%, respectively.Likewise, Martínez-Fernández et al. [14] detected 15.7% lower daily CH 4 emission and 23.7% lower CH 4 yield in sheep supplemented with 3-NOP.According to Duin et al. [3], 3-NOP acts through inhibiting the activity of the enzyme methyl-coenzyme M reductase (MCR), which is necessary for the formation of CH 4 because it catalyzes the last step of methanogenesis [8].Therefore, the lower CH 4 emissions observed in the current study can be explained by this 3-NOP's mechanism of action.Furthermore, 3-NOP can inhibit methanogenic archaea's growth without negatively affecting other non-methanogenic rumen bacteria [3].
Subgroup analyses revealed that the reduction in daily CH 4 emission and CH 4 yield was 49.8 and 47.8% greater, respectively, with high doses (101-329 mg/kg DM) of 3-NOP than with low doses (35-100 mg/kg DM) of 3-NOP.This effect was expected since a previous study in cattle [21] estimated that, for every 10 mg/kg DM increase in the dose of 3-NOP, the emission of enteric CH 4 decreases by 2.56%.This variation in the effects of 3-NOP doses on CH 4 emission could be related to the differential effect that the 3-NOP dose has on the rumen microbiota [7].For example, Romero-Pérez et al. [17] reported that low doses (≤109 mg/kg DM) of 3-NOP do not affect the rumen abundance of methanogens in beef cattle.In contrast, other studies [17,18,32] show that high doses (>200 mg/kg DM) of 3-NOP decrease between 35 and 60% rumen methanogens in beef cattle.Rumen methanogens have a positive correlation with daily CH 4 emission and CH 4 yield in beef cattle [63].Therefore, a lower population of rumen methanogens may result in a greater reduction in enteric CH 4 emissions.
Yu et al. [7] mention that more research is necessary to identify whether the effects of 3-NOP on enteric CH 4 emissions are maintained in the long term.In the current study, some subgroup analyses showed that daily CH 4 emission and CH 4 yield decreased, regardless of the 3-NOP supplementation period.These effects suggest that 3-NOP could be used to mitigate enteric CH 4 in beef cattle for long periods without the apparent risk of adaptation of ruminal microorganisms to its effects.Similar to our results, previous studies also reported a persistent long-term (>10 weeks) reduction in enteric CH 4 emissions in dairy cows [64,65] and calves [66] supplemented with 3-NOP.In the present metaanalysis, another subgroup analysis showed that the reduction in daily CH 4 emission was 23.05% greater when high-concentrate diets (>700 g/kg DM) were used, compared to low-concentrate diets (≤700 g/kg DM).These effects could be related to the variation in the amount of NDF between high and low-concentrate diets.In support of this hypothesis, it has been reported that the effect of 3-NOP on the daily emission of CH 4 decreases by 1.64% for each increase of 10 g/kg of DM in the NDF content of the diet [21], and the level of NDF in ruminant diets decreases linearly when the proportion of concentrate in the diet increases [67].
In the rumen, CH 4 is the main sink for H 2 [58].According to Beauchemin et al. [9], the H 2 available in the rumen can be used by methanogenic archaea as a substrate for the formation of CH 4 .Therefore, the increase in daily H 2 emission observed in the current study may be directly related to the detected reduction in daily CH 4 emission.Furthermore, previous studies [17,18,20] detected between a 21.6 and 82.5% greater abundance of rumen protozoans in beef cattle supplemented with 3-NOP.Ruminal protozoans (e.g., Epidinium, Entodinium, Isotricha, and Dasytricha) have hydrogenosomes [68], which are organelles that produce and release hydrogen into the ruminal environment [69].Therefore, an increase in the abundance of rumen protozoans in beef cattle supplemented with 3-NOP could partially explain the greater daily H 2 emission detected in the present study.Although the emission of H 2 also causes energy loss in ruminants, on average, this energy is much lower than the energy lost due to the enteric emission of CH 4 [58].On the other hand, in the present study, dietary supplementation with 3-NOP did not affect the daily CO 2 emission.Similarly, other authors [64,70] also did not detect significant effects of dietary supplementation with 3-NOP (between 40 and 200 mg/kg DM) on CO 2 emission (g/d, g/kg DMI or g/kg milk yield) in dairy cows.

Conclusions
The dietary supplementation with 3-nitrooxypropanol can be used as a nutritional strategy to improve feed efficiency and ruminal fermentation in beef cattle and, at the same time, reduce enteric methane emissions.The greatest reduction in enteric methane emissions is obtained with high doses (101-329 mg/kg DM) of 3-nitrooxypropanol, using long periods (>60 days) of supplementation with 3-nitrooxypropanol and with diets high in concentrate (>700 g/kg DM).However, more long-term studies are necessary to corroborate the anti-methanogenic effect of 3-nitrooxypropanol over long periods.

Figure 1 .
Figure 1.A PRISMA flow diagram detailing the literature search strategy and study selection for the meta-analysis.

Figure 1 .
Figure 1.A PRISMA flow diagram detailing the literature search strategy and study selection for the meta-analysis.

Figure 2 .
Figure 2. Subgroup analysis (subgroup = doses of 3-nitrooxypropanol (mg/kg DM) of the effect of including 3-nitrooxypropanol in beef cattle diets, WMD = weighted mean differences between 3nitrooxypropanol treatments and control.CI: confidence interval of WMD.

Figure 3 .
Figure 3. Subgroup analysis (subgroups = supplementation period (days) and concentrate in diet (g/kg of DM)) of the effect of 3-nitrooxypropanol on the diet of the beef cattle; WMD = weighted mean differences between 3-nitrooxypropanol treatments and control.CI: confidence interval of WMD.

Figure 3 .
Figure 3. Subgroup analysis (subgroups = supplementation period (days) and concentrate in diet (g/kg of DM)) of the effect of 3-nitrooxypropanol on the diet of the beef cattle; WMD = weighted mean differences between 3-nitrooxypropanol treatments and control.CI: confidence interval of WMD.

Table 1 .
Description of the studies included in the meta-analysis database.

Table 2 .
Growth performance of beef cattle supplemented with 3-nitrooxypropanol.

1 Begg Test 2 Control Means (SD) WMD (95% CI) p-Value p-Value I 2 (%) p-Value p-Value
N: number of studies; NC: number of comparisons between 3-nitrooxypropanol treatment and control treatment; SD: standard deviation; WMD: weighted mean differences between treatments with 3-nitrooxypropanol and control (without 3-nitrooxypropanol); CI: confidence interval of WMD; p-Value to χ 2 (Q) test of heterogeneity; I 2 : proportion of total variation in size effect estimates that is due to heterogeneity; 1 : Egger's regression asymmetry test; 2 : Begg's adjusted rank correlation; DMI: dry matter intake; ADG: average daily gain; FE: feed efficiency.
N: number of studies; NC: number of comparisons between 3-nitrooxypropanol treatment and control treatment; SD: standard deviation; WMD: weighted mean differences between treatments with 3-nitrooxypropanol and control (without 3-nitrooxypropanol); CI: confidence interval of WMD; p-Value to χ 2 (Q) test of heterogeneity; I 2 : proportion of total variation in size effect estimates that is due to heterogeneity; 1 : Egger's regression asymmetry test; 2 : Begg's adjusted rank correlation; NH 3 -N: ammonia nitrogen; TVFA: total volatile fatty acids.

Table 5 .
Meta-regression comparing the associations between covariates and measured outcomes.
QM: coefficient of moderators; QM is considered significant at p ≤ 0.05; Df: degree of freedom; R 2 : the amount of heterogeneity accounted for.DMI: dry matter intake; CH 4: methane.

Table 5 .
Meta-regression comparing the associations between covariates and measured outcomes.
QM: coefficient of moderators; QM is considered significant at p ≤ 0.05; Df: degree of freedom; R 2 : the amount of heterogeneity accounted for.DMI: dry matter intake; CH4: methane.